PHYSIOLOGICAL REVIEWS
Vol. 79, No. 3, July 1999
Printed in U.S.A.
Microvascular Permeability
C. C. MICHEL AND F. E. CURRY
Cellular and Integrative Biology, Division of Biomedical Sciences, Imperial College School of Medicine,
London, United Kingdom; and Department of Human Physiology, School of Medicine, University of
California, Davis, California
0031-9333/99 $15.00 Copyright © 1999 the American Physiological Society
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I. Introduction
II. Microvascular Permeability in Normal (Undisturbed) Tissues
A. General characteristics of microvascular permeability to fluid and hydrophilic solutes
B. Different preparations used for measuring permeability coefficients
C. Characteristics of permeability coefficients and some relations between them
III. Principal Pathways for Water and Small Hydrophilic Solutes
A. A fiber matrix forms the molecular sieve in the interendothelial cleft and at fenestrae
B. Pore models of transvascular exchanges of water and hydrophilic solutes
C. Structure-function relations in continuous capillaries: pores across the capillary wall
D. Structure-function relations in continuous capillaries: pores within the interendothelial cleft
E. Structure-function relations in continuous capillaries: limitation of conventional approaches
F. Structure-function correlation: combined serial sections, tracer studies, and new threedimensional modeling of cleft geometry
G. Structure-function correlation: the molecular sieve and role for the glycocalyx
H. Structure-function correlation: modeling water flows through the breaks in the presence of a
fiber matrix
I. Structure-function correlation: fiber-entrance junctional break model of the endothelial cleft.
Limitations and future developments
J. Further consideration of mammalian muscle capillaries
K. The fiber entrance model and Starling forces across the capillary wall
L. Charge effects at the walls of continuous capillaries
M. Barriers to water and solute in fenestrated microvessels
IV. Transcellular Exchange of Water and Small Hydrophilic Molecules
A. Water
B. Glucose and amino acids
C. Urea
D. Conclusion
V. Permeability to Macromolecules
A. Arguments against transport of macromolecules via vesicles
B. Is macromolecular transport convective in nature?
C. General features of vesicular transport
D. Caveolae and the vesicles of endothelial cells
E. Evidence for the involvement of vesicles in the transendothelial transport of macromolecules
F. Receptor-mediated transport of macromolecules
G. Concluding comments on macromolecular permeability
VI. Increased Microvascular Permeability
A. Phenomena of increased permeability
B. Increased permeability in inflammation and with inflammatory mediators
C. Local edema formation during the initial phase
D. Quantitative estimates of increased permeability with histamine-like mediators
E. Ultrastructural basis of increased microvascular permeability
F. Openings in the endothelium associated with increased permeability
VII. Signal Transduction
A. Overview
B. Receptors
C. Individual microvessels: introduction
D. Individual microvessels: experimental studies
E. Individual microvessels: heterogeneity in endothelial cell responses
F. Comparison with in vitro studies: calcium influx
G. Comparison with in vitro studies: NO/cGMP and calcium entry
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Comparison with in vitro studies: cGMP/NO and increased permeability in venular endothelium
Comparison with in vitro studies: cAMP
Comparison with in vitro studies: PKC
Comparison with in vitro studies: summary of acute inflammatory responses
Mechanisms determining long-term increases in permeability: sustained increases in endothelial
barrier permeability
M. Mechanisms determining resting permeability and long-term increases in permeability:
leukocyte-dependent processes to increase permeability
VIII. Summary
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I. INTRODUCTION
In this review we consider both the “basal” permeability of microvessels to fluid and hydrophilic solutes in
normal (undisturbed) tissues and increased microvascular permeability such as occurs during the early stages of
acute inflammation.
Thus, in the first part of this review, we address the
classical question of relating functional measures of microvascular permeability in “normal” tissues to the ultrastructure of the microvascular wall. We extend the discussion to consider the contribution of endothelial cell
membrane permeability to microvascular permeability to
water and small solutes. We also reexamine the question
of how macromolecules are transported through microvascular walls with particular emphasis on recent work
on the endothelial plasmalemmal vesicular system. In the
second part of the review we examine the phenomena of
increased microvascular permeability and consider the
intracellular signaling mechanisms that enable the endothelial cells to bring this about.
With the focus on the cellular and molecular basis of
microvascular permeability, the content of this review
differs from that of two Physiological Reviews articles
that discussed different aspects of microvascular exchange some 5– 6 years ago. In the first of these, Aukland
and Reed (17) considered the exchange of fluid between
the microvasculature and the interstitium and were concerned more with the properties of the interstitium than
with pathways through microvascular endothelium. In the
second review, Rippe and Haraldsson (246a) discussed
how microvascular permeability to macromolecules
could be described in terms of convection and diffusion
through two populations of pores in microvascular walls.
Although they considered how the “large pores” might be
interpreted in ultrastructural terms, since their review
was published there have been considerable advances in
our general knowledge of vesicular transport and of the
molecular constituents of the caveolae (or uncoated vesicles) of endothelial cells. We have, therefore, reexamined
Rippe and Haraldsson’s conclusions in the light of this
recent work.
Many of the general characteristics of basal microvascular permeability appear to be well established. Surprisingly, this information is not so widely known, although it forms a basis for understanding the role of
microvascular exchange in many physiological systems.
We, therefore, begin our discussion of the permeability of
normal vessels with a summary of this basic information.
II. MICROVASCULAR PERMEABILITY IN
NORMAL (UNDISTURBED) TISSUES
A. General Characteristics of Microvascular
Permeability to Fluid and Hydrophilic Solutes
Although changes in microvascular permeability are
often reported in terms of changes in the fluxes of fluid or
solute between the blood and the tissues, the functional
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Michel, C. C., and F. E. Curry. Microvascular Permeability. Physiol. Rev. 79: 703–761, 1999.—This review
addresses classical questions concerning microvascular permeabiltiy in the light of recent experimental work on
intact microvascular beds, single perfused microvessels, and endothelial cell cultures. Analyses, based on ultrastructural data from serial sections of the clefts between the endothelial cells of microvessels with continuous walls,
conform to the hypothesis that different permeabilities to water and small hydrophilic solutes in microvessels of
different tissues can be accounted for by tortuous three-dimensional pathways that pass through breaks in the
junctional strands. A fiber matrix ultrafilter at the luminal entrance to the clefts is essential if microvascular walls
are to retain their low permeability to macromolecules. Quantitative estimates of exchange through the channels in
the endothelial cell membranes suggest that these contribute little to the permeability of most but not all microvessels. The arguments against the convective transport of macromolecules through porous pathways and for the
passage of macromolecules by transcytosis via mechanisms linked to the integrity of endothelial vesicles are
evaluated. Finally, intracellular signaling mechanisms implicated in transient increases in venular microvessel
permeability such as occur in acute inflammation are reviewed in relation to studies of the molecular mechanisms
involved in signal transduction in cultured endothelial cells.
MICROVASCULAR PERMEABILITY
July 1999
TABLE
1. Membrane permeability coefficients
Coefficient
Symbol
Hydraulic permeability
Hydraulic conductivity
Filtration coefficient
Diffusional permeability
Lp
Solvent drag
(ultrafiltration)
Reflection coefficient
Osmotic reflection
coefficient
Pd
sf
sd
Definition
S D
S D
S D
S D
JV /A
when Dp 5 0
DP
Js /A
when Jv50
DC
Js
12
when DC 5 0
JvC
DP
when Jv 5 0
Dp
Jv /A, net flux of fluid (vol) driven through unit area of the membrane by differences in hydrostatic pressure (DP) and osmotic pressure
(Dp) between solutions flanking membrane; Js /A, net flux of solute per
unit area of membrane; DC, difference in solute concentration across
membrane; C, concentration of solute on both sides of membrane when
DC 5 0.
make for large molecules but not for small hydrophilic
molecules, where there may be large differences between
the arterial and mean microvascular concentrations of the
diffusing solute. The difference between Ca and Cc is
determined by the ratio of the permeability-surface area
product to the flow through the microvessels. This difference becomes negligible at high perfusion rates when
clearance does approximate closely to the product of
permeability and the exchange surface area as the arteriovenous concentration difference approaches zero (e.g.,
Renkin, Ref. 237). The interpretation of clearances of
large or intermediate-sized molecules as permeability coefficients is also complicated. Here, transport may be
dominated by convection rather than diffusion so that the
clearance is a function of both sf and Pd and varies with
the net fluid filtration rate from plasma to the tissues.
Thus clearances, though convenient measures of microvascular exchange, need to be interpreted with caution.
B. Different Preparations Used for Measuring
Permeability Coefficients
Microvascular permeability coefficients have been
reported from measurements on intact whole organisms
(including human subjects), on perfused tissues and organs, on single perfused microvessels, and on monolayers
of cultured microvascular endothelial cells.
These different experimental preparations have their
advantages and disadvantages. Thus, although measurements made on the intact regional circulation of a human
subject suffer from uncertainties surrounding the exchange surface area of microvascular wall and the values
of the transcapillary differences in pressure and concentration, they usually involve minimal interference with the
microvessels themselves. Thus these studies can provide
valuable information concerning microvascular exchange
under basal conditions. At the other extreme are measurements on single vessels. Here the surface area of the
vessel can be measured directly, as also can the difference in pressure and concentration across the vessel
walls. The disadvantages of studies on single vessels,
however, are 1) that they involve direct interference with
the vessels involved, and 2) they are usually restricted to
a small number of convenient vessel types (e.g., mesenteric vessels). Direct interference with a vessel whether it
be exposure to light or micromanipulation might be expected to increase permeability. For this reason, the early
measurements on single microvessels in frog mesentery
were regarded with suspicion, particularly as values of Lp
and Pd to small hydrophilic solutes appeared to be an
order of magnitude higher than estimates of Lp and Pd
based on measurements on intact microvascular beds of
skeletal muscle. This concern was allayed, however,
when it was shown that measurements of Lp (52) and Pd
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measures of microvascular exchange that represent the
properties of microvascular walls are the permeability
coefficients. Because microvascular exchange is largely
(if not entirely) passive, the permeability coefficients relate the net fluxes of fluid (Jv) and solute (Js) to the
differences in pressure (P) and concentration (C) that
drive them through microvascular walls. Four permeability coefficients are of interest: the hydraulic permeability
(or hydraulic conductance or conductivity) (Lp), the diffusional permeability (to a particular solute) (Pd), the
solvent drag or ultrafiltration coefficient (sf), and the
osmotic reflection coefficient (sd). For “ideal” solutes
(i.e., those with activity coefficients of unity), the osmotic
reflection coefficient sd is equal to the ultrafiltration coefficient sf. The permeability coefficients are defined in
Table 1. For a full discussion of their significance, the
reader is referred to Curry (48).
In addition to the membrane coefficients, the term
clearance is often used to describe microvascular exchange. The clearance of a substance from one compartment to another is defined as the net flux of material
divided by the solute concentration in the compartment
from which it is being cleared. Thus, if a solute is being
cleared from the blood as it flows through a microvascular bed into the tissues, the clearance of the substance is
equal to the flux of solute from blood to tissues (Js)
divided by the arterial concentration of solute (Ca), i.e.,
Js/Ca. When the flux is unidirectional (as it may be for a
tracer diffusing into a large tissue store), the clearance
may approximate to the product of the diffusional permeability and the surface area of the microvascular walls
through which exchange occurs. This approximation is
only valid if the permeability to the substance is so low
that the mean solute concentration in the microvessels
(Cc) is equal to Ca. This is a reasonable assumption to
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FIG. 1. Relation between permeability (Pd) of microvessels in skeletal muscle to hydrophilic solutes and solute molecular radius. Permeability has been plotted on a logarithmic scale to show range of values,
although it is possible that values of Pd for the largest molecules are
overestimates (see text). [Data from Renkin (238a).]
solutions (D) as their molecular size increases. This is not,
however, the entire story. In Figure 2, the ratio of Pd to D
for each of the molecules shown in Figure 1 has been
plotted against molecular radius. It is seen that Pd /D falls
by more than an order of magnitude as molecular radius
increases from 0.23 to 3.6 nm. If Pd declined only as a
consequence of the fall in D, the ratio Pd /D would be
constant. The decline of Pd /D with molecular size was
first described by Pappenheimer et al. (222) as “restricted
diffusion.” They correctly interpreted it to be a consequence of the diffusion of the solutes through water-filled
C. Characteristics of Permeability Coefficients and
Some Relations Between Them
Figure 1 shows how Pd to hydrophilic solutes in the
microcirculation of skeletal muscle declines as solute
molecular size increases. The values for Pd have been
plotted on a logarithmic scale, and it is seen that the
decline of Pd is maintained until the molecular radius
reaches 3.6 nm (the Stokes-Einstein radius of serum albumin).
Values of Pd for molecules larger than serum albumin
appear to decrease much less rapidly with increasing
molecular size, suggesting that either the pathways or
mechanisms concerned in the transport of macromolecules differ from those for smaller solutes. The decline in
Pd of the smaller molecules is partly accounted for by the
decrease in their free diffusion coefficients in aqueous
FIG. 2. Restricted diffusion of hydrophilic solutes at walls of microvessels in skeletal muscle. Values of Pd shown in Figure 1 have been
divided by free diffusion coefficient (D) of same solute. If fall in Pd with
increasing molecular radius were due to reduction in D alone, then Pd /D
would be a constant. It is seen that Pd /D falls by nearly 2 orders of
magnitude as molecular radius rises from 2.4 to 36.
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to potassium ions (79) in single muscle capillaries were an
order of magnitude lower than their values in mesenteric
vessels and similar to values based on indirect measurements on the intact muscle microcirculation. The differences lay in the different permeabilities of microvessels in
different vascular beds and were not the consequence of
exposure or manipulation of the tissues. Interestingly, the
reflection coefficient of mesenteric capillaries to macromolecules such as serum albumin is similar to that of
muscle microvessels.
Although the rapid growth of endothelial cell biology
is largely a result of experiments on cultured endothelial
cells in vitro, there are serious limitations to the use of
monolayers of cultured endothelial cells for gaining direct
information about vascular permeability. The most widely
reported permeability measurement on monolayers of
cultured endothelium is Pd to serum albumin, and mean
values are usually in the range of 1026 cm/s (11). This is
two orders of magnitude greater than estimates based on
the flux of albumin through the walls of intact microvessels. Estimates of the reflection coefficients of cultured
monolayers of endothelial cells to macromolecules are
too low for plasma proteins to exert a significant osmotic
pressure across them (302). Reports of monolayers of
cultured endothelium with high values of s to macromolecules appear to be based on an erroneous calculation of
s (290, 291). These results clearly indicate that monolayers of cultured endothelial cells do not reflect the permeability characteristics of microvascular endothelium in
vivo. For this reason, we have restricted discussion of
permeability properties and the values of permeability
coefficients to measurements made on either single vessels or on intact microvascular beds. We have, however,
referred extensively to work on cultured endothelium in
section VII, where signaling mechanisms within the endothelial cells are discussed.
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MICROVASCULAR PERMEABILITY
FIG. 3. Reflection coefficient to serum albumin (salbumin) and hydraulic permeability (Lp) in different microvascular beds. Each point
represents mean value of salbumin and Lp for microvessels in a particular
tissue: Œ, cat hindlimb; ●, rat hindlimb; ‚, dog lung; e, dog heart; n, frog
mesentery; m, rabbit salivary gland; L, dog small intestine; E, dog glomerulus; ƒ, rat glomerulus. [From Michel (186).]
FIG. 4. Relations between hydraulic permeabilities (Lp) of microvessels in different tissues and their diffusional permeabilities to small and
intermediate-sized molecules (Pd). Solid circles are values for Lp plotted
against values for Pd to either sodium or potassium from same vessel.
Open circles are corresponding data for Pd to inulin. Values of Lp and Pd
have been plotted on logarithmic scales to show a range of 2 orders of
magnitude. Lines are not regression lines but have been constructed
through points with a slope of unity to indicate direct proportionality.
is that variations in Lp in different microvessels are not
accompanied by variations in their leakiness to macromolecules. If we think of the pathways through microvascular walls as water-filled pores, then it would seem that
the variations in Lp are accounted for by variations in the
number of pores per unit area of wall in different vessels,
but the diameter of the pores (which determines the
reflection coefficients to macromolecules) is fairly constant.
If the differences in permeability of different microvascular beds are the consequence of variations in the
numbers of channels (of constant selectivity) per unit
area of wall in different microvessels, we might anticipate
that Lp is proportional to Pd for small hydrophilic solutes.
This is shown in Figure 4. Here again, each point represents the mean value of Lp for a particular type of microvessel plotted against mean Pd to inulin (open symbols) or mean Pd to sodium or potassium (solid symbols)
for the same vessel. Both scales are logarithmic so that
values covering two orders of magnitude can be shown.
The lines relating Lp to Pd have been drawn to have slopes
equal to unity, indicating direct proportionality. One conclusion from Figure 4 might seem to be that water and
hydrophilic solutes cross microvascular walls by the
same pathways. As we shall see, this is not entirely true,
and a small fraction (10%) of the Lp may be accounted for
by a path from which hydrophilic solutes are excluded. It
does suggest that the dominant pathway for fluid and
solute exchange is one which they share, and this does
appear to be true. Direct proportionality between Lp and
Pd is also consistent with the hypothesis that variations in
these coefficients are the result of variations in pore or
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pores or channels with diameters or widths that were up
to an order of magnitude larger than the diameter of the
diffusing molecule. They pointed out that as the molecular diameter increased, so the degree of exclusion of the
molecules from the pore and the viscous drag on the
diffusing molecule would also increase.
Values for Pd of the largest molecules shown in Figure 1 (and corresponding values of Pd/D in Fig. 2) are
probably overestimates because they are based on measurements of transport between the plasma and the
lymph. This method requires that steady-state transport is
established, and such a steady state is difficult to achieve
in microvascular beds such as those of muscle where Lp
is low (see sect. V and Renkin and Tucker, Ref. 245).
Figures 1 and 2 describe relations between solute
permeability and molecular size in microvessels in mammalian skeletal muscle. Similar relations are found in
other types of microvessels, although the absolute values
of permeability to the smallest molecules may vary by
several orders of magnitude. The microvessels in very
different tissues, however, have rather similar values for
sf or sd to macromolecules. The wide range of values of
Lp and the relatively constant value of s to serum albumin
in different types of microvessel are shown in Figure 3.
Each point in this diagram represents the mean value of
Lp and s to albumin for a different type of microvessel or
microvascular bed.
The Lp value has been plotted on a logarithmic scale
so that values covering three orders of magnitude can be
displayed. It is seen that there is no correlation between s
to albumin and Lp. The value of s is as high in those
vessels with Lp values in the range of 4 3 1026
cmzs21 z cmH2O21 as it is in those vessels with Lp values
of 1028 cmzs21 z cmH2O21. The conclusion from Figure 3
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matical modeling studies have been used to explore these
ideas.
III. PRINCIPAL PATHWAYS FOR WATER AND
SMALL HYDROPHILIC SOLUTES
A. A Fiber Matrix Forms the Molecular Sieve in
the Interendothelial Cleft and at Fenestrae
The fiber matrix model of capillary permeability can
now be understood as providing a molecular understanding of the size selectivity of the wall of microvessels
described in the classical pore theory. In microvessels
with continuous endothelium, the principal pathway for
water and solutes lies between the endothelial cells
through the interendothelial cell cleft. Furthermore, when
combined with estimates of the size, structure, and density of fenestrations, the fiber matrix theory not only
accounts for the similarity in selectivity properties between fenestrated and continuous endothelium but also
for the resistance of the matrix layer to water and solutes
in fenestrated endothelium. In fact, it is likely that a fiber
matrix determines the selectivity of all the possible pathways across the capillary wall shared by water and solutes. In this section, we first review the problems of
interpreting classical pore theory in terms of the ultrastructure of the cleft between adjacent endothelial cells in
continuous endothelium. We highlight the idea that classical pore theory restricted us to a simple one-dimensional interpretation of the cleft geometry leading to difficulties in attempts to understand pore densities in terms
of the fraction of the length of the junction between
adjacent endothelial cells that is effectively open to exchange. We also review the evidence that the molecular
filter is not present at the level of the breaks in the
junctional strand. Thus pore size is not determined by the
space between adjacent endothelial cells at the level of
the tight junction but by the interfiber spacing in a fiber
matrix at the entrance to the interendothelial cleft. Fenestrated microvessels are described at the end of the section
(see sect. IIIM).
B. Pore Models of Transvascular Exchanges of
Water and Hydrophilic Solutes
For water and water-soluble solutes, many of the
principal permeability properties of the capillary wall can
be described in terms of flow through water-filled cylindrical pores or rectangular slits through the vessel wall.
Pore theory describes the resistance to water flow in
terms of the viscous energy dissipation assuming laminar
Poiseuille flow within a pore, the resistance to diffusion in
terms of the additional drag on a spherical molecule
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channel numbers per unit area of vessel wall and not
variations in pore size. If increases in channel dimensions
were responsible for increases in permeability, Lp would
be expected to rise more rapidly that Pd and the slope of
the relations in Figure 4 would be greater than one.
We have indicated that general characteristics of the
permeability coefficients that are illustrated in Figures
1– 4 can be interpreted in terms of pores or channels of
constant selectivity. When we come to consider the real
ultrastructure of the microvascular walls, we are presented with a greater challenge. The walls of the vessels
with high values of Lp (.1026 cm z s21 z cmH2O21) are
fenestrated endothelium, whereas those of vessels with
lower Lp values are continuous endothelium. Very different pathways are primarily responsible for the transport
of fluid and hydrophilic solutes through these two types of
endothelia. In fenestrated endothelia, this pathway is
through the fenestrae, whereas in continuous endothelium, it is through the intercellular clefts. It is, therefore,
very surprising that the clear differences in morphology
are not accompanied by a qualitative change in the properties of the permeability coefficients. The permeabilities
of fenestrated vessels to intermediate-sized molecules are
characterized by restricted diffusion, and their reflection
coefficients to macromolecules are similar to those of
vessels with continuous endothelium. There is little difference in the permeability characteristics of vessels from
the salivary gland (which fall at the bottom end of the
range of Lp for fenestrated vessels) and those of mesenteric capillaries (which have high permeabilities for vessels with continuous endothelium). It would seem that the
presence of fenestrations has an effect that is quantitative
rather than qualitative on the permeability characteristics
of a vessel. Fenestrations increase the Lp and the Pd to
small hydrophilic solutes without changing the Lp/Pd or s
to macromolecules such as albumin. Furthermore, as the
number of fenestrations per unit area of vessel wall increases, both Lp and the Pd to small hydrophilic solutes
increase in direct proportion (152).
Thus the molecular sieving characteristics of microvascular walls appear to be determined by the properties
of some structure that is common to (or very similar in)
both fenestrated and continuous endothelium. Curry and
Michel (56) suggested that it was the luminal glycocalyx
that acted as the molecular filter in both types of vessels,
and it seems likely that this might be so. They also suggested that differences in Lp in different types of vessel
with continuous endothelium might be determined by
differing extents to which the intercellular clefts were
open. It was suggested that in all these vessels, the ultrafiltration properties of the vessel wall (s to macromolecules; degree of restricted diffusion to small solutes) were
largely determined by the luminal glycocalyx. In section
III, we discuss how reasonable this view has proven to be
and how recent experimental work coupled with mathe-
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moving within the pore relative to movement in free
solution, and the selectivity of the membrane in terms of
steric exclusion at the pore entrance (48, 187, 294). For
each population of pores within the membrane forming
the capillary wall, a hydraulic conductivity Lp can be
calculated from Poiseuille’s Law
L p ~pore! 5
npR4
8 h Dx
wpore 5 (1 2 a)2
Dpore
5 1 2 2.10444a 1 2.08877a3 2 0.094813a5 2 1.372a6
Dfree
where L is the total slit length per unit area of vessel wall
and f is the fraction of the length of the slit open to a
width W. The Lp of the whole membrane is the sum of the
individual pathway Lp values each weighted by area of the
pathway relative to total membrane area.
The corresponding relations for the solute permeability coefficients (Pd) are
P d ~pore! 5 n p R 2 3 D pore 3
f
Dx
f
Dx
where Dpore or Dslit are the solute diffusion coefficients
within the pore or slit, w is the solute partition coefficient
and f is the fraction of slit which is open. The whole
membrane permeability coefficient also is the sum of the
individual pathway coefficients. Specific relations for diffusion coefficients and solute partition coefficients in cylindrical pore and rectangular slits are given in Table 2.
Implicit in the relations for Pd are the definitions of the
pore area per unit membrane area (fractional area for
exchange) for each pore population (Ap) equal to npR2 or
WfL and a pore area per unit cleft depth (Ap/Dx) equal to
npR2/Dx or WfL/Dx. A key assumption is that the models
are one dimensional. They do not account for lateral
spreading at the pore entrance or exit, or within the
membrane, and they do not account for interactions between pores. More recent work has highlighted the importance of these interactions in two- and three-dimensional models (see sect. III, F-I).
In a pore, the solute partition coefficient is a measure
of the area available to solute within the pore entrance
wslit 5 1 2 a9
Dslit
5 1 2 1.004a9 1 0.418~a9!3 1 0.210~a9!4 2 0.1696~a9!5
Dfree
w, Partition coefficient; D/Dfree, fractional reduction in free diffusion coefficient; a, solute radius (a)/pore radius (R); a9, solute radius
(a)/slit half width (W/2).
relative to water. The osmotic reflection coefficient (s) of
a porous membrane is a measure of the selectivity of the
membrane to a particular solute that depends only on
pore size, and not pore number or membrane thickness,
and is given by the relation (48)
s 5 ~1 2 f ! 2
When there are several pathways in parallel, the membrane
reflection coefficient is the sum of the individual coefficients
weighted by the fractional contribution of each pathway to
the membrane hydraulic conductivity. A fundamental assumption when using classical pore theory is that the same
structures that determine the selectivity of the membrane
also determine the primary resistance to water flow and
solute diffusion. Thus a constraint on the interpretation of
measured values of the reflection coefficient is that for a
pore or slit within the endothelial barrier to be the primary
selective barrier, it must also be the largest diffusion barrier
in the pathway (with a corresponding large concentration
drop across the barrier) (50).
There is now abundant data demonstrating that the
magnitude of transcapillary flows of water, and the selectivity properties of many microvessels, to solutes ranging
in size from electrolytes to plasma proteins and having
both continuous endothelium and fenestrated endothelium, can be described in terms of three porous pathways
in parallel: an exclusive water pathway across the endothelial cells, a population of small pores with a radius of
4 –5 nm, and a population of larger pores with 20- to 30-nm
radius (246a, 294). Estimates of the relative number of
small pores to large pores fall in the range of 4,000 to 1
(skeletal muscle) to ,1,000 to 1.
There are also well-documented limitations to these
idealized pore models. For example, the pore sizes and
pore densities that describe the water flows and the selectivity of the wall to macromolecules do not always
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LfW 3
12 h Dx
P d ~slit! 5 fWL 3 D slit 3
Cylindrical pores
Rectangular slits (L .. W)
where n is the density of pores (number/cm2) of radius R,
h is water viscosity, and Dx is the membrane thickness.
The equivalent relation for long rectangular slits used to
describe the space between adjacent endothelial cells,
and with Dx measured as the depth of the cleft from
lumen to tissue, is
L p ~slit! 5
2. Solute partition coefficient and diffusion
coefficients in pores or slits
TABLE
710
C. C. MICHEL AND F. E. CURRY
C. Structure-Function Relations in Continuous
Capillaries: Pores Across the Capillary Wall
As a first step to relate these pore structures to the
ultrastructure of the microvessel wall, it is useful to update calculations by Renkin and Curry (240) describing
the exchange properties of a typical microvessel within
mammalian skeletal muscle. Given a typical hydraulic
conductivity of the order of 1 3 1028 cm z s21 z cmH2O21
for skeletal muscle (see sect. I), pore theory enables the
calculation of the pore density, which accounts for the
permeability properties of the porous pathways in a typical vessel 5 mm in diameter and 1,000 mm in length, with
a total surface area for exchange of ;16,000 mm2, formed
by 32 endothelial cells each with a mean area of 500
mm2/cell. The estimate of pore density depends on the
value of the length of the exchange pathway across the
wall, Dx, which characterizes the porous pathway. For a
value characteristic of a straight channel across the average thickness of an endothelial cells (0.2 mm), this vessel
would contain the equivalent of 9,000 –11,000 small pores
of 5-nm radius and 2.2 large pores of 30-nm radius in
parallel with an exclusive water pathway. For a longer
pathway (up to 0.8 mm or longer through the cleft between cells), the vessel would have to contain four times
this pore density. These pore densities correspond to pore
areas ranging from ,0.01% to close to 0.04% of the total
capillary surface area.
D. Structure-Function Relations in Continuous
Capillaries: Pores Within the
Interendothelial Cleft
As a further step to understand the cellular and molecular basis for the permeability properties of continu-
ous endothelium, we review attempts to relate these calculated pore sizes and densities to the structure of the
microvessel wall. Pappenheimer et al. (222) estimated
that the fractional area of the small pores (Ap/As) was
,1% of the total microvessel surface area and suggested
that the small pore system lay within the interstices of the
intercellular cement that was assumed to be present in
the intercellular junctions. The model of a fiber matrix
within part of the cleft provides a quantitative description
of almost the same idea and is developed throughout this
section.
One estimate of the maximum area for exchange
occupied by the space between endothelial cells can be
calculated from morphological estimates of the length of
the line of contact per unit area per cell (L) multiplied by
the width of the wide part of the intercellular junction (W)
(see slit pore theory above). The value of W, measured as
the average spacing between the walls of adjacent endothelial cells in the interendothlial cleft (close to 20 nm), is
a remarkably consistent figure in regions more than 30 nm
away from the junctional strand in normal endothelium
(9, 27, 279). This constant spacing has been explained on
theoretical grounds to reflect the presence of spacer molecules that span the wide part of the cleft (120, 278), and
there is some experimental evidence to support the presence of such structures (260, 275). The value of L, estimated to lie in the range 1,200 –2,000 cm/cm2 (5, 28) in
skeletal muscle, heart muscle, and mesenteric capillaries
depends on endothelial cell size and shape and on the
extent of interdigitation of cells.
Together, these values of L and W provide maximum
estimates of the area for exchange between cells of the
order of 0.4% of the total capillary surface area. Thus the
actual area of the cylindrical small pores described above
for water and solutes in skeletal muscle (,0.01– 0.04% of
the total surface area) leads to the estimate that from 2.5
to 10% of the length of cell-cell contact between adjacent
endothelial cells needs to be effectively open to accommodate the surface area for exchange estimated above
(52, 79, 149, 226). The calculation is based on the assumption that flows through the cleft are essentially one dimensional. This means that there is an exact correspondence between the magnitude of flows through the cleft
and the fraction of open junction. Because of the nature
of the structure of the junctional strands, this simple
correlation is no longer thought to exist (see Fig. 5 and
sect. III, F-I).
E. Structure-Function Relations in Continuous
Capillaries: Limitation of Conventional
Approaches
Although the idea that only a small fraction of the
area between adjacent cells was needed for transport was
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account for the measured fluxes of solutes across the
capillary wall (50, 186). Also, macromolecular transport is
not always coupled to water flows as expected in large
pores (242, 244). Furthermore, as shown in section IIIC,
the average number of large pores in any one capillary is
small. It is likely that there are many vessels with no large
pores at all, and the pore model fails to account for the
transport of macromolecules in these vessels. An even
more fundamental problem is that real pores having the
size and properties described by the pore theory (structure offering a uniform resistance to flows across the
entire thickness of the membrane) have not been found
and probably do not exist. However, just as the idea of the
Stokes radius of a molecule undergoing free diffusion
provides an effective description of the overall resistance
to diffusion of complex ions, sugars, and proteins in solution, the pore theory is a useful starting point to evaluate the possible cellular and molecular structures that
actually determine the permeability properties of the wall.
Volume 79
July 1999
MICROVASCULAR PERMEABILITY
711
FIG. 5. Diagrammatic representation
of 3 possible regimes for flow through
intercellular cleft. A: cleft entirely open
and flow at constant velocity through its
entire length. B: 1-dimensional flow limited to area of discontinuities in tight
junction and wide region of cleft immediately above and below break. C: 2-dimensional convergent and divergent flow
through wide regions of cleft above and
below discontinuities in tight junction.
[From Adamson and Michel (9).]
radius in true capillaries and argued that the leakage sites
described using electron-dense tracers in previous experiments were present only in venular capillaries.
It was also recognized by Wissig (318) and Wissig and
Williams (319) that the apparent penetration of a tracer
across a junctional strand when observed in a single
random section did not prove the existence of a break in
the strand at that position. Tracer may have crossed the
strand at a break on either side of the section and spread
by following a tortuous path or by lateral diffusion. The
same arguments also compromise attempts to estimate
break frequency from tracer labeling. On the other hand,
breaks smaller than the thickness of one section are not
likely to be found in the absence of tracer. Thus the
evidence suggesting the presence of breaks, and the ultrastructure of the breaks in the junctional strand, based
on random sections where adjacent serial sections were
not examined, and where vessel type was not known, was
severely compromised. Experiments based on serial sections, in the presence of tracer, and with section thickness
adjusted to match possible break size were required. Both
the sample sizes and technical limitations raise formidable sampling issues in capillaries having the permeability
properties of the continuous endothelium in mammalian
muscle, lung, and skin.
Before 1993, the most detailed evaluation of these
questions in mammalian microvessels has been carried
out by Bundgaard (27), who used serial section methods
in rat heart capillaries and venules, but without the use of
a tracer. In a total of 69 segments within true capillaries
and venules representing a total length of junctional
strand close to 40 mm [calculated from an average of 15
serial sections/segment, each 40 –50 nm (0.04 mm) in
thickness], Bundgaard (27) reported only 6 sections with
detectable breaks in the junctional strand: 3 in capillaries
and 3 in a single venule. The reconstructions demon-
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developed over 25 years ago at the Benzon Symposium on
Capillary Permeability (149), the interpretation of this
result in terms of the ultrastructure of the junction remains a fundamental problem. The simplest interpretation is based on the assumption that the fraction of the
junction that is effectively open is determined by breaks
or discontinuities in the junctional strands. In freeze fracture, the junctional strands are seen as lines of particles in
the membranes of the adjacent cells. These form an interconnected network that is most complex in arteriolar
and true capillaries and less complex in venular capillaries (279, 282). In endothelial cells, the number and complexity of junction arrangement is generally less than in
epithelial membranes. The location of the junctional
strands in an electron micrograph of the cleft between
adjacent cells corresponds to the position of the so-called
“tight junction” region between the cells.
The hypothesis that up to 10% of the junction length
might be open for exchange of solutes leads to the reasonable expectation that breaks in the junctional strand
should be observable in conventional electron microscopy. During the late 1960s and 1970s, investigators demonstrated the penetration of electron-dense molecular
probes (mol wt 10 – 45,000) across the junction strands in
sections from mammalian skeletal muscle (279, 318). In
addition, investigations of junctional ultrastructure suggested that, at the level of the tight junction, membranes
of adjacent endothelial cells did not fuse but were separated by a space 4 – 8 nm wide. All studies were carried
out on randomly selected sections, and none provided
data on the frequency of the breaks. Thus a fundamental
question for over two decades has been whether there are
breaks in the junctional strand with a size and frequency
consistent with the expectations of the pore models described above. For example, Simionescu et al. (282) found
no leakage sites for tracer probes larger than 3 nm in
712
C. C. MICHEL AND F. E. CURRY
within the wide part of the cleft; the resulting concentration gradients (a type of unstirred layer effect on either
side of the junctional strand) mean that the 6- to 8-nm slit
is much less effective as a molecular sieve than expected
from its dimensions alone (50, 57).
A second problem is that tracer studies in mammalian microvessels continue to show that most of the length
of the junction is effectively impermeable to small electron-dense tracers (197, 232, 309, 310). On the other hand,
it has been recognized recently that the calculation of the
effective area of open junction associated with the breaks
described in conventional sections may be significantly
underestimated. This is because two-dimensional spreading of water and solute flows on the luminal and abluminal sides of breaks increases the effective area for exchange in the breaks by two- to fourfold, as explained
below. Finally, the problem that there is no structure that
might form a molecular sieve at the level of larger breaks
in the junction strand is resolved if structures at the
entrance to the endothelial cleft, formed by the endothelial cell glycocalyx, form the primary molecular sieve in
transcapillary pathways (56, 185, 186). The key to these
new developments has been the use of serial section
methods, with and without tracer, in microvessels of frog
mesentery. These vessels have continuous endothelium,
but the fraction of open junction is larger than in mammalian muscle capillaries and therefore more easy to
analyze. We review this work in the next sections and
then use these new insights to reevaluate studies in mammalian microvessels where the sampling problems remain
unresolved.
F. Structure-Function Correlation: Combined
Serial Sections, Tracer Studies, and New ThreeDimensional Modeling of Cleft Geometry
Adamson and Michel (9) investigated the ultrastructure of true capillaries in frog mesentery using serial
sections. The strategy to use frog mesenteric microvessels exploited the observation that these vessels have
mean hydraulic conductivities and permeabilities to small
solutes that are 3–10 times higher than those in muscle
capillaries of frog and mammals. Thus it was predicted
that the size of the breaks and/or frequency of breaks in
the junctional strand would be larger than in mammalian
heart and skeletal muscle (46, 50). In the microvessels
used for ultrastructural analysis, the hydraulic conductivity was measured before each vessel was fixed under
well-controlled experimental conditions. Thus these studies provide a unique picture of the ultrastructure of vessels of known permeability. In addition, in some vessels,
the low-molecular-weight electron-dense tracer lanthanum, perfused for short periods up to 15 s, was used to
identify specific pathways for small solutes across the
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strated that at a break, the intercellular cleft is open to a
width close to 20 nm at the level of the discontinuity. This
is the same as the width in the cleft far from the junctional
strand and shows no restrictive structure at the level of
the break.
The break frequency observed by Bundgaard (27)
using conventional thin sections (1/13.3 mm in true capillaries or 1/6.9 mm overall) was far too high to represent
large pores, even though they were close to the size of
large pores (40 –50 nm long and up to 20 nm wide). On the
other hand, Bundgaard (27) suggested that the observed
break frequency was too low to account for normal permeability of heart microvessels to small solutes. This
argument can be summarized as follows. For a break to
be detected using sections 40 –50 nm thick, it must be
longer that the section thickness. If the break length
detected by Bundgaard is set at 80 –100 nm (twice the
length of a single section), the total length of discontinuities measured in his serial section experiments is no
more than 1.5% of the total strand length. Given that
Bundgaard measured a mean interendothelial cleft depth
close to 0.8 mm, this figure is less than one-third that
estimated previously (96) as necessary to account for the
flux of small solutes across the microvessel wall.
Bundgaard (27) also recognized that conventional thin
sections may fail to detect breaks in the junction strand
that are smaller than 40 –50 nm long. To test the latter
possibility, he reexamined a small sample of 16 ultrathin
serial sections of average thickness 12.5 nm, collected
previously to investigate vesicles in muscle capillaries. In
these ultrathin sections, Bundgaard found two regions,
more than one section thick, where the adjacent endothelial cell membranes were separated by a distance greater
than 4 nm but less than the 20 nm characteristic of the
larger breaks. Because the larger discontinuities apparently did not account for sufficient open junction,
Bundgaard (27) suggested that the open portion of the
junction might lie within very small breaks of the order of
12.5–15 nm long and 4 –20 nm wide distributed along the
junctions.
When published in the early 1980s, Bundgaard’s data
(27) provided new support for the idea that both the
extent of the open junction and the magnitude of the
size-limiting structure of the interendothelial cleft might
reside within the fine structure of the junctional strand.
The model, known as the “constricted slit model,” described tracer penetration through two barriers in series:
the wide portion of the cleft modeled as a parallel sided
slit up to 20 nm wide and accounting for more than
three-fourths of the cleft depth and a narrow slit up to 8
nm wide that was assumed to form the principal sizelimiting structure (46). Subsequent analysis has exposed
limitations in the arguments used to support this model.
One important limitation is that the resistance to diffusion
of molecules larger than 1-nm radius can be significant
Volume 79
July 1999
MICROVASCULAR PERMEABILITY
microvessel wall. The approach extended previous investigations of the ultrastructure of junctions, the distribution of electron-dense tracers in the junction, studies of
transport by vesicles, and investigations of the structure
of cell surface glycocalyx in frog mesenteric microvessels
(7, 37–39, 156, 169). These earlier studies, and independent investigations by Bundgaard and Frokjaer-Jensen
(28), demonstrated that, within the limitations of the
methods available, the continuous capillaries of frog mesentery shared the same ultrastructure characteristics as
continuous capillaries in mammalian vessels.
Figure 6 shows details of serial section reconstructions in vessels with Lp values ranging from 2.2 to 3.6
cm z s21 z cmH2O21, perfused with lanthanum tracer. In
six capillaries perfused with lanthanum, Adamson and
Michel (9) found that the tracer filled the entire depth of
the junction from lumen to tissue to mark clear passage of
this low-molecular-weight tracer at 11 sites within a total
strand length of 23.5 mm. Five of the regions were completely delimited within the serial sections; in five others,
only the first or last section lay within the leaky region,
and at one site, the tracer penetrated the junction for 22
consecutive sections. The breaks account for 2.52 mm or
10.6% of the length of junction examined. In the absence
of the tracer, Adamson and Michel (9) also reconstructed
from serial sections on three capillaries, three breaks of
0.14, 0.14, and 0.17 mm long within a total capillary length
of 13.36 mm. Although the fraction of open junction here
is only 3.4%, it is not significantly different from the higher
figure that was found in the presence of the lanthanum
tracer. Thus Adamson and Michel (9) showed that in frog
mesenteric capillaries, the breaks in the junctional
strands between the endothelial cells are longer and occur more frequently than in capillaries of cardiac or skeletal muscle. This difference in ultrastructure of the tight
junction is consistent with the Lp and Pd to small hydrophilic molecules being higher in mesenteric capillaries
than in the capillaries of either of the other tissues.
Following the lead of Ward et al. (311), Adamson and
Michel (9) also examined the tight junctions between the
endothelial cells on a tilting stage and found that the outer
leaflets of the cell membranes were not fused but separated by an electron-lucent region with a mean width of
2.3 nm. By demonstrating that the lanthanum ions made
their full contribution to the osmolarity of their perfusates, they confirmed that the lanthanum ions were in
solution and that the failure of lanthanum to penetrate
this potential pathway through the junction was not due
to the formation of large ion complexes. They concluded
that if a pathway is present through the tight junction, it
appears to have a lower permeability than the dimensions
of the electron-lucent region would suggest. The principal
pathway through the intercellular cleft lies through the
breaks or discontinuities in the junctional strands. Recent
work on the molecular structure of the tight junction is
consistent with the observations that the outer leaflets of
the cell membranes are not fused. The transmembrane
protein associated with tight junctions has been identified
as occludin (85a). This is a 64-kDa protein, and structural
models, based on its novel 504-amino acid sequence, indicate that four hydrophobic transmembrane helices allow each protein to form two extracellular loops of 44 and
45 amino acids. It is proposed that the tight junction is
formed by the interlocking loops of occludin molecules
from adjacent cells. If this model is correct, then the
distance between the outer leaflets will be determined by
the lipophilic properties of the outer section of each loop.
Such properties may allow the outer section of the loop of
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FIG. 6. Reconstructions of discontinuities in tight junction from
serial sections of intercellular cleft in frog mesenteric microvessels
perfused without lanthanum in perfusate (top) and after 10-s perfusion
with lanthanum before fixation (bottom). [Modified from Adamson and
Michel (9).]
713
714
C. C. MICHEL AND F. E. CURRY
C~ l , 0.5! 5 K@~1 2 a 21 ! 1/2 #/ l 3 K~ a 21/2 !
where K is a complete elliptic integral of the first kind and
a 5 (1 2 cosh pl)2/4 cosh pl. For a slit 20 nm wide which
occupies 10% of the junction length, Lp (Poiseuille) is
3.4 3 1027 cm z s21 z cmH2O21 and C 5 2.14, so the
predicted Lp of the vessel wall is 7 3 1027
cm z s21 z cmH2O21. The measured Lp values of the vessels in these studies fell in the range of 2.2–3.6 3 1027
cm z s21 z cmH2O21. Thus the measured Lp values can be
accommodated by flow through the observed breaks,
even before the additional resistance to water flow associated with structures that may form the molecular sieve
are added. An understanding of the two-dimensional
spreading of flow to amplify the effective area available
for exchange has provided a new way to investigate structure-function relationships in the interendothelial cleft
over the past 5 years. All previous theory was restricted to
“one-dimensional” models such as the pore theory and the
one-dimensional slit model (Fig. 5, A–C).
The diffusion of small solutes through breaks in the
junctional strand is described by a two-dimensional diffusion equation whose form is identical to the equation
for the pressure distribution determining water flows in
Figure 5. Thus the solute permeability coefficients of the
vessel wall in which there is a two-dimensional diffusion
profile are described by the relation
P 5 C 3 P slit
where C accounts for the two-dimensional spread of solute to increase flux flow through the discontinuity and has
the same value as for water flow and Pslit is fLDslit/DX, the
permeability of a rectangular slit assuming one-dimensional diffusion across the capillary membrane. For potassium ion, sodium chloride, and glucose, the calculated
solute permeability coefficients are 50.4 3 1025,
39.2 3 1025, and 14 3 1025 cm/s, respectively. These
values are close to the largest measured values for these
solutes in frog mesenteric capillaries (in cm/s): 67 3 1025
(potassium), 44 3 1025 (sodium chloride), and 10 3 1025
(glucose) (45, 47). These calculations demonstrate that
some of the largest measured permeability coefficients of
the mesenteric microvessels to small solutes can be accounted for if close to 10% of the junction is actually open
in these vessels and the effective open area approaches
close to 30% taking into account spreading. On the other
hand, the permeability coefficient to albumin predicted
from this model is 7 3 1026 cm/s, which is 20 –30 times
larger than the measured values (54). Thus, to account for
the measured permeability coefficients to larger solutes,
the molecular sieve must significantly restrict albumin but
have only a minor resistance to small solutes.
G. Structure-Function Correlation: The Molecular
Sieve and Role for the Glycocalyx
The principal hypothesis to describe the molecular
filter within the pathway associated through the breaks in
the junctional strands is the fiber matrix model of capillary permeability (56). Specifically, the molecular filter is
assumed to be a fiber matrix associated with the endothelial cell glycocalyx and possibly extending into the
intercellular cleft. On the luminal side of the cleft the
presence of a glycocalyx layer on the endothelial cell
surface was first described based on staining experiments
using ruthenium red and Alcian blue for cell surface glycoprotein (158). These experiments suggested the layer
extended into the luminal caveolae and outer regions of
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an occludin molecule to enter the bilayer of the adjacent
cell. If the loops of occludin molecules from adjacent cells
remain entirely extracellular but interlock snugly, then
the separation of the outer leaflets would be ;2 nm. It is
of considerable interest that this is the value of cell separation reported by Adamson and Michel (9).
In the region of the discontinuity, the adjacent endothelial cell membranes were separated by 20 nm, which
was the same value as that for the rest of the wide part of
the cleft. As in rat heart muscle microvessels (27), there
was no obvious structure to restrict solute movement at
the level of the discontinuity in tight junction when the
junction break was at least 40 –50 nm long. Figure 5C
shows streamlines for the two-dimensional water flow
through the discontinuities in the junctional strand compared with the one-dimensional models considered previously. The key feature is that the water flow is a twodimensional regime that converges on the discontinuity in
the junctional strand on the luminal side of the junction
and diverges from the discontinuity on the abluminal side.
The flow on either side of the discontinuity is described
by the hydrodynamics of flow through a thin layer of fluid,
rather than Poiseuille flow in a pore and is obtained by
solving the Laplace equation for two-dimensional pressure distribution within the cleft in the region of a discontinuity in the junctional strand. It is also assumed that the
flows through individual breaks do not interact. Phillips et
al. (228) expressed the Lp of capillary wall through which
fluid permeated by this pathway as C 3 Lp (Poiseuille),
where C is a factor .1 that depends on the size of the
discontinuity relative to the depth of the cleft (l) and the
depth of the strand within the junction expressed relative
to junction depth (m). The Lp (Poiseuille) described the
flow through the parallel sided slit in which there is no
spreading of flow (1-dimensional model) and was calculated from the one-dimensional slit theory. The expression for C (when the strand is at the center of the cleft,
m 5 0.5) is
Volume 79
July 1999
715
MICROVASCULAR PERMEABILITY
ability (Pd) and reflection coefficients (s) were then described by the relations
s 5 ~1 2 w ! 2
L p 5 ~A fiber /Dx! 3 ~K p / h !
P d 5 ~A fiber /Dx! 3 ~D fiber /D free ! 3 w
where Afiber is the area of fiber filled pathway, Dx is the
length of the pathway, and the term Kp is a measure of
the resistance of the fibers to water flow and was
calculated from the semi-empirical relation for Kp in
Table 3 known as the Carman Kozeny equation. The
main assumption in this form of fiber matrix theory was
that the fiber matrix formed the primary resistance to
solute and water movement. Thus, in contrast to the
pore theory, where a given pore radius accounted for
both the resistance of the pore to flow, and also the
area available for exchange (taking into account pore
density), the geometry of the water pathway could be
described in terms of the area available for exchange (A
TABLE 3. Solute partition and diffusion coefficients
and specific hydraulic conductance in a fiber matrix
Random fiber arrangement (stochastic theory)
F
w 5 exp 2~1 2 e!
S
DG
2a a2
1
rf r 2f
Dfiber
5 exp@2~1 2 e!0.5~1 1 a/rf!#
Dfree
Kp 5
e3r 2f
~1 2 e!24G
Ordered fiber arrangement (stochastic theory)
w 5 1 2 Vf (11a/rf)2
S
F
DG
2a
Dfiber
5 1 2 ~1 2 e!0.5 1 1 0.5
Dfree
p rf
Parallel array of cylindrical fibers within a rectangular slit
(hydrodynamic theory)
wfiber/slit 5
1 2 b1Vf ~1 1 a/rf!2
1 1 b1Vf ~1 1 a/rf!2
S DF
G
Dfiber/slit
Dslit
a
1a2
5
z 1 1 0.5 1
Dfree
Dfree
3Kp
Kp
21
w, Solute partition coefficient; D/Dfree, fractional reduction in free
diffusion coefficient; Kp, specific hydraulic conductance; a, solute radius; rf, fiber radius, Vf, fractional fiber volume, e is fractional void
volume 5 1 2 Vf; G, Carman-Kozeny coefficient set equal to 5 by Curry
and Michel (56) but actually a function of Vf (see Ref. 150); b1, coefficient
of leading term of a doubly periodic Wierstrasse expansion series used
by Weinbaum et al. (315) to describe flow in a slit containing fibers. A
detailed review of stochastic theory is in Reference 52.
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the intercellular clefts. Electron micrographs of microvessels perfused with solutions containing native ferritin suggested that, where the luminal contents had been adequately fixed, the ferritin concentration was greatly
reduced close to the luminal surface of the endothelial
cells. Quantitative evidence that ferritin was excluded
from the luminal caveolae was reported by Loudon et al.
(156) as well as Clough and Michel (40), strengthening the
idea that the glycocalyx could act as a barrier to the
diffusion of macromolecules. More accurate estimates of
the possible thickness of the endothelial cell glycocalyx
were provided by Adamson and Clough (7) in frog mesenteric capillaries. Using cationic ferritin, they visualized
the outer surface of the glycocalyx that was up to 100 nm
from the endothelial cell surface (average thickness 60
nm) when the vessel was perfused with plasma. The
glycocalyx was thinner in the presence of albumin-Ringer
perfusate (31 nm). These observations were consistent
with the hypothesis that plasma proteins were adsorbed
to the endothelial cell glycocalyx and form part of the
structure forming the molecular filter at the cell surface
(49, 185, 186, 263, 264).
Further evidence for the role of absorbed macromolecules on the cell surface in the formation of a molecular
filter was the observation that perfusion of microvessels
with cationized ferritin in Ringer solution, without albumin, formed a matrix layer on the cell surface and restored the hydraulic conductivity and selectivity of the
vessel wall to that when albumin was present (192, 303).
Adamson (4) also demonstrated that enzymatic removal
of the glycocalyx, using pronase, increased the hydraulic
conductivity of frog mesenteric capillaries by 2.5-fold.
Thus, in the first quantitative description of the fiber
matrix model of capillary permeability, Curry and Michel
(56) accounted for the exclusion of solutes from the
matrix in the principal hydrophilic pathway in terms of
solute exclusion by a network with fibers characteristic of
glycoproteins on the endothelial cell surface. The nature
of fibers associated with the endothelial cell surface and
the cleft entrance is not well understood, but the side
chains of glycosaminoglycans that are likely to form part
the cell glycocalyx have a characteristic molecular radius
close to 0.6 nm. Another possible site for this ultrafilter
could be just within the luminal aspect of the intercellular
clefts. Regularly arranged electron densities have been
demonstrated in this region by Schultze and Firth (275),
and these could represent fibers of a molecular filter.
Using the stochastic theory of Ogston et al. (209a),
Curry and Michel (56) described the solute partition coefficient w and the reduction in solute diffusion coefficients of the matrix relative to the free diffusion coefficient (Dfiber/Dfree) in terms of the fraction of the matrix
volume occupied by fiber (Vf) and the fiber radius (rf), as
shown in the expression in Table 3 (random fiber arrangement). The membrane coefficient and the solute perme-
716
C. C. MICHEL AND F. E. CURRY
FIG. 7. Junction break surface fiber matrix model. A: plane view of
interendothelial cell cleft. Junction contains periodic discontinuities of
length 2d and spaced 2D apart. Regular array of fibers, radius r f, of depth
Lf is at entrance to cleft. Average spacing between fibers is distance D.
Parameters L1, L2, and L3 measure position of junction strand. B: 3-dimensional sketch of a single periodic unit of width 2D shows central discontinuity in strand and a possible narrow slit pathway for very small solutes
,1.5 mm diameter. X is distance from lumen to tissue, and Y is distance
along cleft in direction of strand. [From Fu et al. (82).]
K p 5 0.057r 2f ~D/r f ! 2.377
Here D is the spacing between the fibers, rf is the fiber
radius, and D is related to the fractional fiber volume by
the relation D 5 [(p/Vf)1/2 2 2] 3 rf . When the fibers lie
within a rectangular slit of width W, the effect of the cleft
wall must also be taken into account. Tsay and Weinbaum
(299a) showed that the effective conductivity of the matrix within the walls (Keff) was related to the value of Kp
for the unconstrained fibers described by the relation
F
tanh~W/2!/K 1/2
K eff
p
5 12
Kp
~W/2!/K 1/2
p
G
This relation predicts the specific conductance of a regular array of fibers, 0.6 nm in radius, all lying perpendicular
to the direction of flow, and spaced to just exclude albu-
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matrix) and membrane thickness Dx independently of
the fiber parameters (Vf and rf). The principal water
pathway was assumed to be via the interendothelial
cleft, but other structures (including fenestrations or
pathways formed by the coalescence of cytoplasmic
vesicles) could be included in the area for exchange.
Using these relations, Curry and Michel (56) found
that a network of random fibers occupying 5% of the
pathway volume accounted for the measured reflection
coefficient to albumin and measured Lp of frog mesenteric capillaries if the fiber matrix was present throughout
the interendothelial cleft (i.e., the junction strands was
open to 50 –90% of the line of its length and the matrix
filled the whole cleft depth). Further calculations showed
that the measured permeability coefficients for solutes
(ranging in size from 0.2 to 3.5 nm) were also consistent
with this model with a cleft length close to 0.8 mm (50).
Thus, although the initial idea for the model suggested the
matrix lay only at the endothelial surface, the first quantitative analyses argued for an extension of a similar type
of matrix throughout the cleft.
These calculations were made before the observations of Adamson and Michel (9) that breaks in the junctional strand would account for only 10% of the actual
length of the junction, and a maximum of only 20 –30% of
the junction being effectively open, taking into account
two-dimensional spreading in the absence of matrix. Thus
a junction break model with only 20 –30% of the strand
open for exchange and with matrix throughout the cleft
would underestimate measured water flows at least threefold. In addition, a more critical evaluation of flows
through fiber matrices, and the predictions of the CarmanKozeny Equation by Levick (150), indicated that the Carman Kozeny equation underestimated the resistance to
flow in matrices with fiber volumes in the range 1–10%.
One problem was that the geometric factor in the equation (the Kozeny coefficient) was not a constant value
[equal to 5 as used in Curry and Michel (56) but increased
significantly at void volumes between 90 and 99%]. These
considerations led to a revised form of the fiber matrix
model for water and solute flows through the interendothelial cleft of frog mesenteric capillaries that incorporated the junction geometry of Adamson and Michel (9)
and the fiber matrix only at the cleft entrance.
Figure 7 shows an extension of the junction-break
model of Figure 5 to include a fiber matrix layer 100 nm
thick at the surface of the capillary. The matrix is shown
as a highly idealized ordered array of fibers spanning the
cleft entrance and with a fiber spacing (D) of 7 nm, close
to the size of albumin (Stokes radius, 3.5 nm). The most
accurate description of the resistance to water flows
through an array of cylinders that are not constrained
within a cleft is given by Sangani and Acrivos (258). For
fiber volume ,0.7, Tsay and Weinbaum (299a) summarized the results from these authors using the relation
Volume 79
July 1999
MICROVASCULAR PERMEABILITY
H. Structure-Function Correlation: Modeling
Water Flows Through the Breaks in the
Presence of a Fiber Matrix
The model in Figure 7 provides a quantitative description of flows through a real endothelial barrier (microvessels in frog mesentery) in terms of directly measured geometry of the junctional strand and reasonable
FIG. 8. Effect of fiber matrix on pressure drop from luminal to albuminal side of an intercellular cleft centered on a discontinuity in junctional
strand. In absence of a fiber matrix, pressure distribution through cleft on
either side of a break in a strand halfway through cleft is symmetrical. In
presence of a ordered fiber matrix at cleft entrance (parameters as in Fig.
7), 80% of pressure drop occurs on luminal side of discontinuity. X 5 0 is
luminal surface; x 5 L12 is on luminal side of discontinuity in junction
strand; x 5 L11 is on abluminal side of discontinuity; y is distance along
strand.
estimates of glycocalyx structure. Furthermore, the configuration of the junctional strands is simple enough that
the effect of changes in the size and frequency of breaks
in the strand and the distribution of the strands can be
systematically studied. The primary effect of the fiber
matrix at the cleft entrance is to modify the two-dimensional spread of water flows through the cleft. In the
absence of a matrix, the pressure falls symmetrically
about a break in the junctional strand located halfway
between the lumen and the tissue, but in the presence of
the matrix up to 80% of the pressure drop occurs across
the luminal part of the cleft (Fig. 8). The result shows that
over a break, the matrix accounts for 80% of the resistance to water flows. With the combined effect of the
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min (fractional fiber volume of 1.7% is 7 3 10214 cm2 and
is reduced to 5.5 3 10214 cm2 when the fibers are within
a rectangular slit 20 nm in width). These values are close
to Kp value of 6.2 3 10214 cm2 calculated by Curry and
Michel (56) using the Carman-Kozeny relation for a random distribution of fibers in a matrix of higher fractional
fiber volume (5%). These comparisons show that the Carman-Kozeny equation underestimated the resistance to
water flow. They also demonstrate that a thin matrix at
the cleft entrance, capable of restricting albumin, can be
a major determinant of the permeability properties of the
capillary wall.
The observation that a highly ordered array as in
Figure 7 occupying only 1.7% of the volume excludes
albumin (w ;0) more than a random array of fibers
occupying 5% of the volume (w 5 0.1) is one example of
the principle that, in addition to fiber volume and fiber
radius, the selectivity properties of a matrix are determined by the degree to which the matrix is ordered.
The equations describing exclusion and diffusion in an
ordered array are in Table 2. These relations describe
the case where the fibers are less regular than in Figure
7 but are ordered in the sense that they do not overlap.
Such a matrix will completely exclude albumin when
the fiber volume is 2.1%. Michel (184a, 185) suggested
that one of the mechanisms whereby albumin increases
the selectivity of the matrix is to bind in a way that the
fibers are ordered, and the albumin occupies additional
space in the matrix.
It is noted that the units of specific conductivity are
square centimeters so K1/2
has units of centimeters, a
p
characteristic pore radius. Thus the resistance to water
flow through a matrix with a given value of Kp may be
characterized by Poiseuille flow through a pore of radius R, where R 5 (8Kp)1/2. It follows that the Lp of a
barrier with a specific conductivity of 5–7 3 1024 could
be described by the resistance to water flow through
cylindrical pores of radius 6.5–7.5 nm. If barriers in
addition to the matrix offered up to one-half the resistance to water flows, then a smaller effective pore size
(4 –5 nm) may account for water flows. This calculation
may provide some insight into the original observation
in pore theory that a pore radius characteristic of the
limiting size for albumin was also a reasonable description of the structure determining water flows.
717
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C. C. MICHEL AND F. E. CURRY
abluminal membrane (48, 185–187). The observations that
there are variations in permeability (Lp and solute permeability coefficients) from capillary to capillary while the
selectivity of the membrane measured as the reflection
coefficient to albumin or myoglobin remains constant
(see Fig. 3) are also readily accounted for as variation in
the fraction of open junction (number and size of junctional breaks), and other areas for exchange all having a
common selective matrix structure associated with the
endothelial cell surface.
The molecular basis for the passage of molecules at the
level of the breaks in tight junctions is more likely to be the
localized absence of cell-cell contacts with corresponding
loss of a closely regulated molecular sieve as suggested by
Weinbaum et al. (315). Thus the junctional break-surface
matrix model suggests independent mechanisms to regulate
the permeability properties of the microvessel wall. The
junctional break size and frequency is likely to involve regulation of cell-cell attachment via occludin and other junctional proteins including the cadherins associated with tight
junction (66 and Fig. 9). On the other hand, the regulation of
glycocalyx density and organization is likely to involve interaction of the molecules forming the cell surface with the
cytoskeleton, and with circulating plasma proteins. Some of
the cellular mechanisms underlying these interactions are
reviewed elsewhere (see sect. VIII; Drenckhahn and Ness,
Ref. 66). The arrangement of the molecular components of
intercellular junctions is currently best understood for junctions between epithelial cells (see Anderson and van Itallie,
Ref. 13a). Thus models of the epithelial tight junction have
been used to interpret the molecular structure of junctions
between endothelial cells. There are, however, some important differences between the junctions between endothelial
cells and those found in epithelia. The separation of the
adherens junction and the tight junction is less well defined
in endothelial cells than in epithelial cells, and the interactions between the directional cytoskeleton with the junction
molecules are different at least in detail. Drenckhahn and
Ness (66) have recently reviewed this subject, and Figure 9
is an updated diagrammatic summary based on their views.
I. Structure-Function Correlation: Fiber-Entrance
Junctional Break Model of the Endothelial Cleft.
Limitations and Future Developments
Much more work is needed to develop and understand the junction break-fiber entrance model for transport through junctional pathways for a variety of junctional configurations and matrix properties. We illustrate
current approaches to the more detailed evaluation of
model in this section. Although the model with close to
10% of the junctional strand open for exchange, and with
a matrix 100-nm thick at the cleft entrance described the
mean Lp, and the permeability coefficients of small sol-
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matrix and the junctional breaks taken into account, the
Lp of a microvessel with the ultrastructure described by
Adamson and Michel (9), with 10% of the junction open,
and the matrix present would be reduced from a value of
7 3 1027 cm z s21 z cmH2O21 in the absence of the matrix
to 2.5 3 1027 cmzs21 z cmH2O21 when the matrix was
present.
In contrast to the resistance of the matrix to water
flow, the thin matrix offers little resistance to small solutes, and the permeability coefficients to solutes with
,1-nm radius are only slightly less than those predicted in
the absence of a surface matrix. Thus the two-dimensional spread of small solutes is relatively unaffected by
the matrix, and their permeability coefficients are the
same as those calculated using the simpler junction break
model without matrix described above. On the other
hand, as the solute size increases to approach the fiber
spacing of 7 nm, the matrix resistance increases to become the major resistance to larger solutes, the size of
albumin. To account for the resistance due to both fibers
in the cleft and the presence of the cleft wall, Weinbaum
and colleagues (85, 314, 315) derived new expressions for
the diffusion and exclusion within the matrix. With the
use of the hydrodynamic theory for solute diffusion and
solute exclusion by a matrix contained within a rectangular slit (85, 314, 315), the permeability coefficient to
albumin is predicted to fall below a value of 2 3 1027
cm/s, two orders of magnitude smaller than cleft without
the matrix. Most of the concentration difference between
plasma and the tissues for molecules the size of plasma
proteins is across the surface matrix, which therefore
forms the primary molecular filter for these solutes.
In summary, these calculations demonstrate that the
fiber matrix-junctional break model provides an interpretation of the original pore theory in terms of molecular
structure of the endothelial glycocalyx and the distribution of discontinuities in the junctional strand. Specifically, the effective pore radius, a measure of the selectivity of the pathways, is determined by the interstices in the
fiber matrix, reflecting the composition and arrangement
of the fiber matrix, whereas the effective number of pores
is determined by the size and frequency of discontinuities
in the strand in capillaries with continuous endothelium.
In some respects, it is similar to the original idea that the
capillary pores may be within the interstices of the intercellular cement (222). The theory suggests that although
the endothelial glycocalyx is the primary molecular sieve,
the effective area for exchange of water and solutes
through porous pathway is determined by the size and
frequency of porous pathways across the endothelial barrier, which may or may not contain a secondary molecular
sieve. These pathways include breaks in the junctional
strand (regulated by cell-cell contact mechanisms), but
they may also include pathways such as fenestrations,
transcellular breaks, or vesicles fused to both luminal and
Volume 79
July 1999
MICROVASCULAR PERMEABILITY
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FIG. 9. Model of molecular machinery associated with junction between endothelial cells and junction-associated
filament system. Details of these interactions are an area of active investigation, and figures of this type are constantly
updated. The following brief description of molecular components is also based on Reference 66 (for another review, see
Ref. 197a). In this figure an idealized arrangement of junctional components is shown with tight junction (ZO, zonula
occludens) spatially separated from adherens junction (AJ). Tight junction is formed by integral membrane adhesion
protein occludin. Peripheral membrane proteins associated with tight junction include ZO-1, ZO-2, cingulin, antigen 7H6,
and a small GTP-binding protein Rap 13. Most of these do not bind directly to actin, although ZO-1 binds to spectrin and
cingulin and may organize or tether actin (197a) main site of attachment of junction-associated actin filaments to plasma
membrane appears to be adherens junction. Calcium-dependent adhesion molecule, VE-cadherin, is clustured at this site
and attaches to actin via additional protein complexes. Actin, a-actinin, and vinculin (311a) bind to b-catenin which, in
turn, is linked to cytoplasmic domain of VE-cadherin by b- and g-catenin. ZO-1 can also act as a cross-linker between
catenin/cadherin complex and actin-based cytoskeleton. Recent experiments suggest that, in endothelium, distinction
between tight junction and adherens junction may not be as clear as suggested by this figure and as described in
epithelial membranes. These experiments show colocalization of tight junction protein occludin and adherens junction
proteins b-catenin and VE-cadherin in endothelial cells (66). Other cadherins that may contribute to junctional adhesion
include P-cadherin and N-cadherin, although VE-cadherin is the dominant form. Another adhesion molecule associated
with junction is platelet endothelial cell adhesion molecule (PECAM-1), a transmembrane adhesion molecule belonging
to calcium-independent immunoglobulin superfamily. This adhesion molecule appears capable of maintaining junction
integrity when calcium-dependent adhesion molecules have been destabilized (265a). It appears to be excluded from
adherens juction. Additional components of intercellular junction shown include the following: connexins 37, 40, and 43,
forming gap junctions (GP) (in some but not all endothelial barriers) and integrins a2b1 and a5b1. Membrane binding site
of desmosomal proteins desmoplakin I and II found in cultured umbilical vein endothelial cells is not known. Vinculin
is also enriched along junction. Some of these components may be present only in specific endothelial barriers, or only
under cultured conditions. Figure does not include molecular structures associated with endothelial glycocalyx that may
extend into luminal aspect of junction or structures associated with focal adhesion sites on abluminal side of endothelial
cells. Although considerable progress has been made to identify molecular components of junctions, contributions of
various molecular complexes to permeability properties of barrier and its regulation are not well understood. [Modified
from Drenckhahn and Ness (66).]
719
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C. C. MICHEL AND F. E. CURRY
J. Further Consideration of Mammalian
Muscle Capillaries
On the basis of the above calculation, it is of interest
to reexamine water and solute flows associated with path-
ways across mammalian heart muscle described by
Bundgaard (27). Specifically, for short breaks in the junctional strand (80 –100 nm long as described by Bundgaard
using conventional thin sections) separated by distances
of 6.9 –13 mm, the values of the correction factor that
accounts for two-dimensional spreading of water flows
when one of the junction strands is the principal determinant of flow patterns are close to 3.0, and the Lp values
of the vessel estimated in the absence of matrix (1.6 –
2.0 3 1027 cm z s21 z cmH2O21) are close to three times
larger than values measured for heart. Thus all the measured water flow could be accommodated within the very
small area available for exchange indicated by break frequencies as small as those observed by Bundgaard using
conventional sections. Additional reduction in Lp would
be accounted for by surface glycocalyx or the presence of
more that one junctional strand within each cleft.
For small solute diffusion across heart muscle capillaries, the two-dimensional spread of solute would also
result in an increase in the effective area for solute exchange from values close to 1% of open junction to close
to 3% (accounting only for 2-dimensional spreading) or to
values between 5 and 10% if the tendency for serial sectioning in the absence of tracer to underestimate the
number of breaks (as found by Adamson and Michel, Ref.
9) is also taken into account. It is therefore reasonable to
expect that a modified surface matrix-junctional pore
model might also account for the measured permeability
properties of mammalian muscle and skin microvessels.
These calculations show that the larger breaks in the
junctional strand observed by Bundgaard (27) may be
sufficient to account for measured fluxes of small solutes
if the geometry of the junction strand allows spreading of
the solute and water flows. This does not necessarily rule
out additional pathways through a constricted slit, but it
does indicate that the contribution of such additional
pathways to exchange may be smaller than anticipated by
Bundgaard. It is also noted that pathways through a constricted slit that are selective for larger solutes, but which
lie in parallel with the larger breaks, will contribute little
to the overall selectivity of the vessel wall. Because the Lp
of breaks 80 –100 nm long may account for up to three
times the normal Lp of the vessel wall, these infrequent
breaks in the junction strand will account for most of the
water flow through the cleft at the level of the strands (9).
Thus, even if there were structures capable of sieving
macromolecules in the strand at sites away from the
breaks, their contribution to the selective properties of
the wall would be diminished by the shunting of flows
through the larger breaks (9, 48, 50). Taken together,
these arguments suggest that the junctional structures
(such as those suggested in the constricted pore models)
are not likely to be the principal determinant of the selectivity properties of the capillary wall in the heart.
Similar estimates of the fraction of open junction in
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utes in frog mesenteric capillaries, the ability of the model
to account for the measured permeability coefficients to
albumin depends on the sharp cut-off in predicted permeability coefficients as solute size approaches the interfiber
distance in the ordered array. The measured values of
permeability coefficient for solutes of intermediate size
(1- to 3.5-nm radius) are not well described by this model
with such a sharp molecular size cut off, and further
revision of the hydrodynamic theory of solute diffusion
and exclusion in a matrix as solute size approaches the
interfiber spacing is needed. Fu et al. (85) described a
better fit of the model to the measured solute permeability
coefficients for solutes ranging in size from 1 to 3.5 nm in
radius using a lower estimates of open junction (5.7%,
calculated as the product of average break size and observed frequency) instead of 10%. However, because the
reduced area for exchange was too small to account for
the measured permeability coefficients to small solutes,
they suggested that an additional pathway for solutes
smaller than 0.75-nm radius may be present within the
junction. Recent experiments (83) to test the contribution
of a pathway for very small solutes in parallel with the
large break pathway demonstrated that the contribution
of this pathway to the diffusion of sodium fluorescein
(radius 0.45 nm) in frog mesentery microvessels was
small. However, these results do not rule out the contribution to exchange by a pathway for even smaller solutes
such as glucose, potassium ions, and other electrolytes.
Fu and co-workers (82, 84) have also extended the
model to describe the distribution of larger molecular
weight tracers up to the size of horseradish peroxidase (6
nm) within the cleft when there was superimposed water
flow through the cleft. For horseradish peroxidase (3-nm
radius), most of the tracer concentration difference between the lumen and the tissue was developed across the
matrix layer. This observation may explain the common
finding in electron microscopy that no large tracer is
found in the cleft. The result indicates that failure to find
tracer in the cleft does not necessarily mean that the
tracer does not cross the cleft pathway, but that, under
the experimental conditions of most tracer experiments,
the tracer concentration downstream from the matrix is
too small to be detected (82, 314). A logical extension of
this model to describe the local Starling forces determining water flows across the sieving matrix in the cleft is
outlined below. Before we evaluate these developments, a
brief application of the fiber matrix-junction pore model
to heart and skeletal muscle microvessels is given below.
Volume 79
July 1999
MICROVASCULAR PERMEABILITY
K. The Fiber Entrance Model and Starling Forces
Across the Capillary Wall
According to the fiber-matrix junction-break model
above, the concentration of the plasma proteins behind
the sieving matrix (i.e., the tissue side of the matrix)
would be determined by the flux of water and solute
across the matrix, and also by the back diffusion of solutes from the tissue through the breaks in the junctional
strand when the average concentration of the plasma
proteins in the tissue (close to 40% of the plasma concentration Cp) is larger than that crossing the matrix. If most
of the macromolecular flux from blood to tissue occurs
via another pathway across the capillary wall (see Fig. 10
and sect. V), the back diffusion component is expected to
be small.
In fact, Michel (188) estimated that, even when the
effective filtration pressure was as low as 1 cmH2O, the
plasma protein concentration on the downstream side of
the sieving matrix would be close to the limiting value
across a sieving matrix (1-s)Cp. This is because the fluid
velocity through the breaks is sufficiently high to prevent
back diffusion of the tissue proteins into the cleft. This
view of events within the cleft builds logically on the
theory of steady-state filtration, where the solute concentration on the downstream side of the sieving matrix is set
equal to Js /Jv as reviewed by Michel and Phillips (185,
192a).
It follows that the effective colloid osmotic pressure
difference across the sieving matrix may be larger than
that expected for the global blood to tissue concentration
of plasma proteins. These ideas are beginning to be explored. For example, a corollary is that local hydrostatic
and colloid osmotic pressure difference across the sieving
matrix within the cleft determines the effective filtration
pressures in the Starling balance, and not the global differences of colloid and hydrostatic pressure between
plasma and tissue. This hypothesis may account for the
discrepancy, pointed out by Levick (151), that the effective filtration pressure across most capillary beds, estimated from the most recent measurements of tissue protein and colloid osmotic pressure, is far larger than that
required to account for the observed lymph flows. Specifically, the effective filtration pressure needed to account
for the measured flows should be only 1–2 cmH2O, but the
estimated values are much greater in the majority of
tissues. Although other global heterogeneities (differences in capillary pressure due to vasomotion in different
units of a microvascular bed, and differences in permeability properties in different microvessels within each
bed) may contribute to the discrepancy described by
Levick (151), the idea that the Starling forces are determined by the local interaction of osmotic and pressure
difference within the cleft links this fundamental regulation of transvascular water exchange to the fundamental
ultrastructure of the endothelial cleft in a new way that
requires further study.
The preliminary calculations by Michel (188) to de-
FIG. 10. Diagram of microvascular endothelium to show how separate pathways
for fluid (through intercellular cleft containing a fiber matrix at luminal entrance)
and protein (through vesicular system)
can lead to differences between mean osmotic pressure between plasma and interstitium (and lymph) and plasma and fluid
in intercellular cleft downstream from
sieving matrix. [From Michel (188).]
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mammalian skeletal muscle based on serial sections are
not available but are likely to be similar or smaller than in
mammalian heart muscle. Recent experiments by Vink
and Duling (308) indicate a thick glycocalyx structure on
the surface of the cremaster muscle capillaries. These
investigators demonstrated that red blood cells and neutral dextrans (mol wt 70,000) were excluded from the
vessel wall by a layer 0.4 – 0.5 mm thick extending from
the endothelial cell surface. This exclusion layer was
destroyed by exposing the vessel to epi-illumination for
1–5 min and by enzymatic degradation of the cell surface.
The quantitative evaluation of the effects of a matrix of
this thickness in series with breaks occupying as little as
1% of the length of the junctional strand remains to be
evaluated.
721
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C. C. MICHEL AND F. E. CURRY
L. Charge Effects at the Walls
of Continuous Capillaries
Solutes, 1- to 2-nm radius and larger, that carry a net
positive charge accumulate in the tissue more rapidly
than similar-sized solutes carrying a net negative charge,
suggesting that walls of microvessels carry a net negative
charge (48). The relative magnitude of the effect of charge
interactions is small compared with the steric interactions. For example, in single perfused capillaries (58), the
highly anionic glycoprotein, orosomucoid, reduced the
solute permeability coefficient of negatively charged
a-lactalbumin (2-nm Stokes radius, net charge) by a factor
of 2 when added to an albumin perfusate at concentrations similar to those found in plasma (0.1–1 mg/ml). The
twofold reduction can be compared with reduction in
permeability due to steric interactions (exclusion and
reduced diffusion) due to the presence of a fiber matrix of
at least 10-fold. The investigations in single capillaries
confirmed and extended the original observations in isolated perfused rat hindlimb that orosomucoid reduced the
clearance of anionic plasma macromolecules (102). Orosomucoid also contributes to negative charge on the glomerular membrane (103a). Using a Donnan model for
solute exclusion, Huxley and co-workers (125, 126) calculated that the additional exclusion due to charge could
be accounted for by an increase in the negative charge on
the microvessel wall from an effective density of 11.2
meq/l in the presence of albumin-Ringer to 34 meq/l in the
presence of plasma. If similar charge exclusion mechanisms act on albumin, the osmotic reflection coefficient to
albumin is predicted to increase from a value to close to
0.9 due to steric exclusion to values close to 0.97 in the
presence of plasma. These calculations show that exclusion due to charge interactions may reduce both the
contribution of both diffusive transport and solvent drag
for charged solutes across the walls of continuous capillaries.
Another mechanism whereby charge interactions
modify the permeability of the microvessel wall is the role
of electrostatic interactions between a fiber matrix and
macromolecules to order the surface matrix. The interaction of albumin with the capillary wall to reduce permeability and increase selectivity depends on the presence of
positively charged arginine groups in albumin (194).
It is noted that the low concentration of albumin in
the renal tubular fluid is usually interpreted as the combined effect of size (steric exclusion) and the electrostatic
repulsion of the negatively charged albumin by glomerular membrane. The charge densities calculated for the
glomerular membrane are three to five times larger than
those estimated for peripheral continuous capillaries.
These models of the glomerular membrane have been
challenged by Comper and colleagues (42, 211) who have
used both theoretical and experimental studies to suggest
that the negative charge densities may be lower than
previously calculated on the basis of the clearance of
charged dextrans. Although independent experiments of
the renal clearance of plasma proteins are consistent with
smaller charge densities on the glomerular membrane
(154), other observations by Comper and colleagues (42,
211) have not been confirmed. These include the possibility that albumin concentration in the glomerular filtrate
may normally be as high as 8% of that in plasma and that
large amounts of albumin are normally reabsorbed intact
across the renal tubule (154).
M. Barriers to Water and Solute
in Fenestrated Microvessels
An important generalization of the fiber matrix model
is that it is not restricted to the junctional slit model. The
role of a matrix layer associated with fenestrae has been
analyzed in detail by Levick and Smaje (152). The size and
frequency of fenestrations varies from 50- to 60-nm radius
in intestinal mucosa, synovium, and submandibular gland
with a density of 2–5 per mm2 to 60- to 88-nm radius with
densities of 20 –30 per mm2 in the renal vasculature (excluding the glomerulus). Levick and Smaje (152) estimated that, for the typical thickness of the diaphragm in
fenestrations of 5 nm, the values of exchange area per
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scribe gradients within the cleft have been confirmed by
Weinbaum and colleagues (121, 313) using an extension
of the model in Figure 7 which involves the use of numerical techniques to account for the pressure drop along the
tissue side of the sieving matrix and the nonlinear coupling of water and large-molecular-weight protein fluxes
in the region of the cleft downstream of the sieving matrix
in the cleft. Thus the stage is set for a detailed analysis of
the permeability properties of microvessel using a much
more sophisticated set of modeling tools than has been
available previously. The results of Hu and Weinbaum
(121) show that coupling of water flow to albumin flux on
the tissues side of the matrix could give rise to a nonuniform distribution of albumin concentration and a corresponding nonuniform distribution of effective osmotic
pressure. The interaction of these osmotic forces with the
pressure difference across the matrix and the junctional
strands distal to the matrix requires further investigation.
Furthermore, recent experiments using analogs of cAMP
demonstrate that elevated endothelial cell cAMP increases the number of junctional strands within the cleft
(88). The importance of the strand structure to further
modify the two-dimensional flow patterns within the cleft,
reduce the effective area available for exchange, and
modify the microstructure of the osmotic gradients within
the cleft are also important areas for future research.
Volume 79
July 1999
MICROVASCULAR PERMEABILITY
detailed review of flow through the interstitium and
fiber matrix structures in relation to the chemical composition of the interstitium is given by Levick (150).
IV. TRANSCELLULAR EXCHANGE OF WATER
AND SMALL HYDROPHILIC MOLECULES
In section III, the exchange of water and small hydrophilic solutes was considered to occur through microvascular walls via an extracellular route. Because water and
many small solutes can enter and leave the endothelial
cells, it is possible for them to exchange by pathways
through the cells. We now consider the possible contribution of these transcellular exchange pathways to the
overall permeability of microvascular walls to water and
small hydrophilic solutes such as glucose, amino acids,
and urea.
Although small ions, such as sodium, chloride, and
potassium, enter and leave the cells, it is unlikely that this
pathway makes a significant contribution to microvascular exchange. Electrical resistance measurements on isolated endothelial cells are in the range of 25,000 V z cm2
(210), whereas the resistance of microvascular walls in
situ range from 1,870 V z cm2 for brain microvessels to
24 –70 V z cm2 for microvascular endothelia of skin and
muscle. Thus, although 10% of the exchange of ions
through the walls of cerebral capillaries might occur
through the endothelial cells, the contribution of the transcellular pathway to ion exchange at other microvascular
walls would appear to be negligible.
A. Water
Evidence for a pathway that is exclusively available
for the exchange of water (and not small hydrophilic
solutes) was presented 30 years ago by Yudilevich and
Alvarez (330). Comparing the unidirectional transport of
3
H2O and 22Na1 from the perfusate to the tissues of the
dog heart, they demonstrated that exchange of water was
more than twice as rapid as the exchange of sodium ions
after differences in their free diffusion coefficient had
been allowed for. This led to speculation as to the contribution of such a transcellular pathway to Lp of microvascular endothelium (153, 221, 298). At that time, the Lp of
endothelial cells was not known, but it was known that
cells with high permeabilities to water, such as human
erythrocytes, had Lp values in the range of 1028
cm z s21 z cmH2O21. In 1976, Curry et al. (55) provided
evidence for the presence of a pathway through the walls
of frog mesenteric capillaries that was available to water
alone in addition to one which water shared with small
hydrophilic solutes. They showed that in these vessels the
values of s for NaCl, urea, sucrose, and cyanocobalamine
increased only slightly with molecular size. Extrapolation
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path length (Afen/Dx) (7,000 –17,000 cm21) are close to
two orders of magnitude higher than required to account
for measured small solute permeabilities and hydraulic
conductivities of fenestrated capillaries. Even when a
correction was made for a reduction in the available area
to account for the thin electron-dense diaphragm that
appears to be arranged as a series of spokes radiating
from a central hub, within each fenestration, the reduction in area is not sufficient to account for the decreased
permeability. Levick and Smaje (152), therefore, concluded that most of the resistance to water and solute
movement across fenestrations must lie within fiber matrix structures more than an order of magnitude thicker
than the diaphragm. The diaphragm, therefore, might support an overlying glycocalyx and underlying basement
membrane containing a mixture of thick and thin fibers.
The relative resistance of the surface glycocalyx, the
basement membrane, and the tissue in fenestrated microvessels has been evaluated. As was observed using
conventional fixation methods, the glycocalyx on the surface of a fenestrated capillary is of the order of 25 nm
thick. If such a layer were formed of fine fibers (0.5- to
0.6-nm radius) spaced to exclude albumin, it would account for ,10% of the measured resistance to water flow,
and a glycocalyx thickness closer to 300 nm would be
required to account for all the resistance to water flow.
On the other hand, there are several possible structures
associated with the basement membrane that may account for a significant resistance to water flow. For example, Levick and Smaje (152) estimated that a 100-nmthick basement membrane, formed by fibers 2.7 nm in
radius and with fibers occupying 50% of the volume of the
basement membrane, would have a reflection coefficient
to albumin of 0.8 and appropriate resistances to water and
solute flows. Alternatively, a 100-nm-thick layer formed
from type IV collagen fibers (0.75-nm radius which occupied 12% of the volume) would account for the resistance
to flow. Thus the permeability properties of a fenestrated
microvessel may be the result of flows through a mixture
of coarse and fine fibers in the basement membrane in
series with a glycocalyx layer on the cell surface.
Levick (151) has suggested that, in general, endothelial barriers should be described as a cellular layer
sandwiched between surface glycocalyx and an interstitial matrix. In the case of an endothelial barrier with
relatively low resistance (for example, fenestrated endothelium), the interstitial resistance may be large
enough to form a significant fraction of the resistance
to water flows between blood and lymph. For example,
in synovial joint, Levick (151) has estimated that the
capillary wall and interstitium contribute approximately equally to the resistance to fluid movement. A
similar analysis may apply to the endothelium when
gaps form between endothelial cells or through the
cells in the presence of inflammatory mediators. A
723
724
C. C. MICHEL AND F. E. CURRY
FIG. 11. Relationship between osmotic reflection coefficient (s) and
molecular radius of small hydrophilic molecules at walls of single frog
mesenteric capillaries. Line describing trend extrapolates to a value of s
.0 when molecular radius corresponds to that of water molecule. [From
Michel (184a). Copyright 1981 The Alfred Benzon Foundation, Hellerup,
Denmark.]
a pathway through the tight junction. Strong evidence for
the exclusive water pathway being transcellular has come
from the isolation of a family of molecules that are the
water channels of cell membranes. A 28-kDa protein
found in the membranes of human red blood cells (232)
was first called CHIP-28 and demonstrated to act as a
water channel when expressed in Xenopus oocytes. The
same protein was identified as the principal water channel
of renal proximal tubules (10) and subsequently shown to
be present in a wide range of tissues. Most relevant to the
present discussion was its identification in continuous
microvascular endothelia, although not apparently in fenestrated endothelia. CHIP-28 was soon recognized to be
one of a family of water channels that are referred to as
the aquaporins, and it is now designated as aquaporin
(AQP)-1. Aquaporin-2 was subsequently shown to be the
water channel of renal collecting ducts where its presence
in the apical membrane of the epithelial cells is regulated
by vasopressin. Aquaporin-3 is found in the basolateral
membranes of the collecting ducts and other epithelia,
and AQP-4 is found predominately in the brain and may be
involved in osmoreception. A fifth aquaporin (AQP-5) has
been associated with secretion of tears, saliva, and sputum, and a sixth channel, AQP-O, which was identified as
early as 1984, is present in the lens epithelia and has a low
Lp. The hydraulic conductances of AQP-1, AQP-2, and
AQP-3 are all blocked by mercuric chloride and p-chloromecuribenzene sulfonic acid (p-CMBS).
The importance of AQP-1 in determining the exclusive water pathway through endothelium has been clearly
demonstrated by Pallone et al. (217) working on isolated
descending vasa recta from the outer medulla of the rat
kidney (OMDVR). The OMDVR have high Lp values
(12 3 1027 cm z s21 z cmH2O21) for vessels with continuous endothelium. Turner and Pallone (304) have shown
that the magnitude of this Lp is largely determined by a
pathway that water shares with small hydrophilic solutes,
since variations in Lp from vessel to vessel correlate with
the variation in their diffusional permeabilities to raffinose. Serum albumin has a high reflection coefficient at
the shared pathway (;0.8) since s for albumin of the
vessel wall is 0.89. In addition to this pathway, there is a
pathway exclusively available to water. Pallone et al.
(217) have shown that water can be drawn across the
walls of the OMDVR by gradients of NaCl concentration and
that these osmotic flows can be abolished by p-CMBS so
they presumably occur through AQP-1 channels, particularly
as AQP-1 has been shown to present in the OMDVR. In
contrast, flows generated by oncotic pressures set up by
differences in albumin concentration across the OMDVR are
not significantly reduced by p-CMBS. The Lp of this p-CMBSsensitive pathway is ;9 3 1028 cm z s21 z cmH2O21, i.e.,
;7% of the overall Lp of the OMDVR. Pallone et al. (217)
went on to show that AQP-1 was present at a concentration
of 2.53 3 109 molecules/mm OMDVR. With an average diam-
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of the relation (Fig. 11) to a molecule the size of water
gave a value that was significantly greater than zero. This
would be possible only if a fraction of the water that
flowed through the vessel wall traversed a pathway from
which the other hydrophilic solutes were excluded. Curry
et al. (55) estimated that this “water only” pathway had an
Lp value of 1.1 3 1028 cm z s21 z cmH2O21, which was
less than 10% of the overall Lp for frog mesenteric microvessels. They noted that much of the variation of Lp
between different capillaries could be accounted for by
variations in the extent of the shared (paracellular) pathway rather than by variations in that available to water
alone.
More recently, Wolf and Watson (322) have estimated
s to small hydrophilic molecules in the exchange vessels
of cat skeletal muscle. Although their values for s were
higher than those reported for frog mesenteric capillaries,
the Lp of the exclusive water pathway calculated from
their data is 0.8 3 1028 cm z s21 z cmH2O21 which is
remarkably close to that of Curry et al. (55). A slightly
lower value for the Lp of the exclusive water pathway
(0.27 3 1028 cm z s21 z cmH2O21) can be calculated from
the data of Rippe and Haraldsson (246), who estimated s
for a large number of small hydrophilic solutes at microvascular walls in the isolated rat hindquarter preparation.
In contrast to the findings of Wolf and Watson (322), the
values of s for NaCl, urea, sucrose, and cyanocobalamine
reported by Rippe and Haraldsson (246) were close to
those of Curry et al. (55).
Although Curry et al. (55) and Wolf and Watson (322)
were able to predict the magnitude of the exclusive water
pathway, they were unable to say where it was located.
Like others, they thought it was most likely to be a transendothelial pathway, but Curry et al. (55) did not rule out
Volume 79
July 1999
MICROVASCULAR PERMEABILITY
the AQP-1 channels make to the total Lp of the microvascular wall (i.e., small).
In most vascular beds, “the exclusive water pathway”
appears to be 10% or less of the overall Lp (240), and this
figure is consistent with the linear relation between Lp
and Pd to small hydrophilic solutes (Fig. 4). A rather
larger figure of 45% of the overall Lp in skeletal muscle
capillaries is suggested by the data of Wolf and Watson
(322) from their estimates of the reflection coefficients to
small hydrophilic solutes. Furthermore, when a point representing data from the same group (312) for Lp and Pd to
sodium is plotted on the graph shown in Figure 4, it is
found to have a higher Lp for its value of Pd in comparison
with the other points. This would be consistent with a
higher contribution of the AQP-1 channels to the overall
Lp in their preparation of cat skeletal muscle capillaries.
Schnitzer and Oh (271) have reported that AQP-1 is
located within the caveolae of rat pulmonary capillaries.
If all the AQP-1 of continuous endothelium is restricted to
the caveolae, the question arises as to whether transcellular flow by this route would be rapidly buffered by
opposing osmotic gradients. Thus net movement of water
from a caveola into the cell would leave a more concentrated solution within the caveola cavity, the full osmotic
pressure of which would be felt across the AQP-1 channels and would oppose further water influx into the cell.
The significance of this phenomenon of osmotic buffering
depends on whether the small solutes such as sodium,
which largely determine the total osmotic pressure of
extracellular fluid, can equilibrate with extracellular fluid
at the entrance to the caveola more rapidly than they can
be concentrated by the efflux of water. A simple calculation suggests that dissipation of concentration gradients
of sodium by diffusion is very much more rapid than
concentration by convection, provided that there are no
severe restrictions to the movement of sodium across the
caveolar neck. The velocity at which sodium can diffuse
over distances of 60 nm (the average diameter of endothelial caveola) is in the range of centimeters per second.
The velocity of convection through the neck of a caveola
is 0.1 mm/s or less.1 Over the very small distances that
appear to be involved, diffusion of ions such as sodium is
so rapid that osmotic buffering should not occur. Thus it
seems that a fraction of the fluid movements between the
blood and the tissues that are driven by the conventional
Starling forces may pass through the cell. At present, the
balance of evidence suggests that this component of fluid
flux is small.
1
This calculation is based on an Lp for the transcellular aquaporin-1 (AQP-1) pathway of 1028 cm z s21 z cmH2O21, a density of
caveolae at the luminal and abluminal surface of 15 mm22, a uniform
distributionof AQP-1 per caveola, and a driving force across the entire
endothelial cell of 80 cmH2O.
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eter of the OMDVR taken as 20 mm, the osmotic permeability of AQP-1 as 10213 cm3 z s21 z molecule21, and with the
assumption that AQP-1 molecules are in equal numbers on
the luminal and abluminal membrane, an Lp value of
7 3 1028 cm z s21 z cmH2O21 can be calculated for this
pathway. The figure is remarkably close to the measured
value of Lp for the p-CMBS-sensitive pathway. It provides
powerful support for the view that the exclusive water pathway through the OMDVR involves aquaporin channels in the
endothelial cell membranes. Furthermore, when taken with
the presence of AQP-1 in the continuous endothelium of
other microvascular beds, it is most likely that these molecules are responsible for the exclusive water pathways
through the walls of all microvessels with continuous endothelium.
Net water movements occur through the AQP-1 channels of the descending vasa recta under physiological
conditions. In antidiuresis, fluid is lost from the plasma as
it flows from the corticomedullary junction to the papilla
of the medulla, concentrating the plasma protein 1.4- to
1.8-fold (218). This fluid loss is driven by the concentration difference of small solutes (sodium and urea) across
the walls of the descending vasa recta (DVR) and not by
conventional Starling forces (transendothelial differences
in hydrostatic and oncotic pressures) that actually favor
fluid uptake from the medullary interstitium (220, 259).
If the concentration of small solutes in the medullary
interstitium is reduced toward isotonic values by treatment with furosemide, the plasma proteins are no longer
concentrated as they flow down the DVR, indicating that
net water efflux from the plasma through the AQP-1 channels has ceased (215, 220). It is tempting to suggest that
the concentration of the plasma proteins in the DVR
during antidiuresis increases the efficiency of fluid uptake
from renal interstitium into the blood returning to the
cortex via the ascending vasa recta (AVR). At present, a
rigorous argument for believing this cannot be made.
There is good evidence, however, that fluid uptake into
the AVR is driven by the conventional Starling forces (i.e.,
differences in hydrostatic and oncotic pressures). The
endothelium of the AVR is highly fenestrated and does not
appear to contain AQP-1.
Although the primary physiological role of the AQP-1
channels probably concerns water exchange between the
endothelial cells and their extracellular environment,
transcellular water movements may contribute to net
fluid fluxes between the plasma and tissues in microvascular beds outside the kidney. Transendothelial differences in the concentrations of small solutes develop in
skeletal muscle during exercise (180) and are responsible
for some of the fluid that leaves the plasma and enters the
muscle. A fraction of the fluid that is driven across microvascular walls by differences in hydrostatic and oncotic
pressure may also pass through AQP-1 channels. This
fraction would be in proportion to the contribution that
725
726
C. C. MICHEL AND F. E. CURRY
B. Glucose and Amino Acids
meability, indicating that the transcellular pathway is of
little importance.
A similar argument can be developed for the transport of amino acids. Whereas Mann et al. (165) expressed
much of their data for amino acid uptake into endothelial
cells in terms of relative transport, absolute values are
given for the uptake of leucine. Thus the influx of leucine
into endothelial cells grown to confluency on microcarrier beads packed into 0.5 ml in the presence of 25 mM
leucine was 2.1 nmol/min. This is equivalent to a permeability of 1.33 3 1028 cm/s, three orders of magnitude less
than the predicted permeability of capillaries in skeletal
muscle.
From these calculations, it appears that the contribution of transcellular transport to the passage of glucose
and amino acids from the plasma to the interstitial space
of skeletal muscle is negligible, and the same is obviously
true for more permeable vessels. This, of course, is not to
say that transcellular transport of these nutrients may be
a feature of one or two microvascular beds where the
transport proteins are expressed at a higher density on
the endothelial cell membranes. The only microvessels
that have been identified so far to show such properties,
however, are the microvessels of the central nervous
system.
C. Urea
In most microvascular beds where measurements
have been made, the permeability to urea is very similar
to that of sodium chloride. This finding is consistent with
the similar diffusion coefficients of urea and NaCl and
with both solutes exchanging through microvascular
walls via a shared water-filled pathway in the intercellular
clefts or through the water-filled pores of the fenestrae. In
1994, Pallone and colleagues (216, 219) reported that the
DVR of the outer medullary regions of the kidney were an
exception to these general findings. Whereas the permeabilities of the DVR and AVR of the inner medulla to
sodium and urea were almost identical, the permeability
of single perfused OMDVR to urea was very much greater
than their permeabilities to sodium chloride. Thus the
mean value for permeability of the OMDVR to NaCl was
76 3 1025 cm/s, whereas the mean value of their permeability to urea was 360 3 1025 cm/s (219). The great
enhancement of urea permeability was shown to be the
result of carrier-mediated transport, since high concentrations of unlabeled (“cold”) urea or thiourea reduced the
passage of [14C]urea through vessel walls by one-third.
Exchange of urea was also greatly reduced by phloretin
and p-CMBS in a concentration-dependent fashion. Complete reduction of the urea permeability to values equal to
those for sodium were not achieved, however. Pallone
(216) suggested the enhanced transport was achieved by
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The classical view that glucose and amino acids exchange across microvascular walls via a paracellular
pathway is reinforced by a comparison of the values of
microvascular permeability to these molecules and their
rates of uptake into cultured endothelial cells.
Estimates of microvascular permeability to hexoses
(glucose, fructose, mannitol) in skeletal muscle range
between 1 3 1025 and 5 3 1025 cm/s (299, 312), assuming
that the perfused capillary beds had surface areas in the
range of 104 cm2/100 g tissue. There do not appear to be
estimates of microvascular permeability to amino acids
except for the endothelium of the brain. If, however,
amino acids and glucose pass through microvascular
walls by diffusion through an aqueous paracellular pathway, then it is reasonable to assume that their permeabilities will be similar. Thus a figure in the range of 1025 cm/s
may be taken as microvascular permeability to both types
of molecule.
Measurements of glucose uptake in cultured endothelial cells show much slower rates of transport. Because the transport processes show saturation kinetics,
they have not been described in terms of permeability, but
for purposes of comparison, we will do so here by assessing the permeability of the cell membrane as the transport
rate per unit area of cell membrane divided by the concentration of the solute in the extracellular fluid. Thus
Gerritsen et al. (88) measured maximum rates of glucose
uptake of 0.5 nmol/min when monolayers of 2 3 105
microvascular endothelial cells from rabbit heart were
exposed to 5.5 mM glucose in the presence of insulin. If
the surface area of a monolayer of 2 3 105 cells is ;1 cm2,
this flux translates into an equivalent permeability of
1.5 3 1029 cm/s. A value in the same range can be calculated from the data of Mann et al. (165). These authors
measured the uptake of glucose and amino acids into
human umbilical vein endothelial cells (HUVEC) that had
been grown to confluence on microcarrier beads. The
beads, which had radii of 100 mm, were packed into a
volume of 0.5 ml in a 1-ml syringe barrel. The surface area
of the endothelium may be estimated by assuming the
beads occupied 0.35 ml of the 0.5 ml (optimal close packing of spheres would give 74% of the volume) and noting
that the ratio of area to volume of a sphere is 3/radius; this
leads to a value of 100 cm2 for the area of the endothelium
per column. Mann et al. (165) reported a glucose uptake
of 0.28 mM z min21 z column21 from a solution containing
11.1 mM glucose, and these figures translate to a value of
4 3 1029 cm/s for glucose permeability. If transport were
to occur between the blood and the tissues through the
endothelial cells, the apparent permeability of the vessel
wall would be no more than half the cell membrane
permeability, i.e., ;1029 cm/s. This is four orders of magnitude less than measured values for microvascular per-
Volume 79
July 1999
MICROVASCULAR PERMEABILITY
urea transporters in the endothelial cell membranes, and
these transporters were similar to those found in the
collecting duct epithelium of the inner medulla.
What is most surprising about the urea permeability
of the OMDVR is that such high rates of transport are
achieved by a membrane transporter. The permeability of
the OMDVR to urea is higher than any other value that has
been reported for microvascular permeability to small
hydrophilic solutes. The physiological importance of this
high urea permeability of the OMDVR in trapping urea in
the inner medulla during antidiuresis seems fairly clear. It
is fascinating to discover that it is achieved by such a
highly selective mechanism.
It would seem that while the transcapillary exchange
of water and small hydrophilic solutes can occur through
the endothelial cell membranes, the quantitative contribution of this pathway to the net flux of water is small and
to that of glucose and amino acids it is negligible. There
are, however, important exceptions. The specialized
transport systems across the blood-brain barrier are well
known, but the significance of transcellular exchange in
the endothelia of the OMDVR is a new development. The
presence of AQP-1 channels in these vessels ensures that
during antidiuresis water is drawn from the plasma entering the outer medulla into its hypertonic interstitial fluid
and not from the interstitial fluid into the blood (which
would follow if only the shared pathways for water and
solutes were available). Furthermore, the rapid exchange
of urea across the walls of the OMDVR reveals that where
transporters are expressed in endothelial membranes at
higher than normal density, their contribution to microvascular transport can be very large indeed.
V. PERMEABILITY TO MACROMOLECULES
For over 40 years, there has been evidence that the
low but finite permeability of microvascular walls to macromolecules involves a pathway through the endothelium
that is additional to that responsible for the exchange of
water and small hydrophilic solutes. Thus, although the
clearance from the plasma to the tissues of small solutes
declines rapidly as their molecular size increases, the
clearance of molecules larger than serum albumin falls
only slightly with increasing molecular radius. This difference was first clearly described by Grotte (95) from studies on the transport of dextran between the plasma and
the lymph. To account for it, Grotte proposed that there
were two sets of pores in microvascular walls: one set,
with radii in the range of 4 –5 nm, was available to small
and intermediate-sized molecules as suggested by Pappenheimer et al. (222), and the other set was large pores
with radii of 40 – 60 nm and responsible for macromolecular permeability. Grotte (95) estimated that there would
be several thousand small pores for every large pore (see
sect. IV). Thus, although small molecules such as glucose
might pass through the large pores, their permeation
through the microvascular wall would be dependent on
the frequency of the small pores per unit area of microvascular wall. In contrast, the permeability to macromolecules would be entirely determined by the frequency and
dimensions of the large pores.
Within a short time, an alternative hypothesis to large
pores had been proposed for the permeation of macromolecules. The early electron micrographs of capillary
endothelium revealed that the endothelial cells contained
many small vesicles (213). Palade (214) suggested that the
vesicles were involved in transport, ferrying small volumes of plasma and interstitial fluid in opposite directions
across endothelial cells, a process which has become
known as “transcytosis” (280). Evidence for transcytosis
accumulated as electron microscopists were able to show
that the vesicles could be labeled by electron-opaque
tracers injected into the blood (e.g., Refs. 24, 25, 35, 36,
137). A further development in the potential involvement
of vesicles in transport came when Simionescu et al. (281)
suggested that vesicles might fuse to form transendothelial channels and act as a pathway through which water
and small hydrophilic solutes might exchange by diffusion and convection.
Measurements of the transport of large molecules
between the plasma and the tissues, however, indicated
that this had a large convective component, and reviewing
the subject in 1994, Rippe and Haraldsson (246a) concluded that there was little positive evidence in favor of it
being achieved by transcytosis. Since this time, however,
much has been learned about endothelial vesicles and
about vesicular transport in general. Before discussing
these new findings, it is important to consider the arguments that Rippe and Haraldsson (246a) used to discount
the importance of transcytosis in microvascular permeability to macromolecules. Although it now appears that
vesicles may indeed be involved in the transendothelial
transport of macromolecules, it is still possible that the
mechanism, itself, might not be transcytosis.
A. Arguments Against Transport
of Macromolecules Via Vesicles
Rippe and Haraldsson (246a) considered four lines of
evidence that appeared to be inconsistent with transcytosis and could easily be interpreted in terms of convection
of macromolecules through large pores.
1) The transport of macromolecules from the
plasma to the tissues or to the lymph has a large convective component even for very large molecules such as
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D. Conclusion
727
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C. C. MICHEL AND F. E. CURRY
Most of the evidence which Rippe and Haraldsson
considered to favor the “large pore” hypothesis and to be
inconsistent with transcytosis was concerned with
whether the transport of macromolecules was convective
in nature, and we shall consider their arguments under
this heading. The arguments based on the morphology of
the vesicles is discussed in section VE.
B. Is Macromolecular Transport Convective
in Nature?
A large number of studies have reported that the
transport of macromolecules from the circulating plasma
into the lymph is enhanced in proportion to the fluid
filtration rate and hence to the lymph flow (243, 294, 295).
The interpretation of these observations as evidence for
convective transport of macromolecules across microvascular walls rests heavily on the assumption that a steady
state is established between the newly formed filtrate
entering the interstitial space and the lymph that is draining it. A rough calculation of the time required to achieve
a steady state between the composition of the capillary
filtrate and the lymph following a step increase in filtration rate gives us immediate insight into how well the
assumption of a steady state has been fulfilled in many of
the published experiments. In a tissue such as the small
intestine where the microvessels are fenestrated and the
Lp is high, a mean value for the capillary filtration
capacity of the tissue would be of the order of 0.1
ml z min21 z mmHg21 z 100 g tissue21. Thus, if the microvascular pressure is raised by 10 mmHg, filtration rate
is increased to 1.0 ml z min21 z 100 g tissue21. The interstitial fluid volume of such a tissue is 15–20 ml/100 g, and
if we assume that the lymph has the composition of
well-mixed interstitial fluid, the washout of macromolecules should follow a single exponential with a time constant of 15–20 min. After a step increase in filtration rate,
it should take 45– 60 min for the lymph protein concentration to be within 5% of its new steady-state value. If
lymph is sampled before this time and it is assumed to
represent the composition of the filtrate that is being
formed at the capillary walls, it will have a higher protein
concentration than that of the filtrate so that even if the
flow of the lymph is equal to the net filtration rate (an
additional confounding factor), the transport of protein
from plasma to lymph will be overestimated.
In tissues such as muscle, the capillary filtration capacity is an order of magnitude lower than in small intestine, and although the interstitial fluid volume may be
slightly lower, the time constant describing the approach
to a new steady state is increased into the range of
120 –150 min. Thus, in these tissues after an increase in
microvascular pressure of 10 mmHg, the protein concentration of the lymph may take 6 – 8 h to be within 5% of its
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fibrinogen. Although convection of macromolecules is to
be expected for transport through large pores, convection
appeared to be inconsistent with transcytosis.
2) In experiments on the perfused hindquarter
preparation of the rat, they found that, under conditions
of fluid balance (i.e., when there was neither net filtration
nor net reabsorption) lowering the tissue temperature
from 36 to 14°C reduced the clearance of albumin from
the perfusate to the tissues by one-third. This was a much
smaller effect of temperature than would be anticipated if
albumin transport was effected by transcytosis but almost
exactly what might be predicted if transport occurred by
convection through large pores.
3) In other experiments with the same preparation,
they found that, under conditions of fluid balance, the
transport of albumin from the perfusate to the tissues was
proportional to oncotic pressure of the perfusate and
hence to the mean hydrostatic pressure in the microcirculation. This fit the picture of the transport of macromolecules through large pores but was not easily accounted
for in terms of transport via vesicles.
4) Haraldsson and Johansson (100) examined the
transport of labeled albumin from the perfusate to the
tissues of the perfused hindquarter preparation of the rat
after the tissues had been fixed with glutaraldehyde. The
reduction in albumin clearance appeared to be in proportion to the reduction in fluid filtration capacity and to the
reduction in the permeability to the small hydrophilic
solute CrEDTA. They concluded that because glutaraldehyde fixation, which immobilizes the movements of the
vesicles, did not reduce the transport of albumin more
than the transport of fluid or small hydrophilic solutes,
vesicular transport did not contribute significantly to the
transport of albumin.
Although it had been repeatedly shown that the endothelial vesicles were labeled in a progressive fashion by
electron-opaque tracers circulating in the blood, Rippe
and Haraldsson (246a) discounted this as evidence for
transendothelial transport of macromolecules by transcytosis on two grounds. First, from the morphological data
of Bundgaard et al. (29) and Frokjaer-Jensen (78, 80, 81),
it appeared that the vesicles were arranged in fused clusters that were continuous with the caveolae at either the
luminal or the abluminal surface of the endothelium.
There were no free vesicles in the endothelial cell cytoplasm, and Rippe and Haraldsson (246a) argued that free
vesicles should be present in the cytoplasm if transcytosis
were taking place. Second, the progressive labeling of the
vesicles could be accounted for by the percolation of the
tracer into the racemose vesicular structures from caveolae on the luminal surface and by “back-filling” of caveolae at the abluminal surface after the tracers had crossed
the endothelium either via the intercellular clefts or possibly via occasional transendothelial channels formed by
the fusion of vesicles.
Volume 79
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MICROVASCULAR PERMEABILITY
sA is 0.97 and six times the convective coupling that is
present when sA is 0.99.
In the perfused hindquarter preparation, the clearance of albumin from plasma into muscle tissue when net
fluid exchange was zero (isogravimetric conditions) was
very much greater than in the muscles of intact rats.
Rippe and colleagues (246a, 247) had provided evidence
to suggest that even under these conditions transport of
albumin from the plasma to the tissues was by convection
through large pores. The isogravimetric condition was
arrived at by balancing the mean pressure difference
across the microvascular walls against the effective oncotic pressure. They argued that if a small number of large
pores were present in microvascular walls as well as the
small pores, the isogravimetric condition would represent
a state when there was filtration of fluid through the large
pores where the effective oncotic pressure would be low
(in view of their low s to macromolecules), and this was
exactly balanced by the reabsorption of fluid through the
small pores where s was high. This would have to represent a balance throughout a microvascular bed. As we
have argued previously, individual microvessels may have
no large pores. In support of their interpretation, Rippe et
al. (247) demonstrated that when the tissue temperature
was lowered from 36 to 14°C, the albumin clearance fell
from 0.029 to 0.019 ml z min21 z 100 g21. A fall in temperature of this magnitude would be expected to reduce
filtration and hence macromolecular clearance through
the large pores in inverse proportion to the resulting
increase in fluid viscosity. The ratio of the clearances
reported by Rippe et al. (247) was almost exactly equal to
the inverse of the fluid viscosities at these two temperatures. They argued that a very much larger decrease in
albumin transport would be observed if this were
achieved entirely by transcytosis.
Their third experiment was also consistent with this
picture. They noticed that as the oncotic pressure of the
perfusate was raised, the transport of albumin to the
muscle tissues of the perfused limbs was increased in
almost exact proportion under isogravimetric conditions.
They pointed out that as the perfusate oncotic pressure
was increased, a new isogravimetric state was achieved
by raising the mean microvascular pressure proportionately and so increasing the filtration of fluid and macromolecules from plasma to tissues through the large pores.
When taken with the initial demonstration of the
filtration dependence of protein clearance, it is difficult to
think of any satisfactory interpretation of these experiments other than that the transport of macromolecules
through the walls of microvessels in the perfused hindquarter preparation occurs by convection through large
pores. The question then arises as to why the findings of
Reed, Renkin, and colleagues (235, 242, 244, 245, 301)
should be so different from those of Rippe and colleagues
(101, 247). The clearance of albumin in the perfused hind-
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new steady-state value with an increased probability of
overestimates of their concentration in the filtrate and the
convective component of their transport.
Although these calculations simplify the real situation and do not take into account the increase in volume
of the interstitial space and the adjustments of the microvascular and interstitial hydrostatic and oncotic pressures
that may occur shortly after fluid filtration has increased,
they do not underestimate the magnitude of the problem.
In a critical review of the lymph clearance technique,
Renkin and Tucker (245) describe a hitherto unpublished
experiment of Renkin and Kramer in which the flow and
composition of the lymph was followed for a period of 8 h
after a step rise in microvascular pressure. From this it is
clear that changes in the composition and the flow of the
lymph continued until 7 h had elapsed. If samples of
lymph taken after 6 h were used to estimate protein
transport through the microvascular walls, the contribution of convection would have been exaggerated considerably. Although many investigators are aware of these
problems, few have been prepared to wait for more than
an hour for a new steady state to be established. Thus
evidence for the convective transport of macromolecules
through microvascular walls based on the passage of
these molecules between the plasma and the lymph
should be viewed critically from this point of view.
To overcome the problem of establishing a steady
state, Reed (235) and Renkin et al. (242) revived and
updated the tissue uptake method for estimating the
transport of macromolecules between the plasma and the
tissues. Independently both groups developed the method
to give a minimum estimate of the reflection coefficient
(sA) of microvascular walls in rat hindlimb skeletal muscle. The values they obtained were high: 0.975 was reported by Reed (235) and 0.99 was reported by Renkin et
al. (242). Both groups reported lower values for sA in rat
skin capillaries (0.94 and 0.98). From these and subsequent studies using the tracer uptake technique, Renkin et
al. (242) concluded that the coupling of the transcapillary
transport of albumin to fluid filtration is low. They have
also obtained evidence to suggest that microvascular albumin permeability may be subject to regulation by atrial
natriuretic peptide (244, 301).
This conclusion based on the transport of albumin
into the muscle tissues of anesthetized but otherwise
intact rats contrasts with findings obtained by Rippe and
colleagues (101, 247) using the perfused hindquarter preparation. Here, they demonstrated a clear linear dependence of albumin uptake into the muscle tissue on the
fluid filtration. The slope of this relation indicated that sA
was between 0.92 and 0.94. Although these values may not
appear to be very much less than of those Reed (235), it
must be remembered that the coupling coefficient is
(1 2 sA), which means that a value of sA of 0.94 represents twice the convective coupling that is present when
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C. C. MICHEL AND F. E. CURRY
C. General Features of Vesicular Transport
Over the past decade, much has been learned about
transport vesicles in cells other than endothelial cells.
Most of this information has come from studies on the
vesicles transporting proteins from the Golgi stacks to the
endoplasmic reticulum and from the vesicles involved in
endocytosis and secretion. Although the details of these
processes vary from one situation to another, several
general features have been identified that appear to be
common to all types of vesicular transport. Excellent
reviews of work in this rapidly moving field have been
published (e.g., Refs. 252, 253). A brief account of them is
given here as a background to discussion of vesicular
transport in endothelial cells.
The first stage in the formation of vesicles from a
membrane involves the assembly of a coat of polymeric
protein and of assembly (ARF) proteins. The ARF proteins bind to the cytoplasmic processes of transmembrane proteins (often receptor proteins) and act as binding sites for the polymeric coat proteins. In endocytic
vesicles, the coat protein is clathrin, whereas at the Golgi
membranes the coat proteins are called cotamers or COPI
or COPII. The assembly process involves the breakdown
of GTP, with energy being required to mold the membrane
and its underlying structures into a flask-shaped invagination.
The bud, so formed, pinches off from the parent
membrane by a process called periplasmic fusion. In
COPI-coated vesicles, this involves the fatty acyl CoAA
and in the clathrin-coated vesicles, the GTPase dynamin.
When GTP is hydrolyzed, the assembly proteins dissociate leaving a coated but less stable vesicle. The coat
proteins then dissociate, and an uncoated transport vesicle is left. This then moves, either by diffusion or by an
active process, along cytoskeletal fibers toward its target
membrane.
Fusion of the transport vesicle with its target involves three general groups of proteins. These are proteins on the cytoplasmic face of the vesicle membrane
(v-SNAREs), proteins on the target membrane (tSNAREs), and a series of cytoplasmic factors called
SNAPs. These acronyms reveal the importance of the
discovery that the process of fusion of a transport vesicle
with its target membrane can be inhibited by N-ethylmaleimide (NEM). SNAP stands for soluble NSF attachment
protein, where NSF, which is an ATPase, is an abbreviation for NEM-sensitive factor. SNAREs are SNAP receptors.
Uncoating the transport vesicle exposes the vSNAREs, which can then bind to the t-SNAREs on the
target membrane. Binding and fusion involve both NSF
and the SNAPs. The SNAPs bind the SNARE complex,
which is then disrupted by the hydrolysis of ATP by NSF
so that membrane fusion can occur. The details of the
fusion process are not clear at present, and it is possible
that they differ slightly in different vesicle systems.
Previously, it was argued that the pairing of the vand the t-SNARES was regulated by low-molecular-weight
proteins that are known as Rabs and hydrolyze GTP. After
the v- and t-SNAREs have paired, it was believed that NSF
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quarter preparation in the isogravimetric state is greater
than that observed in the intact animal by more than an
order of magnitude, and although some of this difference
may be accounted for by underperfusion of the muscle
vascular beds in intact animals and maximal vasodilation
in the perfused preparation, the question arises as to
whether there are large pores in the isolated perfused
hindquarter preparation that are not present in the intact
animal. It is possible that such large pores while being
relatively unselective to albumin may restrict the passage
of a very much larger molecule. A series of experiments
by Rutledge (254) is interesting in this respect. These
studies were carried out on single perfused frog mesenteric microvessels, and the permeability to sodium fluorescein, FITC-labeled albumin, and fluorescently tagged
low-density lipoprotein (LDL) was compared in the same
vessel at 18 –21 and at 4 – 6°C. Lowering the temperature
reduced the permeability to all three solutes: sodium
fluorescein permeability was reduced to two-thirds of its
value at the higher temperature, albumin to one-half, and
LDL to one-fifth. The predicted effects of the increase in
fluid viscosity on diffusion through aqueous channels fitted the reduction in permeability to sodium fluorescein
almost exactly, but the reduction in permeability to LDL
was very much greater than this. Furthermore, whereas
the albumin clearance from the perfused microvessels
could be increased at both 18 –21 and 4 – 6°C by raising the
microvascular pressure, no effect of microvascular pressure upon LDL clearance could be detected at 4 – 6°C,
although a slight correlation was observed at 18 –21°C. To
account for his findings, Rutledge (254) suggested that
LDL might pass through microvascular walls by lateral
diffusion in the lipid membranes of large transendothelial
channels. Rutledge (254) suggested that at 18 –21°C fluid
filtration through the channels might “vectorially enhance” the diffusion process, but at 4 – 6°C, either the
fluidity of the lipid membranes was too low for diffusion
to occur or the channels could not form. A further possibility is that transport of macromolecules through microvascular endothelium involves the vesicles and is enhanced by raised microvascular pressures. The
assumption that vesicular transport is independent of the
pressure gradient across the endothelium is one based on
intuition. Circumstantial evidence, however, is against it
since the labeling of endothelial vesicles by ferritin appears to be enhanced as microvascular pressure is raised
in frog mesenteric capillaries (198).
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MICROVASCULAR PERMEABILITY
D. Caveolae and the Vesicles of Endothelial Cells
Until recently, only morphological information was
available about the vesicles of endothelium. These organelles, which constitute 15–25% of the volume of many
endothelial cells, are relatively small (mean diameter 70
nm) and do not possess a clathrin coat. Differential staining and reconstructions from serial sections had revealed
that the majority of vesicles were present in fused clusters
that communicated with vesicles opening onto either the
luminal or the abluminal surface of the endothelium. That
the fused clusters of vesicles were not artifacts of chemical fixation was demonstrated in studies by FrokjaerJensen (81), who examined the ultrastructure of vesicles
in frog mesenteric capillaries prepared by rapid freezing
and freeze substitution techniques. Serial sections of the
endothelial cells not only confirmed that the vesicles were
arranged in fused clusters but showed that in some cases,
adjacent vesicles of a cluster might be separated from
each other by a single plasmalemmal membrane. The
relative scarcity of free vesicles in the endothelial cytoplasm was used as an argument against transcytosis by
Crone (44).
Flasklike invaginations of surface membranes to
form apparently uncoated vesicular structures were described in other cells and referred to as caveolae (325). In
some cases, these caveolae were found to be arranged in
fused clusters, and the absence of a conspicuous coat
from both the caveolae and the plasmalemmal vesicles of
endothelial cells gave credence to the idea that they differed from transport vesicles and might be static structures. In 1985, however, morphological evidence for a
coat covering the cytoplasmic aspect of endothelial
caveolae was reported (227). This was subsequently confirmed for caveolae of cultured fibroblasts by Rothberg et
al. (251), who identified one of the major components of
the coat as a protein which they named “caveolin.”
At about the same time, a 21-kDa protein, which was
called VIP21, was isolated from the detergent-insoluble
low-density fraction of vesicles of the trans-Golgi network of epithelial cells. Cloning and sequencing revealed
that VIP21 and caveolin were the same protein, which is
now known as caveolin-1 (see Ref. 224 for review). Unlike
clathrin or cotamers, caveolin-1 is an intregral membrane
protein. It has a hairpin structure with the amino and
carboxy terminals being cytoplasmic and the loop of the
hairpin being an unusual 33-amino acid intramembrane
domain. Its molecular organization is shown schematically in Figure 12. Recent work has shown the existence
of a multigene family of caveolin-related proteins that
show similarities to one another in structure but may
differ in specific properties and tissue distribution. Thus
caveolin-2 shows the same distribution as caveolin-1 and
is expressed in the same cells, but caveolin-3 (originally
called M-caveolin) is found only in muscle where it appears to be the major caveolin. Because caveolin-1 is an
integral membrane protein, current views on the structure
of the caveolar coat suggest that it is more an extension of
the cytoplasmic leaflet of the caveolar lipids rather than a
separate enclosing structure like the clathrin and cotamer
coats. This may account not only for the difficulty in
resolving the caveolar coat but may also allow the v- and
t-SNAREs of caveolae to be exposed when the coat is in
place. Thus fusion of coated vesicles may occur to form
coated clusters that are inherently more stable than might
be the case if the coats had to be shed before fusion
occurred. It should be emphasized, however, that the
membrane structure of caveolae is not understood at
present. It could be that like clathrin and cotamers, caveolin is only necessary for caveola formation and once
FIG. 12. Schematic representation of structure of caveolin molecule
indicating how it might be arranged in caveola membrane. Caveola
cavity is above, and endothelial cytoplasm is below. NH2 terminal of
caveolin molecule extends into cytosol, and COOH terminal is palmitoylated (straight line passing in inner leaflet of bilayers). Smallest
components of bilayer are cholesterol molecules. [Redrawn from Parton
and Simons (225). Copyright 1995 American Association for the Advancement of Science.]
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bound to the SNARE complex through a-SNAP. Hydrolysis of ATP by NSF then led to reorganization of the
complex and membrane fusion. Recent work on yeast
(206), however, suggests NSF and a-SNAP may participate before the v- and t-SNARES become entangled, possibly by inducing a conformational change that activates
the SNAREs (or at least the t-SNARE). Nichols et al. (206)
suggest that once the SNARE complex is formed, NSF
then catalyzes its breakdown so that docking and fusion
take place. It is possible that a-SNAP and NSF may act to
disentangle the SNARE proteins, segregating them to vesicular and target membranes for another round of docking and fusion.
The recent work has also shown that both v-SNAREs
and t-SNAREs are present on vesicle membrane. This
provides a molecular basis for the clustered configuration
of vesicles seen particularly in endothelial cells.
731
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C. C. MICHEL AND F. E. CURRY
immunolocalization using anti-caveolin-coated magnetic
microspheres. The caveolae isolated by shearing have
been shown to contain caveolin, Ca21-dependent ATPase,
and inositol 1,4,5-triphosphate (IP3) receptor as identified
in caveolae in situ (272) and the v-SNARE vesicle-associated membrane protein-2, monomeric and trimeric GTPases (including dynamin), annexins II and IV, NSF, SNAP,
and AQP-1 (268, 270).
Evidence that caveolae may undergo fission and bud
away from the isolated luminal membrane has been reported by Schnitzer et al. (273) in a cell-free system. The
budding process was measured as the release of caveolin
from silica-coated membrane fragments and was dependent on the presence of GTP and an extract of cytosol.
Thus, after 17 years of doubt, the endothelial caveolae appear to have most of the molecular credentials for
transport.
E. Evidence for the Involvement of Vesicles in the
Transendothelial Transport of Macromolecules
Rippe and Haraldsson (246a) suggested that the progressive labeling of vesicles with electron-opaque macromolecular tracers could be accounted for by percolation
of tracer through the racemose structures that opened
onto the luminal surface of the endothelial cells combined
with back-filling of abluminal caveolae by tracer. Evidence against this interpretation and in direct support of
a transendothelial pathway via the vesicles was provided
by Wagner and Chen (310). Using terbium as a tracer for
transport in the capillaries of rete mirabile of the eel,
these workers showed that reconstruction from serial
sections of the endothelium revealed localized deposits of
tracer in the pericapillary spaces associated with labeled
vesicles but separate from the intercellular clefts (or any
other pathway).
Although Rippe and Haraldsson (246a) and before
them Crone (44) argued that the absence of free vesicles
from the endothelial cytoplasm was an argument against
transcytosis, an alternative means of vesicular transport,
consistent with this morphology, had been suggested
from different evidence by Clough and Michel (38). They
proposed that vesicles are continually fusing and separating from their immediate neighbors. Mixing of their contents during periods of fusion meant that macromolecules
could be transferred through the vesicles across endothelium without the need for single “unattached” vesicles.
One can speculate that where the endothelium is thin, the
repeated fusion and separation of vesicles to each other
and to the luminal and abluminal membranes might occasionally lead to the formation of channels passing
through the cells (281). Such channels, of course, could
be conduits for convective transport of macromolecules.
If this were so and the channels remained open when the
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formed the caveolae persist. In support of this view is the
observation (284) that treatment of fibroblasts with cholesterol oxidase (which converts cholesterol to cholesterone) leads to the migration of caveolin from the caveolae
at the cell surface to the Golgi membranes, without a
reduction in the total number of caveolae.
The discovery of caveolin-1 initiated the molecular
investigation of the caveolae, and two important features
were recognized at an early stage: 1) that the caveolae
were found in regions of the cell membrane where high
concentrations of sphingolipids and cholesterol rendered
the membrane insoluble to detergents such as Triton
X-100 and 2) that certain ligand binding membrane proteins were concentrated in these detergent-insoluble regions. Still viewing caveolae as static structures, Anderson et al. (14) proposed they were involved in a form of
membrane transport that they referred to as potocytosis.
Here, the ligands linked to their receptor proteins are
concentrated within the caveolae together with specialized membrane transporters for the ligand. The environment within the caveola cavity is modified (e.g., pH reduced) leading to release of ligand from its receptor and
its rapid removal into the cell by the transporter as local
concentration of ligand within the caveola rises well
above that of the extracellular fluid. Evidence for potocytosis was based on uptake of folate into cultured fibroblasts. The folate receptors are glycosylphosphatidylinositol (GPI)-linked proteins and are found concentrated
in the detergent-insoluble extracts of membrane. These
studies, together with evidence that caveolin-1 may occur
in association with regions of membrane where caveolae
are absent, led to the definition of caveolae as membrane
domains that are Triton insoluble and associated with
high concentrations of GPI-linked proteins and cholesterol with the addendum that they may form “uncoated”
pits at the plasma membrane surface. This view is controversial. Not only is the separation of the membrane
component from the morphology of the caveolae open to
dispute (225, 283), but the GPI-linked proteins have been
found to be absent from caveolae isolated from endothelial cells (269). Simons and colleagues (225, 283) have
retained the term caveola to describe invaginations of the
cell membrane while using the term rafts to describe
membrane domains rich in cholesterol and sphingolipids.
The isolation of caveolae from endothelial cells of the
microvessels of rat lung followed the development of a
method described by Jacobson et al. (130) that involved
perfusion of the vasculature with a suspension of cationic
silica particles. These particles coat the luminal endothelial membranes allowing them to be separated from tissue
homogenates by centrifugation (130, 269, 272). Caveolae
attached to the cytoplasmic surfaces of the luminal membranes are then stripped away by shearing during sonication at 4°C in the presence of Triton X-100 (269). In a
recent development (288), caveolae have been purified by
Volume 79
July 1999
MICROVASCULAR PERMEABILITY
indeed report that NEM reduces transport of sucrose
from perfusate to tissues of the heart. The pathway
shared by these molecules would normally be assumed to
pass through the intercellular clefts, but Predescu et al.
(232) suggest that the transport of all hydrophilic solutes
occurs via the plasmalemmal vesicles with selectivity being a temporal phenomenon. Thus they believe that a
vesicle or vesicular transendothelial channel may act as a
large pore for a small fraction of time and a small pore for
much of the remainder of its existence. Although the
hypothesis cannot be discounted, the evidence on which
it is based has to be examined more critically before it is
considered in detail. Predescu et al. (232) do not believe
that the intercellular clefts are pathways for these molecules. Their evidence for this view is that they failed to
detect tracers filling the luminal aspects of the intercellular clefts in their electron micrographs. From the arguments presented in the earlier part of this review, the
present authors do not agree with this interpretation (see
section IIIE). Furthermore, the absence of effects of NEM
on other stages of the transport process has to be demonstrated more convincingly. The report that NEM reduced the transport of sucrose from perfusate to tissues
of the heart by 12% requires follow up because it suggests
that NEM might be reducing the perfusion of the microvascular bed. Because NEM is reactive with components
of the cytoskeleton in addition to those known to be
involved in vesicular fusion, it has to be shown that NEM
is reducing the transport of the sucrose and small proteins
by its effects on the plasmalemmal vesicles and not on
something else in the endothelium or smooth muscle of
the microvasculature. If the latter were so, the reduced
transport of sucrose into the tissues of the heart would
merely reflect reduced delivery of sucrose to the microcirculation due to vasoconstriction of the arterioles.
F. Receptor-Mediated Transport
of Macromolecules
The demonstration of binding sites for plasma macromolecules on the surface of endothelial cells has led to
suggestions that transcytosis might involve receptor-mediated transport. Specific binding molecules have been
identified for transferrin (131), insulin (140), ceruloplasmin (293), and albumin (89, 197, 231, 267, 270). In the case
of albumin, three separate binding proteins have been
described from the membranes of rat microvascular endothelial cells. These molecules have molecular masses of
60, 30, and 18 kDa and are known as gp-60 or albondin,
gp-30, and gp-18, respectively. Albondin is expressed in
those microvascular beds where the endothelium is continuous with the exception of the specialized endothelium
of brain capillaries (270). It is not expressed in microvessels where the endothelium is predominantly fenestrated
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tissues were cooled, they would represent the large pores
of Rippe and Haraldsson (246a). It is possible that such
channels might also remain open after the tissues had
been fixed by glutaraldehyde, but the experiments of
Haraldsson and Johansson (100) should be interpreted
with caution. Measurements of permeability of single perfused vessels before and after fixation suggest that permeation of the interstitium might be reduced by the fixation process (39). There are recent reports that
transcellular channels involving vesicles and vacuoles are
opened by mediators that increase permeability, and this
is discussed in section VIE.
More persuasive evidence for the involvement of vesicles in macromolecular transport across endothelium
has come from applying knowledge of their molecular
constitution. Cells lose their caveolae if they are treated
with the cholesterol scavenger filipin (251). Schnitzer et
al. (274) showed that not only did filipin remove caveolae
from cultured pulmonary microvascular endothelial cells,
but it also inhibited transport of albumin across monolayers of these cells. Even more convincing to physiologists
was their demonstration that the transport of albumin
from microcirculation to tissues of the isolated perfused
lung was greatly reduced by filipin treatment of the tissues, whereas the clearance of inulin, a molecule which is
believed to pass through the intercellular clefts, was unaffected by filipin.
We have seen that the fusion of transport vesicles
with their target membranes is inhibited by the NEM
which binds to NSF and SNAPs. Predescu et al. (230)
reported that NEM reduced both the net transport of
dinitrophenylated albumin from perfusate to tissues of
the mouse heart and the labeling of caveolae with albumin
in the myocardial capillaries. Similar effects of NEM on
albumin transport from perfusate to tissues in the rat lung
and across monolayers of cultured microvascular endothelial cells were subsequently reported by Schnitzer et al.
(266). The latter group also showed that NEM did not
affect the clearance of inulin from the perfusate into the
rat lung tissues, indicating that NEM was not affecting
transport via the paracellular pathway. Thus it appears
that both NEM and filipin, which are expected to inhibit
vesicular transport, have been found to reduce the transport of albumin from the microvessels into the tissues of
the rat lung while not affecting the clearance of inulin, a
molecule which is thought to pass through endothelium
via the paracellular route.
Recently, Predescu et al. (232) reported that NEM
induced a 85–90% reduction in the transport of small
proteins [dinitrophenol (DNP)-labeled myoglobin and
DNP-labeled a-lactalbumin]. This is a surprising finding.
The permeability and reflection coefficients of molecules
of this size (molecular diameters 3– 4 nm) indicate that
they share the same type of pathway as that used by inulin
and sucrose (e.g., see Fig. 1), and Predescu et al. (232) do
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C. C. MICHEL AND F. E. CURRY
G. Concluding Comments on
Macromolecular Permeability
Rippe and Haraldsson (246a) argued that because
macromolecular transport between the blood and the
tissues appeared to be linked so closely with net fluid
filtration, macromolecules were carried through micro-
vascular walls by convection via large pores, and vesicular transport could play no more than a very minor role. It
is therefore worth reconsidering their arguments in the
light of the preceding discussion.
Although transport of macromolecules as large as
fibrinogen between the plasma and the lymph appears to
be convective, the data from those tissues where the
microvascular endothelium is predominantly continuous
should be viewed critically. In these vascular beds, the
failure to reach a steady state between the filtrate leaving
the microvessels and the lymph leaving the tissues is
likely to give the appearance of convective transport of
macromolecules when it does not occur. This criticism
applies less to studies on plasma to lymph transport of
macromolecules in microvascular beds where the endothelium is predominantly fenestrated. Even here, however, a steady state is unlikely to be reached in much less
than an hour.
The experiments of Rippe et al. (247) on the isolated
rat hindquarter prepraration appear to be entirely consistent with convective transport of serum albumin. There is,
however, a conflict between the magnitude of convective
transport of albumin in the microvascular beds of perfused muscle of this preparation and in the very much
smaller degree of convective coupling reported by Renkin
et al. (242) in rat muscle perfused by an intact circulation.
A possible interpretation would be that in microvessels of
perfused tissue, there are a few large pores that are not
present in intact microvascular beds. If this were true, it
would account for discrepancies between the observations on the rat hindquarter preparation and those made
on intact microvascular beds.
Haraldsson and Rippe (103) discounted the potential
role of vesicles in microvascular permeability to macromolecules on the grounds that they believed this would
not involve convective coupling. The evidence now seems
to suggest that vesicles or caveolae are involved, particularly in those microvascular beds where the endothelium
is continuous. Suggestions that the labeling of vesicles do
not represent a pathway through the endothelium can be
discounted as a result of the work of Wagner and Chen
(310). The question as to whether this evidence represents transcytosis or the passage of tracer through transendothelial channels remains unanswered. The report
that filipin, which removes the endothelial caveolae (or
small plasmalemmal vesicles), inhibits the transport of
albumin across microvascular walls of the perfused rat
lung (and also across monolayers of cultured microvascular endothelium) reinforces the evidence for the importance of vesicles in macromolecular transport particularly
when filipin does not appear to influence permeability to
inulin in these preparations. This is further evidence that
NEM inhibits albumin transport in rat lung and mouse
heart but here, as we have seen, more controls are nec-
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or sinusoidal (adrenal, pancreas, liver, and small intestinal mucosa). Albondin binds both native (rat) albumin
and BSA and appears to be expressed only in endothelial
cells. It has homologies with SPARC, a protein secreted
by endothelial cells, fibroblasts, and smooth muscle cells.
Both antibodies to albondin and unlabeled BSA inhibit the binding of 125I-BSA to cultured bovine lung microvascular endothelium and reduce its transport across
monolayers of these cells. They do not, however, appear
to reduce the internalization or transport of albumin-gold
complexes or of BSA that has been chemically modified
by exposure to maleic anhydride (Mal-BSA).
The two smaller albumin binding proteins, gp-18 and
gp-30, bind albumin-gold complexes and Mal-BSA but not
native albumin. Unlike albondin, gp-18 and gp-30 are expressed in fibroblasts and smooth muscle cells as well as
in endothelium. Because they do not bind to native albumin, Schnitzer and Oh (270) have suggested that gp-18
and gp-30 are scavenger receptors involved in the endocytosis and degradation of modified forms of albumin.
They believe that albondin, however, is involved in receptor-mediated transcytosis of albumin. Extending their observations on BSA transport across monolayers of cultured endothelium, they have shown that both unlabeled
BSA and antibodies to albondin reduce tissue uptake of
125
I-BSA in the isolated perfused rat lung preparation. In
contrast, the uptake of Mal-BSA into the perfused rat lung
is not reduced by native BSA or by antibodies to albondin.
There are, however, two problems concerning receptor-mediated transcytosis. First, the transport of macromolecules from blood to the tissues has not been demonstrated to follow the expected type of saturation kinetics.
Second, although binding of albumin to endothelium has
been demonstrated, the affinities appear to be high with
apparent dissociation constants in the range of 1 mg/ml
(1.5 3 1025 M). It is difficult to see how transport can be
affected through the endothelium by a carrier with such a
high affinity when the concentration of albumin in the
pericapillary space may be one-third to one-half of its
concentration in the plasma. The available binding curves
suggest that albumin would not dissociate from its carriers unless the albumin concentration at the abluminal
surface was one-tenth of the plasma concentration. Thus,
until there is stronger evidence to support it, the question
of receptor-mediated transport of macromolecules across
microvascular endothelium remains open.
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July 1999
MICROVASCULAR PERMEABILITY
pothesis becomes more reasonable if macromolecular
transport through the endothelium is nonconvective.
A similar conclusion regarding the importance of
nonconvective transport macromolecules was reached by
Xie et al. (323a) in their model of blood tissue fluid
balance for the whole organism. Their conclusion was
that measured values of interstitial fluid pressure and
oncotic pressure can only be accounted for if the transport of macromolecules from blood tissue was not convectively coupled.
VI. INCREASED MICROVASCULAR
PERMEABILITY
A. Phenomena of Increased Permeability
Increased vascular permeability is usually thought of
in terms of the large increase in permeability to fluid and
plasma proteins that occurs in acutely or chronically inflamed tissues. It is this type of increased permeability
that has been investigated most frequently, and about
which most is known. Before considering it in detail, we
might note that generalized small increases in microvascular permeability have been said to occur in a number of
systemic diseases (e.g., diabetes, hypertension, and rheumatoid arthritis). In addition, there are physiological variations in microvascular permeability.
Although these physiological variations are poorly
understood, we consider them first before discussing recent work on the mechanisms of permeability changes
induced by inflammatory mediators. Studies on amphibians and on rats have shown that microvascular permeability is increased by atrial natriuretic peptide (ANP).
Working on single perfused frog mesenteric capillaries,
Meyer and Huxley (181) have shown that ANP increases
Lp by a mechanism that raises particulate guanylate cyclase in the endothelium. They have also suggested that
the fluid volume status of frogs affects the expression of
receptors on microvascular endothelial cells. The increase in Lp that can be induced in frog mesenteric capillaries is greatly attenuated in animals where fluid volumes are chronically expanded (183). Renkin and Tucker
(243) report that expansion of plasma volume in rats
enhances transport of plasma protein from the vascular to
the interstitial compartment by a mechanism that is dependent on ANP. Because it does not appear to be accompanied by a fall in s, Renkin and Tucker (244) suggest that
it might involve either an increase in the porous area of
the capillary wall without loss of molecular selectivity or
an increase in the transport of macromolecules by transcytosis.
A rather different type of physiological stimulus,
namely, wall shear stress, has also been reported to increase vascular permeability. The most detailed investi-
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essary to be sure that the paracellular pathway is not also
affected.
Thus the current evidence suggests that microvascular permeability to macromolecules is linked to the integrity of endothelial vesicles and is increased by increasing
microvascular pressure. This could mean either 1) that
macromolecules pass through the endothelium via large
pores formed by the transient fusion of vesicles or 2) that
macromolecules are carried through the endothelium by
transcytosis (or in clusters of caveolae), and transcytosis
itself is pressure sensitive.
On the first hypothesis, the progressive labeling of
the endothelial vesicle population with macromolecular
tracers must represent the continual formation and closure of these vesicular channels. Cooling the tissue restricts the labeling to a subpopulation of vesicles that
could include those that are components of channels at
the time of cooling. Filipin treatment, by removing the
small vesicles, makes the vesicular route unavailable. By
inhibiting fusion, NEM prevents channels from forming,
and its presence must lead to the breakdown of channels
that have already formed. If the latter is true, then the
clustered groups of fused vesicles are presumably also
broken down in its presence.
On the hypothesis that transcytosis is pressure sensitive, either the loading of vesicles or their translocation
between luminal and abluminal membranes is accelerated
as the transmural pressure is raised. As noted earlier,
some evidence exists for this. In frog mesenteric capillaries microperfused with ferritin, the fraction of the vesicle
population labeled with ferritin molecules increases as
microvascular pressure increases (198). Furthermore, the
endothelium becomes thinner as transmural pressure is
increased, and this might be expected to reduce transit
time between luminal and abluminal membranes. If this
second hypothesis were true, it would mean that macromolecular transport, while sensitive to microvascular
pressure, was independent of convection.
There is some attraction to this second hypothesis
when one considers the coupling of fluid and protein
movements between the plasma and the interstitial fluid
and the distribution of fluid between the blood and the
tissues (see sect. IIIK). In tissues, where the microvessels
have continuous endothelium, measurements of the socalled Starling forces (e.g., the microvascular and interstitial hydrostatic and oncotic pressure) suggest that they
should favor fluid filtration at a level that is greatly in
excess of the local lymph flow (151) (see sect. IV). The
discrepancy is not present (or very much smaller) in
microvascular beds where the vessels are fenestrated. A
possible explanation is spatial separation at microvascular walls of pathways for the net movements of fluid and
protein from plasma into the tissues, allowing a different
set of Starling forces to be maintained across the primarily fluid-conducting channels (188) (see Fig. 9). This hy-
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C. C. MICHEL AND F. E. CURRY
B. Increased Permeability in Inflammation and
With Inflammatory Mediators
The pattern of increased microvascular permeability
in inflammation was first described from evidence based
on the leakage into the tissues of plasma proteins labeled
with dyes. After mild thermal or chemical injury to the
skin, there was an initial increase in permeability lasting
for 15–30 min, after which leakage was reduced for a
short period before increasing for a second sustained
phase that might last for several hours (317). This pattern
of response varied considerably depending on the species
of the animal and the nature of the stimulus to the tissues.
Mediators such as histamine, serotonin, and bradykinin
have effects that are similar to the initial phase of inflammation. In many cases, the initial phase can be inhibited
by antihistamines (H1 antagonists) (317). Using light microscopy and carbon labeling, Majno et al. (163a) showed
that the increased permeability induced by histamine and
serotonin was confined to the venules and did not involve
the true capillaries. Subsequent electron microscopy by
Majno and Palade (163) revealed that leakage from the
venules was associated with the development of openings
or gaps in the venular endothelia. Subsequent work in a
number of different laboratories confirmed that histamine, serotonin, bradykinin, and many other mediators
opened gaps in the endothelium of the postcapillary
venules but did not appear to influence the ultrastructure
of the capillaries.
The later phase of increased permeability, after thermal or chemical injury, does involve both capillaries and
venules. Once again, this appears to involve the development of gaps or openings in the endothelium (43). More
recently, it has been shown that increased capillary (as
well as venular) permeability occurs as a result of contact
with dead tissue (133). Gaps and fenestrae in capillary
endothelia are also induced by the cytokine, vascular
endothelial growth factor (VEGF) (248).
C. Local Edema Formation During the
Initial Phase
When human skin is injured by burns or by stings
from insects or plants, there is a rapid extravasation of
fluid into the tissues often resulting in the formation of a
blister. Formerly it was believed that this resulted from a
rapid increase in microvascular permeability combined
with a rise in microvascular pressure as a result of arteriolar dilatation. Although a quantitative study (16) suggested that a change in permeability alone was inadequate
to account for the rapid development of edema in tissues
subjected to burns, it is only within the last 10 years that
a clear explanation has emerged. Working initially on
burns to the skin of anesthetized rats, Reed and co-workers (142, 160, 161) reported that a rapid fall in interstitial
fluid pressure (Pi) occurred shortly after the injury was
inflicted. The fall in Pi was enormously amplified if the
circulation to the tissue was arrested, with values of Pi
approaching 100 mmHg below atmospheric pressure. In
the presence of an intact circulation and with the subsequent development of edema, the fall in Pi was to only
210 to 215 mmHg. Reed and co-workers have since
demonstrated similar rapid decreases in Pi in skin after
chemical injury (250), after treatment with carageenan
(249), and in the respiratory tract submucosa after degranulating the local mast cells (142). More modest
changes were seen after application of histamine and
serotonin to the tissues.
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gation of this phenomenon that has been published so far
was carried out on isolated perfused coronary venules of
the pig (327). It was shown that in this preparation transport of fluorescently labeled albumin across the vessel
wall was enhanced by increased flow through the vessel.
Estimation of the vessel wall permeability coefficient
showed that this increased with flow rate. Special precautions were taken to ensure that the mean pressure within
the vessel was constant even though the pressure difference along the vessel increased with increasing flow
rates. Yuan et al. (327) showed that the phenomenon
could be inhibited by the L-arginine analog NG-monomethyl-L-arginine, which is known to block nitric oxide (NO)
synthase (NOS). This finding greatly strengthens the evidence for the phenomenon, demonstrating that it is not
merely a consequence of the effects of flow upon the
estimation of permeability. There have also been three
additional reports of the phenomenon. The first of these
was by Friedman and de Rose (77) who noted that the
scatter of estimates of permeability of single frog mesenteric capillaries to potassium ions could be reduced by
plotting permeability against the flow through the vessel.
Friedman and de Rose concluded that potassium permeability was flow dependent. Their evidence, however, was
circumstantial, but very recently, Kajimura et al. (136)
used a microperfusion technique to vary the flow velocity
in frog mesenteric microvessels and demonstrated that
permeability to potassium ions was indeed increased as
flow velocity increased. In a very different preparation,
Pallone et al. (219) and Turner and Pallone (304) have
shown that the permeability of isolated perfused descending vasa recta to small hydrophilic solutes increases with
increasing perfusion rate.
If permeability is generally flow dependent, it has
important physiological implications particularly for the
transport of small hydrophilic solutes to and from the
tissues. In muscle, for example, it could account for the
large increase in glucose uptake that occurs during exercise without having to invoke large increases in the number of capillaries perfused within the tissue.
Volume 79
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MICROVASCULAR PERMEABILITY
D. Quantitative Estimates of Increased
Permeability With Histamine-Like Mediators
Early attempts to assess the magnitude of an increase
in permeability were based on estimates of leakage of
macromolecules (labeled with dyes or radioactivity) or
fluid from the plasma into the tissues (196, 317). This line
of approach was followed at a microscopic level (147)
where leakage was observed to occur from single microvessels. Arfors et al. (15) showed how this approach
could be made quantitative by counting the numbers of
leakage sites that could be seen to open in a prescribed
microvascular field. Working on the microvascular bed of
the hamster cheek pouch, they demonstrated a linear
relation between the number of leakage sites induced by
topically applied bradykinin and the logarithm of the bradykinin concentration. From this it would seem that openings have “all or none” properties; increasing the concentration of a mediator at the endothelial surface increases
the number of openings rather than increasing the size of
those that are already open.
Attempts to measure permeability coefficients when
permeability is increased have been made in a variety of
whole organ experiments. In an early study, Diana et al.
(62) showed that histamine increased the capillary filtration capacity (the mean value of LpS for a vascular bed)
without increasing the equivalent pore radius in microvascular walls of the isolated perfused dog hindlimb preparation. Essentially similar conclusions were reached
about the effects of histamine by Korthius et al. (143), and
Renkin et al. (239) reported a significant increase in
plasma protein flux from plasma to lymph in the perfused
dog paw without a significant fall in osmotic reflection
coefficients. Renkin (237) pointed out that some of these
experiments (particularly those of his own group) were
carried out over a longer period than would have coincided with the opening of gaps in the venular endothelium. Rather different results have been reported by McNamee and Grodins (179) and Wolf et al. (321), who found
that increased filtration was accompanied by a transient
loss of barrier properties to macromolecules. A similar
conclusion might be drawn from the study of Rippe and
Grega (245a), who found a large increase in the filtration
capacity of microvascular walls in the rat hindlimb after
histamine infusion, while the diffusional permeability to
chromium-labeled EDTA remained constant.
There have been many studies of the changes in
permeability coefficients on single perfused microvessels.
Most of these experiments have been carried out on frog
mesenteric microvessels, which do not respond to histamine, bradykinin, or serotonin (241) but do increase their
permeability in response to ATP (107, 114), ANP (181),
calcium ionophores (111, 193), and removal of plasma
protein (124, 168, 194, 303). Following the changes in Lp
alone, transient increases have been seen after calcium
ionophores (111) and ATP (107, 114). Removal of plasma
protein, however, brings about a sustained increase in Lp.
Relatively few studies have been carried out where
measures of two coefficients have been followed in a
single microvessel when permeability has been increased.
Changes in the reflection coefficient to macromolecules
and Lp have been measured in frog microvessels following mild thermal injury (41) and calcium ionophore (193).
Recently, Michel and Kendall (189) have reported a similar study comparing the effects of histamine and serotonin on the Lp and effective oncotic pressure exerted by
macromolecules across the walls of single rat venules.
Although both these mediators are believed to increase
permeability by opening gaps in the endothelia of the
vessels, histamine appeared to increase Lp more and reduce the macromolecular oncotic pressure less than serotonin. Using a simple two-pore model to analyze their
data, Michel and Kendall (189) concluded that although
the effects of serotonin were consistent with the opening
of pores at which macromolecules exerted an oncotic
pressure of zero (i.e., gaps in the endothelia), histamine
increased permeability by opening pores that still restrained the leakage of macromolecules, although to a
lesser extent than the same vessel in the absence of
mediators. Both the effects of histamine and those of
serotonin were transient, with peak increases in permeability being seen within 3 min of microperfusion with
serotonin and between 6 and 9 min after microperfusion
with histamine. Permeability returned to control levels
between 12 and 30 min despite the continued presence of
the mediator in the perfusate. Thus the time course of the
permeability changes correspond approximately with the
opening and closing of gaps in the venular endothelium.
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Thus it appears that the rapid development of edema
at the site of an injury may be partly, if not largely, a
consequence of the action of the interstitium. Just how
the reduction in Pi is brought about is not understood at
present. Reed et al. (236) have shown that antibodies to
connective tissue integrins also induce a fall in Pi. This
observation led them to suggest that normally the interstitial space is actively compressed from within. It is
proposed that fibroblasts attach themselves to the collagen network, pulling the component molecules closer
together and concentrating the macromolecules within
the network. It is possible that attachments are also made
between sites on the flexible chains of some of these
molecules and the more rigid structure of the matrix. It is
thought that breaking the bonds between the fibroblasts
and the matrix allows the interstitium to expand, lowering
Pi. At the same time, the release of parceled chains of
flexible polymers within the interstitial space should increase the osmotic effects of these molecules, and these
will be detected as a further fall in Pi.
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C. C. MICHEL AND F. E. CURRY
The quantitatively different effects of serotonin and histamine on the permeability to fluid and macromolecules,
however, suggest that histamine opens different pathways
to those opened by serotonin. Further evidence for different mediators opening different permeability pathways
has been reported by Schaeffer et al. (261). Investigating
the permeability of monolayers of cultured endothelial
cells, they have found that thrombin increased permeability to large molecules, whereas bradykinin increased permeability to small molecules.
E. Ultrastructural Basis of Increased
Microvascular Permeability
F. Openings in the Endothelium Associated With
Increased Permeability
When Majno and Palade (163) first described openings or gaps in the endothelium of the postcapillary
venules, they noted the correspondence in light microscopy between the accumulation of carbon in inflamed
vessels with the “silver lines” that are believed to indicate
the junctions between the endothelial cells. They also
observed that in their electron micrographs the cell membranes bounding the gaps were well formed and showed
no signs of disruption. In some micrographs, carbon or
mercuric sulfide could be seen between the abluminal
surface of the endothelium and the basement membrane
in the vicinity of an intact intercellular cleft. They interpreted these images to indicate that either the cleft had
reformed after being the site of an intercellular gap or that
the cleft opened into an intercellular gap either above or
below the plane of section. These observations were rapidly confirmed in many different laboratories, and a belief
in the intercellular location of gaps became widely accepted as the most reasonable interpretation of the available evidence. Complementing this hypothesis, Majno et
al. (164) proposed that the gaps were formed by adjacent
endothelial cells contracting away from each other. The
evidence for this was entirely morphological. Regions of
the endothelial cells bordering a gap were often greatly
attenuated with the cytoplasm bunched around the center
of the cell where the nuclei were wrinkled. Such changes
in shape might be expected to accompany cell contraction (164).
Subsequent work on isolated and cultured endothelial cells revealed the association of myosin with actin and
boosted the cell contraction-cell separation theory of gap
formation (67, 265a). Large openings form between the
cells with the rearrangement of the actin cytoskeleton
(265a) and the development of tension. Until recently,
however, the evidence for the intercellular location of the
gaps that arose in vivo remained largely indirect. A small
number of openings induced by histamine in the venules
of rat and cat mesentery were examined by computer
reconstruction of electron micrographs of serial sections
by Fox et al. (76). They revealed that the openings communicated with the intercellular clefts, but their complex
shape did not seem compatible with simple separation of
the cells that had contracted away from one another.
More recently, a series of investigations by McDonald and
colleagues (117, 176) provided strong evidence based on
light and electron microscopy for the intercellular location of the gaps. Investigating the effects of sensory nerve
stimulation and intravascular substance P on the permeability of venules in rat tracheal mucosa, McDonald (176)
correlated the beaded deposits of silver within the silver
lines of the intercellular clefts with the presence of gaps.
More recently, the same group used a combination of
techniques including scanning electron microscopy and
reconstruction from serial ultrathin sections to show that
most of the openings in the endothelium pass through the
clefts (18). The structure of these gaps, however, was
complex with many fine fingerlike processes linking one
cell to the other across the region of the opening. Reconstruction of gaps from electron micrographs of serial
sections has also been carried out by Braverman and
Keh-Yen (23). In venules of psoriatic lesions of human
skin, they reported that approximately one-half of the
openings in the endothelium passed between the cells,
but one-half of them passed through individual cells remaining quite distinct from the intercellular clefts. In a
parallel study, Braverman and Keh-Yen (23) investigated
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Although the opening of gaps in the endothelium
appears to account for increases in microvascular permeability under many conditions, there are circumstances
where permeability increases and no changes in endothelial ultrastructure can be detected. The perfusion of vessels with solutions devoid of plasma proteins is an example of this phenomenon (9, 169). Here, the failure to
detect openings in the endothelium led to the suggestion
that removal of plasma protein from the endothelial luminal glycocalyx led to increased permeability and loss of
selectivity of this structure (56). The demonstration that
serum albumin (263) and other proteins (303) did bind to
the endothelial luminal surface coat and reduce its permeability to other macromolecules supported this view.
Albumin and other plasma proteins were believed to occupy space within the glycocalyx, increasing its hydraulic
resistance (56) and to organize the molecules of the cell
surface into a more regular array that would increase its
effectiveness as a molecular filter (56, 184, 186). It is now
known that the interaction of serum albumin with microvascular endothelia is a more complex phenomenon.
Washing albumin from the vessel wall leads to an influx of
calcium into endothelial cells that can be prevented and
even reversed by depolarizing the cell with high concentrations of extracellular potassium ions (106).
Volume 79
July 1999
MICROVASCULAR PERMEABILITY
cases, the structure of the intercellular openings appears
to be simple, but in others, it involves complicated arrangements of fingerlike processes that may interdigitate
with each other. The transcellular openings, in contrast,
appear to be much less complex, although in some examples, a transcellular opening leads into the wide region of
an intercellular cleft. The latter appearance raises the
possibility that some intercellular gaps may be derived
from transcellular openings.
The importance of determining whether openings in
the endothelium are transcellular or intercellular relates
to how their formation might be investigated most profitably. Both types of opening appear to involve changes in
endothelial cell shape consistent with contraction. From
the effects of raised transmural pressure, it would seem
that increased wall tension alone leads predominantly to
the formation of transcellular gaps. Presumably, for intercellular gaps to form, endothelial cell contraction has to
coincide with uncoupling of the junctions. There is no
clear evidence as to how transcellular openings develop,
but it is possible that they originate from transendothelial
channels formed by the fusion of vesicles or vacuoles
with the luminal and abluminal membranes (202, 204).
Vacuoles are reported to occur more frequently after
endothelium has been activated than when it is quiescent.
Neal and Michel (202) have published images of serial
sections through vacuolar structures, and in one case, a
vacuole appeared to form a pathway through the endothelium. Contraction of endothelial cells to thin their
cytoplasm in the parajunctional region would speed the
process of transcellular channel formation by reducing
the size to which a vesicle or vacuole must expand before
it could form a channel. For a vesicle or vacuole to
enlarge, it must gain membrane, and this must come from
fusion with other vesicles or vacuoles or from the plasmalemmal membranes. Thus fusion of vesicles or vesicles
and vacuoles would seem to be a necessary part of transcellular gap formation. This sort of picture is consistent
with the suggestion that transcellular openings develop
from vacoles and VVO (74, 202). It may, however, involve
a very different mechanism from that proposed for the
formation of intercellular gaps.
VII. SIGNAL TRANSDUCTION
A. Overview
In the past 10 years it has become clear that the
modulation of microvessel permeability involves complex
signaling within endothelial cells. Much of the information about signaling pathways in endothelial cells has
been obtained from cultured endothelial cells where there
is sufficient cellular material to measure changes in the
intracellular composition of signaling molecules such as
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gaps induced in human skin by histamine injection. Here
all the openings in the endothelium appeared to communicate with intercellular clefts.
Braverman and Keh-Yen’s report (23) provided the
first clear evidence that some openings in inflamed
endothelium might pass through endothelial cells (i.e.,
transcellular) as well as between them (intercellular).
Earlier, Hammersen (99) pointed out that the morphology of the endothelium surrounding a gap was often
difficult to interpret in terms of the gap being formed by
adjacent endothelial cells contracting away from each
other. There was also evidence that cells might migrate
through openings that passed through the endothelial
cells of venules in bone marrow (31, 60), lymph nodes
(32), and the brain, in experimental allergic encephalomyelitis (155). Over recent years, Neal and Michel (201–
205) have presented evidence for transcellular openings associated with increased permeability in
mesenteric microvessels. When permeability was increased by mild heating (in the frog), A-23187 (frog and
rat), high transmural pressure (frog), and VEGF (frog
and rat), the openings were predominantly transcellular. With histamine and serotonin, however, both transcellular and intercellular openings were found with a
predominance of them being intercellular (191).
The Dvoraks and their colleagues (69, 72, 73) have
suggested that VEGF, histamine, and serotonin increase
the permeability of venules primarily by opening a transcellular route through the vesiculovacuolar organelles
(VVO). These are clusters of vesicles and vacuoles that
this group has described in the endothelia of tumor microvessels and of normal venules (70, 73). In unstimulated
venules, intravascular ferritin fails to enter the VVO, but
after local injection of certain inflammatory mediators,
the VVO became labeled with macromolecules. Using reconstructions from serial sections, the same group has
also found transcellular openings in venular endothelia
close to and often running into the intercellular clefts
(74). Because the openings are seen more frequently after
particulate tracers such as colloidal carbon have been
injected into the circulation, it has been suggested that
gaps may be derived from VVO and that their frequency is
greatly increased by particulate tracers (74). This challenging proposal needs further support.
At present, it would seem that openings in venular
endothelium may either pass between adjacent endothelial cells or pass through the peripheral cytoplasm of
single cells. The possibility that transcellular openings
might be fixation artifacts has been virtually eliminated by
Neal and Michel (205), who found that their frequency
was unaffected when the primary fixative was changed
from glutaraldehyde to osmium tetroxide. Through their
collaboration with McDonald and colleagues (18), they
confirmed that in rat tracheal mucosa the gaps induced by
substance P are predominantly intercellular. In some
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C. C. MICHEL AND F. E. CURRY
mechanisms that determine some chronic increases in
microvessel permeability.
B. Receptors
At least three receptor types are recognized in endothelial cells: 1) receptors coupled to trimeric G proteins
that activate or inactivate a wide variety of enzymes to
modulate the formation of second messengers; 2) receptors whose cytoplasmic domain is activated when the
receptor is occupied. The cytoplasmic domain may activate one or more specific enzymes to simultaneously
stimulate multiple signaling pathways. 3) There are receptors linked directly or indirectly to ion channels. The
general properties of these receptors are reviewed elsewhere (13, 285).
Many of the agents acting in acute inflammatory responses [including ATP, bradykinin, histamine, plateletactivating factor (PAF)] couple, via seven membranespanning receptors linked to the Gq family of G proteins,
to enzymes that activate phospholipases to release second messengers derived from membrane lipids. Phospholipase C (PLC)-b is activated to release IP3 and diacylglycerol (1, 75, 159, 262), while phospholipase A2 and
phospholipase D initiate arachidonic acid pathways and
phosphatidic acid pathways, respectively (72). Another
important subfamily of G proteins are Gs and Gi, which
activate and inhibit, respectively, adenylate cyclase to
regulate intracellular cAMP levels (166, 167). There are
multiple levels of complexity in the activation of the
signaling pathways. These may include multiple combinations of the G protein subunits (22) and the expression of
multiple isoforms of the key enzymes such as PLC and
adenylate cyclase (167, 289). In addition, a signaling molecule (e.g., diacylglycerol) may arise from more than one
pathway [e.g., from PLC (short acting) or phospholipase
D (long term)] with each source of the signaling molecule
itself activating a different pathway (166). These mechanisms may account in part for different patterns of permeability increase initiated by different inflammatory mediators.
Receptors whose cytoplasmic domains are activated
by phosphorylation can link to a variety of effector molecules including tyrosine kinases, guanylate cyclase, isoforms of PLC, and enzymes that activate or inactivate a
family of monomeric GTPases. These GTPases have a
variety of functions, including regulation of cytoskeleton
arrangement. For example, members of the Rho subfamily of GTPases have been shown to modulate endothelial
barrier permeability (116, 305), possibly by regulating
myosin light-chain phosphorylation and/or cytoskeletal
rearrangements (116, 305). Another function of monomeric GTPases is as transcription factors. Thus receptors
acting via GTPases can activate several signaling path-
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IP3, cAMP, and cGMP (see Refs. 158, 305 for reviews)
changes in protein phosphorylation (86, 87, 91, 276, 307)
and where electrophysiological and imaging methods can
be more easily applied to follow ion fluxes and changes in
intracellular composition (2, 34, 207, 306). Furthermore,
new approaches involving systematic mutational analysis
of receptor proteins, effector molecules, and signaling
molecules can be applied in culture and are less widely
available for studies in vivo (66, 87, 91, 158, 209, 335). On
the other hand, the advantages of working on cultured
endothelial cells are offset by differences between endothelial cells in culture and the endothelia of intact microvessels. Differences include the expression of receptors, ion channels, and contractile proteins due, at least in
part, to differences in cell attachment and different shear
and pressure forces acting on the cell surfaces (59, 297).
These differences may also reflect the absence of cells
such as pericytes, mast cells, and circulating leukocytes
that can modulate endothelial responses. Furthermore, as
discussed in section VII, endothelial cells in culture fail to
develop the permeability properties characteristic of intact endothelium, with permeability properties in culture
often close to the values measured near the peak of an
inflammatory response in an intact microvessel.
Over the past decade, the availability of fluorescent
probes to measure intracellular ionic composition, coupled with the refinement of methods to cannulate and
perfuse individual vessels in thin tissue suitable for intravital microscopy, have made it possible to evaluate the
contribution of particular signaling pathways to the control of permeability in intact microvessels (53, 105, 107–
111, 113, 114, 182, 186, 331). Another approach has been
to dissect individual microvessels from tissues such as the
heart and to cannulate and perfuse these isolated segments (123, 128, 139, 323, 326, 329, 333). The development
of these techniques has made it possible to build on the
knowledge of signaling from investigations in cultured
endothelial cells. In this section, we review investigations
of the regulation of microvessel permeability using results
from both intact microvessels and endothelial cells in
culture. We first give a brief overview of some of the
principal receptors involved in endothelial cells. We then
describe recent investigations of signaling pathways that
modulate acute changes in permeability in individually
perfused venular microvessels. These include the initial
increase in endothelial cell intracellular calcium concentration, a necessary, but not sufficient, requirement to
increase permeability during most acute increases in permeability, and mechanisms acting downstream from the
initial rise in cytoplasmic calcium concentration. In the
third part of this section, we have compared and contrasted results in vivo and in vitro. Finally, we note that
results from investigations of acute changes in permeability provide insights into the mechanisms that maintain
normal permeability properties and also possible links to
Volume 79
July 1999
MICROVASCULAR PERMEABILITY
C. Individual Microvessels: Introduction
Exposure of endothelial cell monolayers to a wide
variety of inflammatory agents results in a rapid increase
in cytoplasmic calcium to a peak in ,1 min when intracellular calcium concentration ([Ca21]i) falls back toward
control values. In endothelial cell monolayers in culture,
there is an increase in tension within 1–2 min, but in many
studies, especially those using thrombin as the agonist,
the onset of an increase in permeability to macromolecules may be delayed by many minutes after the rise in
[Ca21]i and the phosphorylation of myosin light chains,
with the peak increase developing after 20 –30 min or
even several hours. These results suggested that the increase in permeability was dependent not only on the
initial increase in calcium and tension in the endothelial
cells, but also on a cascade of calcium-dependent mech-
anisms acting downstream from the initial rise in [Ca21]i.
The problem with the use of such monolayer experiments
to understand the regulation of permeability in intact
microvessels is that the monolayers do not reproduce the
characteristic permeability increase in intact venular microvessels (51). In intact venular microvessels, the initial
increase in permeability is much more rapid after exposure to acute inflammatory agents, with a peak within 2–5
min. In most cases, permeability returns toward control
and, after washing the agonist from the tissue, the vessels
will respond with a second response within 30 min (53, 93,
171, 173).
D. Individual Microvessels: Experimental Studies
To investigate the initial signaling events and their
relation to the initial increase in permeability in intact
microvessels systematically, Curry and co-workers (111)
modified established methods to cannulate and perfuse
venular microvessels in the mesentery of mammalian and
frog microvessels, to load the endothelial cells with the
calcium-sensitive dye fura 2, and to measure both the
initial calcium influx and the initial increase in permeability under the same experimental conditions. Figure 13
summarizes data on the modulation of the permeability
properties of venular microvessels in frog mesentery exposed to ATP (114). Similar experiments have been carried out in hamster and rat mesenteric venular microvessels and in isolated coronary venules. In the experiments
shown in Figure 13A, the Lp of the wall was a measure of
changes in the permeability properties of the microvessel
wall. The permeability changes for larger solutes followed
the same time course as the changes in Lp because most
of the increase in solute flux was coupled to water flows
(255, 256).
These experiments demonstrated that in single perfused venular microvessels of frog, rat, and hamster mesenteries, the increase in permeability after exposure to
inflammatory mediators was similar to that in the intact
microcirculation. Furthermore, a transient increase in
[Ca21]i, with an initial peak that corresponded to, or
slightly preceded, the time of the initial peak increase in
permeability, was a required step leading to the initial
increase in the permeability of endothelial barriers. The
inflammatory agents used to activate the endothelial cells
in these studies included ATP, ionomycin, bradykinin,
histamine, and VEGF (20, 111, 114) (see Fig. 13A). In the
endothelial cells of intact microvessels, between 50 and
70% of the initial increase in [Ca21]i was accounted for by
calcium influx (114). If [Ca21]i was reduced by decreasing
the electrochemical driving force for calcium entry, the
associated increase in permeability is also attenuated
(Fig. 13B). Thus the magnitude of the initial peak [Ca21]i
was one determinant of the initial increase in permeabil-
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ways in parallel. Examples include the action of the VEGF
R2 (FLK-1 or KDR), receptors to increase cytoplasmic
calcium via a pathway involving PLC-g, and IP3. The same
receptor activated by VEGF could signal via Ras, with
subsequent activation of Raf and the mitogen-activated
protein kinase (MAPK) signaling pathway leading to cell
division (97). Thrombin also activates multiple pathways
leading to calcium release and Rho activation (305).
The third group of receptors is linked to channels for
ionic currents across the endothelial channels. Some are
linked via second messengers (calcium-dependent potassium channels) or via other components of the IP3 signaling pathway (2, 51, 138, 207, 262, 316). Because of the
importance of calcium influx in the regulation of endothelial barrier function, the mechanism of coupling between
receptors and calcium entry channels called “capacitative
calcium entry” is of particular interest. According to one
form of this hypothesis, calcium influx is controlled by a
chemical factor released by intracellular calcium stores
as these calcium stores are depleted. The nature of the
putative chemical has not been identified despite extensive research, and some investigators are now exploring
alternative mechanisms to account for the apparent coupling between receptor activation and channel opening
(33, 223). Recent studies suggest that G proteins may form
part of the coupling mechanism between receptors and
calcium influx. Investigations of the transient receptor
potential (trp) in the Drosophila photoreceptors have
demonstrated that both the putative store-operated channels and other store-independent calcium entry channels
are part of a family of membrane proteins from the trp
and trp-like (trpl) gene products. Expression of these
gene products suggests a direct activation of some channels by G proteins such as G11a (209, 335). The nature of
the link between the G proteins and the channel opening
is not yet understood (see Ref. 223 for review).
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Volume 79
ity, and the change in [Ca21]i was itself determined largely
by the magnitude of calcium influx.
The experiments in Figure 13B show that calcium
ions enter the endothelial cells via a passive conductance
for calcium ions. After exposure of venular microvessels
to ionomycin or ATP with normal bathing solutions, the
endothelial cell membrane was hyperpolarized from the
resting membrane potential of 250 mV by up to 20 mV.
This hyperpolarization is presumably the result of the
opening of calcium-activated potassium channels and increases the electrochemical driving force for calcium entry. Approximately half the increase in calcium influx is
the result of an increase in electrochemical driving force
for calcium entry (107). This normal increase in the electrochemical driving force for calcium entry is greatly
reduced under conditions that decrease the endothelial
cell potassium equilibrium potential (e.g., high potassium
bathing solutions as in Fig. 13B) and attenuate the driving
force for calcium entry. Under resting conditions, exposure of microvessels to isotonic high potassium solutions
(60 mM K1) depolarized the membrane potential of the
endothelial cells from values close to 250 mV to values on
the order of 10 –20 mV (105, 114). These results also
demonstrate that voltage-gated calcium channels do not
make a significant contribution to calcium influx into
endothelial cells in intact microvessels (51).
Although an increase in [Ca21]i is necessary for an
agonist-induced increase in permeability, it is not sufficient. The argument against a direct mechanistic link
between the rise in [Ca21]i and increased permeability,
and in favor of a cascade of calcium-dependent and
calcium-independent mechanisms leading to increased
permeability, is based on experiments that demonstrated increases or decreases in permeability, but with
no significant modification of the initial calcium transient. Examples are given in Figures 14 and 15. With
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FIG. 13. A: comparison of time course of changes in
Lp (open circles, ordinate on right) and endothelial intracellular Ca21 concentration ([Ca21]i) (solid circles,
ordinate on left) as a function of time after vessels were
exposed to ATP (10 mM). Lp is expressed as ratio of test
Lp over control Lp (n 5 14). Endothelial [Ca21]i are
mean values from 8 vessels within 0.5- to 1-min intervals.
Potassium ion concentration in normal Ringer-albumin
perfusate was 2.4 mM. B: same axes as in A. Figure
shows attenuation of increase in both [Ca21]i and Lp
when microvessels are exposed to high-potassium
Ringer solutions to depolarize endothelial cell membranes and exposed to ATP (10 mM). [From He et al.
(114).]
July 1999
MICROVASCULAR PERMEABILITY
Yuan and co-workers (323, 328) have also demonstrated that histamine increases the permeability of isolated pig coronary microvessels by a calcium-dependent
mechanism linked through PLC to NO production and
increased cGMP. Inhibitors of NO synthesis and protein
kinase G inhibited the increase in permeability caused by
histamine with no effect on the calcium transient. They
have also extended their studies to evaluate the role of
protein kinase C (PKC) activation to increase permeability. In isolated coronary venules, stimulation of the endothelium with phorbol 12-myristrate 13-acetate to activate
PKC increased the basal permeability to albumin without
an initial increase in calcium (123). This increase in permeability was attenuated by inhibitors of NOS and protein
kinase G. However, the increase in permeability due to
FIG. 14. Effect of nitric oxide synthase (NOS)
inhibitors on ATP-induced increase in Lp and [Ca21]i.
A: paired measurements of Lp in a single vessel are
shown as a function of time. Control Lp measured
with albumin-Ringer perfusate was 5.1 3 1027
cm z s21 z cmH2O21. ATP (10 mM) induced a transient increase in Lp. In presence of NG-nitro-L-arginine methyl ester (L-NAME; 10 mM), increased Lp in
response to ATP was significantly attenuated. B: effects of NOS inhibitors on ATP-mediated increase in
endothelial [Ca21]i. Paired measurements of endothelial cell [Ca21]i in response to ATP (10 mM) before
and after administration of NG-monomethyl-L-arginine (L-NMMA; 100 mM) are shown as a function of
time. L-NMMA showed no effect on magnitude and
time course of increase in endothelial cell [Ca21]i
induced by ATP. [Modified from He et al. (110).]
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increased endothelial cell concentrations of cAMP, the
increase in permeability was attenuated without modifying the initial calcium transient (107). The increase in
permeability was also attenuated by NOS inhibitors,
again without modifying the initial calcium transient.
Inhibition of cGMP formation using an inhibitor of
guanylate cyclase also blocked the increase in permeability. On the other hand, pretreatment of the microvessels with membrane-permeable cGMP analogs to
increase endothelial cell cGMP concentrations potentiated the increase in permeability induced by ATP, again
without modifying the initial calcium transient. These
agents suggest that increased NO/cGMP act downstream of the calcium influx to increase permeability,
whereas cAMP acts to decrease permeability.
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C. C. MICHEL AND F. E. CURRY
Volume 79
histamine was not blocked by agents that block PKC.
These results suggest that PKC acts at least in part by
modulating the Ca21-dependent NO/cGMP pathway in
these vessels. The regulation of the permeability of the
wall of small coronary arterioles (mean diameter 44 mm)
isolated from pig heart has also been investigated by
direct microperfusion techniques (128, 129).
The general features of permeability increases in Figures 13–15 are representative of the actions of many acute
inflammatory mediators that directly increase permeability
in venular microvessels. For example, Haraldsson et al.
(101a) demonstrated, in the isolated perfused rat hindlimb,
that histamine did not increase the whole organ capillary
filtration coefficient when the perfusate was calcium free.
Furthermore, the calcium channel antagonists verapamil
and felodipine, which blocked calcium entry into vascular
smooth muscle, had no effects on the histamine-induced
changes. These observations can be understood in terms of
the mechanisms described in Figure 13, A and B, demonstrating calcium entry through a passive conductance pathway as one of the key steps in the signal transduction
cascade leading to increased permeability. Investigations in
the hamster cheek pouch also demonstrated attenuated responses on venular microvessel to inflammatory mediators
with reduced extracellular calcium (174). Furthermore,
acute increases in venular microvessels are attenuated by
agents that increase intracellular cAMP in endothelial cells
(see sect. VIII and Refs. 93 and 94 for reviews). More recent
work has demonstrated that the acute increase in microvessel permeability due to histamine, ADP, bradykinin, leukotrienes, ionophore, ATP, PAF, and substance P are all attenuated by NOS inhibitors (145, 170–172, 208, 234). Also
agents that increased cGMP increase permeability in these
vessels and in whole organs (12, 127, 182, 301).
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FIG. 15. Effect of cGMP analog 8-bromo-cGMP
(8-BrcGMP; 2 mM) on ATP-induced increases in Lp and
[Ca21]i in individually perfused frog mesenteric
microvessels. A: paired measurements of Lp in a single
vessel are shown. Control Lp was 4.3 3 1027
cm z s21 z cmH2O21. In presence of 8-BrcGMP, increase in Lp in response to ATP (10 mM) was significantly potentiated. B: paired measurement of [Ca21]i
in response to ATP (10 mM) before and after exposure
to 8-BrcGMP. 8-BrcGMP had no effect on magnitude
and time course of increase in endothelial cell [Ca21]i
induced by ATP. [Modified from He et al. (113).]
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MICROVASCULAR PERMEABILITY
E. Individual Microvessels: Heterogeneity
in Endothelial Cell Responses
The data in Figure 13 represent the average change in
[Ca21]i in a measuring window containing an average of
;20 endothelial cells. These methods have been extended
to measure calcium in the individual cells making up a
segment of the microvessel wall. The main feature of
investigations using both ionomycin and ATP to increase
[Ca21]i is the variation in the initial peak [Ca21]i from cell
to cell (178, 212). This observation suggests that the calcium responses in endothelial cells in venular microvessels are not closely coupled. Furthermore, in microvessels exposed to ionomycin, the largest increases in
permeability are localized to the region of cells with the
largest initial peak values of [Ca21]i (212). Thus, at the
level of individual cells, or groups of cells, the magnitude
of the initial peak increase in permeability is also propor-
tional to the initial increase in [Ca21]i. The reasons for the
differences in the magnitude of response from cell to cell
within a single microvessel are not understood. The lack
of coupling between endothelial cells in venular microvessels appears to contrast with results in arterial
endothelium. It may be important to reduce the spread of
injury responses throughout a microvascular segment.
F. Comparison With In Vitro Studies:
Calcium Influx
There is no pharmacological or electrophysiological
evidence for functional voltage-gated calcium channels in
the endothelial cells forming the wall of venular and true
capillaries with continuous endothelium (114). Neither
the resting levels of calcium in vessels exposed to highpotassium Ringer solutions nor the initial calcium peak in
vessels exposed to ATP was modified by L-type calcium
channel-blocking agents (nifedipine) or the voltage-gated
calcium channel agonists BAY K 8644 (114, 331). These
experiments are consistent with experiments in endothelial cells in culture. Several recent reviews of the ion
channels in large vessel endothelial cells show how the
membrane potential is part of the mechanism controlling
calcium influx (2, 51). In large-vessel endothelial cells,
many of the same agents that increase the permeability of
venular microvessels also regulate endothelium-dependent vasodilation (51).
G. Comparison With In Vitro Studies: NO/cGMP
and Calcium Entry
In the experiments described above on the role of
NO/cGMP pathways to regulate increased permeability in
the endothelial cells forming venular microvessels, there
was no change in the initial calcium transient as microvessel permeability was either increased or decreased by
modifying the production of NO or the cellular levels of
cGMP. These results may be contrasted with experiments
in human aortic endothelial cells, “microvascular” endothelial cells from human foreskin, and HUVEC when the
agonist was thrombin (65, 305). Pretreatment of the
monolayers with the cGMP analog 8-(4-chlorophenylthio)guanosine 39,59-cyclic monophosphate (8-CPT-cGMP) attenuated the increase of the permeability of the monolayers and, in the case of aortic and foreskin endothelial
cells, reduced (but did not completely block) the magnitude of the initial increase in [Ca21]i. Because protein
kinase G is reported to be selectively activated by the
cGMP analog 8-CPT-cGMP, a protein kinase G-dependent
pathway that modulates calcium influx is implicated in a
feedback mechanism, present in endothelium from aortic
and foreskin vessels, but absent in the endothelium from
HUVEC. This mechanism may also explain the observa-
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The action of inhibitors of NOS activity to attenuate
the increase in permeability of venular microvessels must
be distinguished from hemodynamic effects that are mediated by NO. For example, many of the same agents that
increase the permeability of venular microvessels via calcium-dependent release of NO also act on the endothelial
cells in arterioles to cause endothelium-dependent vasodilation (51, 182). Thus, by increasing the number of
vessels perfused and/or the pressure within microvessels,
NO/cGMP-dependent processes can increase the total
amount of solute transported into tissue with or without
an increase in permeability. Thus, quite apart from the
direct actions of NO to modulate the permeability properties of the microvessel wall, the application of a NOS
inhibitor may decrease total solute flux into a tissue by
decreasing perfusion area and capillary pressure. On the
other hand, basal NO release from endothelial cells is an
important mechanism to maintain a low level of leukocyte-endothelial cell interactions, to maintain mast cell
stability, and to reduce platelet aggregation (145). Inhibition of NO would tend to increase solute flux by activation
of leukocytes, platelets, or mast cell-dependent increases
in permeability (145). Thus it is no surprise that the
application of NOS inhibitors in different vascular beds
gives rise to conflicting observations with respect to the
site of action of NO, depending on the contribution of
hemodynamic changes, leukocyte attachment, and direct
permeability changes due to the response of venular endothelial cells. An important argument in favor of investigations on individually perfused microvessels is the ability to separate direct actions to change the permeability
properties of the vessel wall, from the additional effects in
a microvascular bed to changes in surface area for exchange, microvessel pressure, leukocyte interactions, and
mast cell-dependent responses.
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C. C. MICHEL AND F. E. CURRY
H. Comparison With In Vitro Studies:
cGMP/NO and Increased Permeability
in Venular Endothelium
The mode of action on a NO/cGMP-dependent pathway to increase the permeability of individually perfused
venular microvessels is not well understood. The mechanism lies downstream of calcium entry and probably does
not involve a modification of the contractile force developed in endothelial cells because the known actions of
NO/cGMP pathways on contractile mechanisms all result
in decreased, not increased, contractility (175, 324). One
possible mechanism for cGMP to modify the permeability
in venular microvessels is to regulate endothelial cell
cAMP levels. Levels of cAMP may be reduced when cGMP
stimulates a cGMP-activated phosphodiesterase [phosphodiesterase (PDE) 2] to lower both cAMP and cGMP.
Phosphodiesterase 2 has been demonstrated in some endothelial cells (104, 292). He et al. (113) suggested that
increased cGMP stimulated PDE2, and lowered cAMP, to
increase permeability in frog and rat mesenteric vessels.
An action of cGMP to lower cAMP via PDE2 has been
described in platelets (63). However, the expression of
PDE and their activity appears to differ in endothelial
cells of different sources. Draijaer et al. (64) measured
some reduction in the permeability of monolayers of
HUVEC when treated with 8-bromo-cGMP (rather than
the cGMP analog specific for protein kinase G) and related this to the presence of a PDE3 isoform that is
inhibited by cGMP, thereby increasing intracellular cAMP
levels. It is noted that, depending on the level of cGMP,
there may be a rapid switch between the relative activities
of PDE2 and other phosphodiesterases (195).
Another action of cGMP in endothelial cells is phosphorylation of VASP, a protein associated with focal adhesion sites and adherens junctions in cultured cells (64),
but the contribution of this mechanism to changes in
permeability remains to be evaluated. It is also possible
that a NO/cGMP-dependent mechanism may regulate
cells other than the endothelial cells. For example, NO
released by the venular endothelium may modulate the
contractile state of pericytes associated with postcapillary venules. One possibility is that pericyte “relaxation”
may be required for the additional tension developed in
endothelium exposed to inflammatory mediators to cause
sufficient cell deformation to increase permeability. This
idea runs counter to the concept that “contraction” of
pericytes may contribute to increased permeability (277).
Although it is known that many vasoactive agents acting
directly cause pericytes to contract, NO donors cause
bovine retinal pericytes to relax (98, 332). Furthermore,
pericytes of most organs express a high level of cGMPdependent protein kinase (134, 135), which plays a key
role in the relaxation of vascular smooth muscle. The idea
that pericyte relaxation may be part of the mechanism to
increase venular microvessel permeability has not been
tested because of the technical difficulties of in vivo experiments on pericytes.
I. Comparison With In Vitro Studies: cAMP
In cultured endothelial cell monolayers, increased
intracellular cAMP levels decrease the basal permeability
properties on the barrier and attenuate the increase in
permeability when the monolayers are exposed to inflammatory agents. The effect of increased cAMP alone on
basal permeability of intact microvascular bed is small,
but the acute increase in venular microvessel permeability by a wide variety of inflammatory stimuli is attenuated
by agents that increase endothelial cell intracellular
cAMP concentration (activators of adenylate cyclase, in-
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tions by Suttorp et al. (292) that agents that increase
cellular cGMP levels (NO donors, ANP, and dibutryl
cGMP) blocked the H2O2-induced increases in permeability in porcine pulmonary artery endothelial cell monolayers. One exception to observations that increased cGMP
attenuates the initial calcium transient in large-vessel endothelium is the observation of Piper and co-workers
(119) that increased intracellular cGMP potentiates the
calcium influx into aortic endothelial cells induced by
ionomycin. There was a corresponding potentiation of
monolayer permeability.
The action of cGMP to attenuate the initial calcium
transient in large-vessel endothelial cells is similar to the
action of increased cGMP to reduce cytoplasmic calcium
in vascular smooth muscle, one of the main mechanisms
leading to vasodilation by NO. However, it is not clear that
the mechanism of action is the same. In vascular smooth
muscle, a reduced calcium influx through L-type calcium
channels and membrane hyperpolarization has been demonstrated (175, 324). In the absence of voltage-gated calcium channels, neither of these mechanisms is expected
to account for reduced calcium influx in large-vessel endothelium. In summary, it is difficult to generalize the
observations of van Hinsberg et al. (305) and Drenckhahn
and Ness (66) to suggest that cGMP acts to promote
barrier function in endothelial layers. This mechanism
may play a role to protect the permeability of some endothelial barriers in large vessels, but it does not account
for the action of cGMP in venular microvessels, the primary site of action of acute inflammatory responses. In
fact, the principal generalization that is emerging from
studies of cGMP-dependent processes is that the action of
cGMP depends on the distribution of expression of cGMPdependent kinases and phosphodiesterases in endothelial
cells, and perhaps the cells closely associated with the
endothelium (see sect. VIIH).
Volume 79
July 1999
MICROVASCULAR PERMEABILITY
epithelial barriers (68). Furthermore, the loss of junctional strand complexity and particle orientation within
the strand, which occurred spontaneously in primary culture of brain endothelial cells, was blocked by increasing
endothelial cell levels of cAMP by treatment with forskolin and PDE inhibitors (320). The mechanisms underlying
these changes are not known but are likely to involve
changes in the transport of preformed junctional proteins
to the membrane and phosphorylation of components of
the adhesion complex at the tight junction.
The recent experiments in intact microvessels that
demonstrate increased numbers of junctional strands
in between the endothelial cells after treatment with
both the adenylate cyclase stimulator forskolin and the
PDE4 inhibitor rolipram conform to the hypothesis that
one of the main effects of increased cAMP in venular
endothelium is to increase cell-cell adhesion. Under
experimental conditions to elevate levels of cAMP in
individually perfused microvessels of frog mesentery,
the number of strands in venular microvessels of frog
mesentery increased (preliminary results show an increase in strand mean number from 1.7 to 2.2), and this
was associated with a reduction in the unstimulated Lp
of the vessel wall by two- to sixfold (8). There is no
direct evidence of changes in tight junction distribution
after exposure to inflammatory agents, but this could
be a key step to the opening of the junction or deformation of the endothelial cell in the region of the
junction or the formation of vesicle clusters to form
VVO (e.g., VEGF; Refs. 69, 202, 248).
Indirect evidence for the role of cAMP in the modulation of components in the junction complex comes from
recent studies of the role of tyrosine phosphorylation in
regulating the permeability of cultured brain endothelial
cell monolayers and individually perfused microvessels in
frog mesentery using phosphatase inhibitors. In cultured
bovine brain endothelial cell monolayers, phenylarsine
oxide (PAO), an inhibitor of phosphoprotein tyrosine
phosphatase, increased the permeability of the monolayer
and increased the tyrosine phosphorylation of b-catenin,
a cytoplasmic component associated with the tight junction (6, 287). The permeability of individually perfused
frog microvessels, measured as Lp of the microvessel
wall, was also increased after exposure to PAO (6). Increased PAO concentration did not increase the magnitude of the response but decreased the time to reach the
maximum response. The increased permeability was
maintained while PAO was in the perfusate, but the effect
was rapidly reversed when a reducing agent was used to
counteract the effect of PAO on the phosphoprotein tyrosine phosphatase. In both brain endothelial cells in
culture and in the venular microvessels of frog mesentery,
cAMP did not reduce the maximal permeability increase
after exposure of the endothelium to PAO. However, the
presence of cAMP did increase the time required for the
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hibitors of phosphodiesterases, and cAMP analogs) in
whole organs. In particular, b2-adrenergic agents reduce
plasma leakage due to histamine, bradykinin, substance
P, PAF, allergen, thrombin, and H2O2 (94, 176). In the rat
trachea, the leakage caused by substance P release from
sensory nerves was inhibited by the long-acting b-agonist
formoterol, and this was correlated with a reduction in
the number of gaps between endothelial cell in venular
vessels, but not the size of the gaps. The mechanisms of
action of increased cAMP to reduce permeability have not
been studied in these whole organs, but most investigators have assumed that the main mechanism to attenuate
permeability increases involves reduced tension due to
reduced actin myosin interactions.
As a general rule, in cultured endothelial cells the
increase in intracellular cAMP concentration that results
from combined activation of adenylyl cyclase and inhibition of PDE3 and PDE4 is larger than the increase due to
either activation of adenylate cyclase or inhibition of PDE
alone (148). The result suggests that cAMP is normally
rapidly metabolized in endothelial cells. The increase in
cAMP may range from 2- to 4-fold to .100-fold. Van
Hinsberg et al. (305) has pointed out that the reduction in
permeability is inversely proportional to the increase in
cAMP concentration. Measurement of intracellular cAMP
levels has not been made in intact microvessels in the
basal or stimulated state, although it is in principle possible to measure intracellular cAMP concentration using
ratio imaging (3).
There are at least two mechanisms whereby cAMP
can attenuate the increase in permeability: a reduction in
tension development and an increase in cell-cell attachment. Although Luckhoff et al. (157) found that the increase in [Ca21]i induced in cultured bovine aortic endothelial cells by ATP was attenuated by isoproterenol,
prostacyclin, and dibutyl cAMP, most reports of the action of cAMP on the permeability in endothelial barriers
describe no reduction in the initial calcium transient (Fig.
1 and Refs. 26, 30, 257). Thus the action of cAMP appears
to be downstream of calcium entry. There is some evidence that agents that increase endothelial cell cAMP
concentration reduce the myosin light-chain phosphorylation in the control state and after stimulation with histamine (199). This is expected to reduce tension. However, in a preliminary report, Wysolmerski and colleagues
(90) have shown that although cAMP reduced resting
myosin light-chain phosphorylation and resting tension in
HUVEC, subsequent stimulation of the endothelial cells
with thrombin in the presence of cAMP increased both
myosin light-chain phosphorylation and tension in these
endothelial cells. Thus the primary action of cAMP may
not be to decrease tension in endothelial cells exposed to
inflammatory agents.
Elevated intracellular cAMP concentration has been
shown to increase the number of junctional strands in
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C. C. MICHEL AND F. E. CURRY
J. Comparison With In Vitro Studies: PKC
The use of phorbol esters that increase the activity of
PKC increases the permeability of monolayers in parallel
with, or independently of, an increase in cytoplasmic
calcium (26, 159). Inhibitors of PKC attenuate the increase in permeability induced by inflammatory agents in
a number of organs including hamster cheek pouch stimulated by bradykinin (200) and lung stimulated by free
radicals (132) and phorbol esters (141). Inhibitors of PKC
also decrease the basal permeability of isolated rat coronary venules (123). Another mechanism by which PKC
activity modulates increased permeability in microvessels
involves stimulation of NO production (234). It is not yet
known if the NO-dependent pathway contributes to, or is
separate from, a possible role of PKC pathways to modulate junctions. Thus, although there are multiple sites of
action of PKC, modulated by multiple isoforms of the
enzyme, at this stage there is no consistent pattern to the
action of PKC that would support the suggestion by Lum
and Malik (159) that the activation of PKC-dependent
pathways is a point of convergence of early signaling
pathways for acute changes in permeability.
Investigations of the role of PKC in the regulation of
permeability are also complicated by the different
sources of lipids to activate PKC. The diacylglycerol generated by the activation of PLC-b is rapidly destroyed in
the cell. Thus the activation of the PKC by this pathway
may be partial and short-lived in a cell that has not been
exposed to a stimulus to PKC activation. Longer term
activation of PKC results from the generation of phosphatidylserine due to the action of phospholipases A and D
(72). Most of the studies of the action of PKC involve the
use of the phorbol esters that activate PKC for a longer
time than a single acute stimulus.
K. Comparison With In Vitro Studies: Summary
of Acute Inflammatory Responses
Overall, the investigations of the mechanisms to regulate the permeability properties of the endothelial barrier in venular microvessels carried out in both intact
microvessels and in endothelial cells in culture conform
to the hypothesis that the initial increase in permeability
after exposure to acute inflammatory mediators is determined by a cascade of calcium-dependent mechanisms.
They are initiated by calcium influx through a calcium
channel having the properties of a passive conductance
pathway. Thus the magnitude of the initial increase is
determined by the increase in conductance of the calcium
channels and the electrochemical driving force for calcium entry. The endothelial cell membrane potential, determined largely by potassium conductance of the cell
membrane, plays an important role to regulate calcium
influx by decreasing (depolarization) or increasing (hyperpolarization) the driving force for calcium influx into
the cell. The nature of the calcium channels and their
regulation remains unclear, but it appears likely that they
are linked to receptors via families of G proteins.
Downstream of the calcium influx, calcium modulates tension developed by actin-myosin interactions, controlled by one or more isoforms of myosin light-chain
kinase. A second calcium-dependent pathway that appears to be essential to the increase in permeability of
intact venular microvessels is a calcium-dependent increase in the synthesis of NO and the subsequent generation of increased cGMP. The sites of action of NO/cGMP
pathways that directly modify the permeability of venular
microvessels are not known but possibly involve modulation of cellular levels of cAMP by cGMP-stimulated
PDE. The role of other cells whose function is likely to be
modulated by NO/cGMP pathways (pericytes, mast cells)
remains to be explored, but the acute effects of NO/cGMP
on venular microvessels to increase permeability are independent of the action of NO/cGMP pathways on the
hemodynamics and endothelial cell-leukocyte interactions. The action of NO/cGMP pathways may differ widely
in different endothelial cell types depending on the expression of isoforms of cGMP-dependent kinases and
PDE (Fig. 16).
Another common pathway that modulates the permeability response downstream from the calcium influx involves cAMP. This may modulate contractile properties of
endothelial cells downstream from calcium influx via myosin light-chain kinase, but other important actions involve
increased formation of tight junctional strand proteins and
possible modulation of cytoskeletal arrangements near the
junctions. These processes are assumed to resist changes in
tension. Pathways involving PKC and Rho proteins further
modulate permeability, particularly with respect to longer
term increases in permeability. The contribution of each
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permeability increase to develop. The result suggests that
there may be competition between phosphorylation and
dephosphorylation by a tyrosine kinase/phosphatase and
phosphorylation by cAMP-dependent kinase at one or
more sites on b-catenin or other component of the junction complex. It is noted that the microvessels treated
with PAO showed numerous gaps in the microvascular
walls (8), and although it is reasonable to expect that
these are forming between the endothelial cells, it is
important that further studies are carried out to test this
(73, 74, 201, 202).
In the most striking exception to the usual action of
cAMP to decrease permeability, Piper and co-workers
(115) report that in monolayers of rat coronary microvessel endothelium, isoproterenol, and A2 adenosine receptor agonists increased albumin flux. These effects were
antagonized by stimulators of guanylyl cyclase.
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MICROVASCULAR PERMEABILITY
749
pathway may vary depending on the agonist, receptor expression, and the prior history of activation of the pathway.
These observations also conform to the hypothesis that the
acute increase in permeability resulting from inflammatory
stimulus reflects a change in the balance of forces acting on
the one hand to increase tension and deform the endothelial
cells and, on the other hand, to resist these deformations.
The relative contribution of these opposing forces determines the magnitude and time course of the permeability
change.
L. Mechanisms Determining Long-Term Increases
in Permeability: Sustained Increases in
Endothelial Barrier Permeability
Although most effort has been directed toward investigations of acute changes in permeability characterized
by localized leakiness of venular microvessels, methods
are becoming available to study longer term increases in
permeability in single perfused microvessels. One example is the sustained permeability increase in all types of
microvessels after albumin is removed from the perfusate
(Ringer perfusion, Ref. 185). This state of high permeability is initiated by an increase in [Ca21]i; this increase in
[Ca21]i is transient and returns to values close to control
after 5–10 min. However, in contrast to the transient plus
sustained increase in microvessel permeability found in
intact microvessel after exposure to most inflammatory
mediators, the permeability of microvessels perfused
without albumin or plasma proteins in the perfusate increases rapidly and remains elevated at three to five times
control for as long as the perfusion is maintained. Although these observations suggest that increased mean
values of [Ca21]i are not required to maintain the high
permeability state, the continued influx of calcium is required. Thus, for example, any reduction in the electrochemical driving force for calcium entry (membrane depolarization or removal of extracellular calcium, Ref. 106)
results in a return of the permeability of the Ringerperfused vessel to the control state. The observations
indicate that, on average, calcium influx is balanced by
calcium efflux but that localized areas of increased calcium concentration within the cells (possibly just beneath
the cell membrane) may be needed to sustain the high
permeability state. An area for future investigation is the
use of methods to measure the distribution of [Ca21]i
within individual cells and the relation between calcium influx, efflux, and localized sites for the regulation
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FIG. 16. Summary of main signal transduction pathways leading to increased permeability in venular microvessels
of frog and rat mesentery. Solid lines represent calcium-dependent pathways leading to activation of actin-myosin, a
nitric oxide (NO)/cGMP pathway acting via a nitric oxide synthase, and guanylate cyclase. Hypothesis that increased
intracellular cGMP concentration acts to stimulate a cGMP-dependent phosphodiesterase to reduce intracellular levels
of cAMP is shown as a solid line. Solid lines also represent hypothesis that decreased intracellular cAMP level may
modulate integrity of cell-cell adhesion and activity of myosin light-chain kinase. Dashed line shows a feedback loop
whereby increased intracellular cGMP concentration may attenuate initial rise in Ca21 concentration. This pathway is
reported in cultured human aortic and foreskin endothelial cell monolayer but does not appear to be present in
microvascular venular endothelial cells (see Ref. 306). Dotted line is a pathway described in cultured human umbilical
vein endothelial cells in which increased cGMP concentration inhibited a phosphodiesterase (PDE) and raised intracellular cAMP levels (306). Not shown are possible actions of diacylglycerol to activate protein kinase C isoforms that
may also modulate NO production, proteins in junctional complex, and myosin light-chain kinase (MLCK). CaM,
calmodulin.
750
C. C. MICHEL AND F. E. CURRY
M. Mechanisms Determining Resting Permeability
and Long-Term Increases in Permeability:
Leukocyte-Dependent Processes to Increase
Permeability
A great deal of attention has focused on the mechanisms whereby circulating inflammatory cells attach to
endothelial cells through a series of adhesive interactions
with the endothelial cell surface (286, 296). After initial
rolling and adhesion, leukocytes migrate across the endothelium. The mechanisms involved in leukocyte migration
across the endothelium are not well understood, but some
of the same processes as led to acute increases in permeability after exposure to inflammatory agents may contribute to leukocyte migration. Activated leukocytes secrete oxygen radicals, lipid metabolites, and proteases,
each of which has the potential to increase microvessel
permeability (92, 229). Furthermore, leukocytes may signal directly to the endothelial cells via the sites of attachment. It has been observed in cultured endothelial monolayers that leukocyte-endothelium interactions induce a
transient increase in endothelial [Ca21]i and are associated with an increase in monolayer permeability (122,
334). Hixebaugh et al. (118) have demonstrated that ex-
posure of HUVEC monolayers to chemoattractant-stimulated polymorphonuclear neutrophils results in an increase in tension development by endothelial monolayers.
Furthermore, polymorphonuclear neutrophil adhesion to
tumor necrosis factor-activated endothelial cells appears
to induce the disappearance from endothelial cell-to-cell
contacts of adherens junction components under certain
experimental conditions (61). The relation between increased permeability and increased passage of leukocytes
or other inflammatory cells across the barrier remains
poorly understood.
It is not known whether there is a direct interaction
between the mechanism that initiates acute increases in
permeability and the mechanisms that initiate leukocyte
rolling and attachment before migration and sustained
increases in permeability. The selectin family of adhesion
molecules mediates the initial rolling of leukocytes on
endothelial cells. The principal selectins that are found on
the endothelial cell surface are P-selectin and E-selectin.
P-selectin is constitutively stored in Weibel-Palade bodies
of endothelial cells (and in a-granules of platelets; Refs.
21, 177). A variety of inflammatory mediators (thrombin,
histamine, complement fragments, oxygen radicals, and
inflammatory cytokines) that initiate an acute increase in
endothelial barrier permeability also release P selectin
from Weibel-Palade bodies onto the endothelial surface.
This release is measurable within 1–5 min. Inhibition of
basal NO release may also lead to P-selectin release onto
the endothelial cell surface. Although recent reports indicate that there may be more heterogeneity in vivo than in
vitro of P-selectin expression after exposure to inflammatory agents (71), it is generally not recognized by physiologists that these processes have the same time course
as the early stages of acute increase in permeability, and
the possible interaction between the two processes has
not been investigated. Some of the secreted P-selectin is
recycled back into the endothelial cells. P-selectin may
also mediate some longer term adhesion by mechanisms
involving new protein synthesis.
E-selectin expression requires several hours (4 – 6) to
reach maximal levels after stimulation of endothelial with
a variety of inflammatory mediators (interlukin-1b, tumor
necrosis factor-a, interferon, substance P) as well as with
viruses (such as replicating cytomegalovirus) and bacterial hemolysin and Staphylococcus a-toxin (144). Selectins are linked to the endothelial cell cytoskeleton, and
the molecular complexes linking selectins to the cytoskeleton contain many of the same components as are found
at cell-cell and cell-matrix adhesion. The nature of acute
inflammatory responses in endothelial cells that have previously been exposed to these long-term stimuli is an area
for further investigation. Investigations of both leukocytedependent and leukocyte-independent increases in microvessel permeability are being investigated using a va-
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of permeability within endothelial cells. These experiments demonstrate that calcium-dependent processes
may modulate a far more extensive range of mechanisms to modulate permeability than the formation of
gaps between and through endothelial cells in venular
microvessels (51).
An active area of current research involves investigations of the sustained high permeability state induced
by VEGF. Increased expression of this growth factor is
associated with a number of chronic disease states involving increased permeability as well as the high permeability state induced in some tumors (69). Exposure of venular microvessels in frog mesentery to VEGF causes a
transient increase in cytoplasmic calcium and a transient
increase in permeability lasting only a few minutes. This
increase in permeability is dependent on calcium influx
(20). The method to cannulate and perfuse individual
microvessels and measure their permeability has been
developed further to enable the permeability of a microvessel in situ in the frog mesentery and exposed to
VEGF on day 1 to be examined 24 –72 h later. The permeability of the microvessels exposed to VEGF was found
to have increased after 24 – 48 h and to have returned to
control after 72 h. These observations provide a method
to investigate the regulation of microvessel permeability
by signaling pathways such as the MAPK cascade (19).
They also enable investigation of the role of the initial
calcium signal in the regulation of the longer term response in these microvessels (323).
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MICROVASCULAR PERMEABILITY
riety of techniques in a cell column (145, 146), and in
individually perfused microvessels (112).
VIII. SUMMARY
ture of fluid transport and labeling of endothelial intercellular clefts with tracers). They also suggest new ways of
understanding the disposition of the pressures and oncotic pressures across microvascular walls that are fundamental to interpreting the movements of fluid between
the circulation and the tissues of the whole organism.
Studies on the permeability of the membranes of
isolated endothelial cells have provided data that allow us
to assess the contribution of exchange through the cells
to the overall permeability of microvascular walls.
Whereas diffusion through the cells is presumed to be the
principal route for the exchange of the highly diffusible
lipid-soluble molecules, the permeability of the cell membranes indicates that this cannot be an important pathway
for the exchange of small ions, glucose, and amino acids
through the walls of most microvessels (although the
microvessels of the central nervous system are an obvious
exception in this respect). Because the water channel
AQP-1 is found in continuous (but not fenestrated) endothelium, exchange of water can occur by a transcellular
route. Although there have been few studies on AQP-1
channels in isolated endothelial cells, physiological studies indicate that in most microvessels, the AQP-1 channels
probably account for ;10% of the Lp, although a 50%
contribution to Lp has been suggested for capillaries of
skeletal muscle (322). In the endothelium of the DVR of
the kidney where AQP-1 channels have been shown to be
responsible for only 10% of the Lp, their presence is of
obvious physiological significance for they determine the
direction of fluid movement between the plasma and the
hypertonic tissues of the renal medulla. The DVR of the
outer medulla also have a higher permeability to urea than
any other microvessel with continuous endothelium. Surprisingly, this high urea permeability is due to the expression of a urea transporter in the endothelium. It may be
that transcellular mechanisms of exchange are important
in other specialized microvessels, and we should be cautious before considering that the transport properties of
endothelial cells in situ are restricted to those of cultured
endothelial cells.
In vitro studies have, however, been an essential
component in the development of cell biology, and the
rapid advances in our understanding of transport vesicles
in different cells have drawn attention once again to the
possible role of endothelial plasmalemmal vesicles in microvascular transport of macromolecules. Direct evidence for transport through the vesicular system of endothelial cells was reported in the early 1990s (310).
Within the past 4 years, evidence has been published
which suggests that normal levels of macromolecular
transport through microvascular walls (but not the transport of smaller molecules) are dependent on the presence
of the plasmalemmal vesicles or caveolae (274). The view,
based on the morphology of the caveolae, that the plasmalemmel vesicles are fused into immobile clusters has
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We opened this review by summarizing fundamental
properties of the permeability coefficients of microvascular walls to fluid and hydrophilic solutes. We have described how the classical approach of interpreting these
coefficients in terms of convection and diffusion through
cylindrical pores has been extended to models where
pathways are based on the real ultrastructure of microvascular walls. Investigations of permeability and ultrastructure of the same individual microvessels with continuous endothelium (9) have provided experimental
evidence for a pathway that runs through the breaks or
openings in the junctional strands within the intercellular
clefts. Although these openings are relatively few in number, models of flow and diffusion through this pathway
suggest that it can easily account for the normal permeability properties of the walls of frog mesenteric capillaries and probably those of the rat heart. It seems likely that
such a model can also account for the permeability of
microvessels in skeletal muscle, although the detailed
structure of the junctional strands of the intercellular
clefts of these vessels will have to be determined before
this can be demonstrated. As in earlier reconstructions of
the intercellular clefts of continuous endothelium (27),
the breaks in the junctional strands represent a pathway
that is no narrower than the wide regions of the intercellular clefts (i.e., 15–20 nm). Thus the most important
feature of these realistic models is that the tight junctions
are not the site of the endothelial barrier to macromolecules. This barrier is now provided by a fiber matrix filter
close to the entrance to the clefts from the luminal surface. At present, the balance of evidence is that this
ultrafilter is the luminal glycocalyx of the endothelium.
In microvessels with fenestrated endothelium, there
is a good correlation between the permeability to fluid
and to small hydrophilic molecules and the numbers of
fenestrae per unit area of microvascular wall (152). The
permeability and hydraulic conductivity of the fenestrae,
however, appear to be much lower than one might expect
from considerations of their structure as revealed by conventional electron microscopy. Levick and Smaje (152)
have argued that other structures (which are most likely
to be the glycocalyx and basement membrane) must contribute to resistance of the fenestrae even if the fenestral
diaphragms possess the properties necessary to account
for the sieving of macromolecules during ultrafiltration
through the walls of these vessels.
These new models of microvascular permeability are
able to account for many of the inconsistencies between
functional and ultrastructural studies (e.g., convective na-
751
752
C. C. MICHEL AND F. E. CURRY
favored by hyperpolarization, which is an additional effect of some mediators. Depolarizing the endothelial cell
slows the rise in [Ca21]i and can prevent the increase in
permeability.
Although calcium is known to modulate the actomyosin interactions, a rise in [Ca21]i is also essential for the
activation of NOS. Increases in microvascular permeability induced by mediators such as histamine and ionomycin can be prevented by inhibiting NOS even though the
initial transient rise in [Ca21]i is unchanged. Thus an
NO/cGMP cascade is part of the sequence of reactions
leading to increased permeability. The site of action of
this cascade is not known but may involve modulation of
cellular levels of cAMP by a cGMP-stimulated PDE.
Raised levels of cAMP have also been shown to override the rise in [Ca21]i and prevent increases in permeability. The conventional view is that cAMP phosphorylates the myosin light-chain kinase, but other important
actions of cAMP involve increased formation of tight
junctional strands and the possible modulation of cytoskeletal arrangements near the cell junctions.
Although many candidate mechanisms have been
identified, we do not understand how the intracellular
reactions increase microvascular permeability. Although
the picture is becoming increasingly complicated, the
methodology for comprehending it is also advancing rapidly. There are good reasons for believing that many of the
classical questions of microvascular permeability will
soon be answered in the context in which they were
asked or turned into questions that involve a different
level of complexity.
Research by the authors is supported by The Wellcome
Trust Programme Grant 038904/2/93/2/127 (to C. C. Michel) and
by National Heart, Lung, and Blood Institute Grants R37-HL28607 and RO1-HL-44485 (to F. E. Curry).
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