Am J Physiol Heart Circ Physiol 301: H48–H60, 2011.
First published April 1, 2011; doi:10.1152/ajpheart.00133.2011.
Determinants of valve gating in collecting lymphatic vessels from
rat mesentery
Michael J. Davis,1 Elaheh Rahbar,3 Anatoliy A. Gashev,2 David C. Zawieja,2 and James E. Moore, Jr.3
1
Department of Medical Pharmacology and Physiology, University of Missouri School of Medicine, Columbia, Missouri;
Department of Systems Biology and Translational Medicine, Texas A&M Health Science Center, Temple, Texas; and
3
Department of Biomedical Engineering, Texas A&M University, College Station, Texas
2
Submitted 8 February 2011; accepted in final form 29 March 2011
isolated vessel; lymphatic pump; secondary valve; leaflets; servo-null
pressure; contraction cycle
LYMPH PROPULSION IS DETERMINED by the interplay of active and
passive forces that combine to propel lymph centrally against
a pressure gradient (28, 44, 58). Active lymph propulsion is
achieved by the spontaneous, rhythmic contractions of collecting lymphatic vessels (21, 30, 38, 55). Passive factors influencing lymph flow include Starling forces that favor net fluid
reabsorption at the level of the lymphatic capillaries, tissue
compression from the contractions of nearby skeletal muscle
fibers (19, 45), respiratory movements (7, 41, 43), and gravitational forces (45, 50).
Lymphatic valves are essential for minimizing backflow of
lymph, and two systems of valves have been described previously (50). Primary lymphatic valves are composed of the
overlapping, but discontinuous, junctions of endothelial cells in
Address for reprint requests and other correspondence: M. J. Davis, Dept. of
Medical Pharmacology & Physiology, Univ. of Missouri School of Medicine,
1 Hospital Dr., Rm. M451, Columbia, MO 65212 (e-mail: davismj@health.
missouri.edu).
H48
lymphatic capillaries (8, 9, 40), which function to facilitate
preferential movement of interstitial fluid into initial lymphatics. In contrast, secondary lymphatic valves are typically bicuspid structures, spaced every few millimeters along collecting lymphatic vessels. Valve location and development closely
correspond with the presence of a muscle cell layer in the
vessel wall. Each semilunar valve cusp is lined on both sides
by a layer of endothelial cells and anchored at its base by
collagen fibers in the wall. The downstream insertion point of
a valve leaflet is anchored to the wall by collagen and elastin
(1, 26, 54), forming a buttress to prevent inversion when output
pressure is elevated (50). Competent secondary valves are
essential for ensuring the net proximal flow of lymph toward
the heart. A series of dysfunctional valves would exacerbate
lymphedema by allowing lymph reflux into peripheral networks of lymphatic vessels, thus promoting elevated hydrostatic pressure in the initial lymphatics, which, in turn, would
retard fluid absorption from the interstitium. The consequences
of insufficient or malformed secondary valves are readily
apparent in genetic conditions involving lymphatic valve malformation (6, 47).
Secondary lymphatic valves are thought to gate passively
according to the instantaneous luminal pressure gradient (36,
50), opening and closing with each lymphatic contraction
cycle, as fluid is transferred from upstream to downstream
lymphangions (18, 59). In some species, a role for active force
development by specialized smooth muscle cells or actin filament extensions from these cells into the leaflets (4, 34) has
been postulated to facilitate valve closure (5, 31); however, this
view is based almost exclusively on structural rather than
functional evidence (50). Studies of lymphatic valve ultrastructure abound (1, 26, 54), but we are unaware of published
measurements of the forces required to gate secondary lymphatic valves. A passive mechanism for gating is inferred from
assumptions based on structure (“funnel-like” shape) and size
(37, 50), in which a very low Reynolds number for lymph flow
predicts that valve closure is strictly controlled by pressure and
viscous forces. Passive movements of the valve leaflets are
consistent with the results of micropipette occlusion and fluid
injection protocols (27, 29, 52, 53).
Although there is no reason to question the basic assumptions regarding passive lymphatic valve gating (50), several
observations in isolated, pressurized collecting lymphatic vessels from rat mesentery led us to suspect that other factors
significantly influence this process. First, the valves in passive,
single-valve segments are open when pressures in the two
cannulation pipettes are equal. A valve can remain open even
during a spontaneous contraction because the intraluminal
pressure spike associated with systole is blunted by the low
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Davis MJ, Rahbar E, Gashev AA, Zawieja DC, Moore JE Jr.
Determinants of valve gating in collecting lymphatic vessels from rat
mesentery. Am J Physiol Heart Circ Physiol 301: H48 –H60, 2011.
First published April 1, 2011; doi:10.1152/ajpheart.00133.2011.—
Secondary lymphatic valves are essential for minimizing backflow of
lymph and are presumed to gate passively according to the instantaneous trans-valve pressure gradient. We hypothesized that valve
gating is also modulated by vessel distention, which could alter leaflet
stiffness and coaptation. To test this hypothesis, we devised protocols
to measure the small pressure gradients required to open or close
lymphatic valves and determine if the gradients varied as a function of
vessel diameter. Lymphatic vessels were isolated from rat mesentery,
cannulated, and pressurized using a servo-control system. Detection
of valve leaflet position simultaneously with diameter and intraluminal pressure changes in two-valve segments revealed the detailed
temporal relationships between these parameters during the lymphatic
contraction cycle. The timing of valve movements was similar to that
of cardiac valves, but only when lymphatic vessel afterload was
elevated. The pressure gradients required to open or close a valve
were determined in one-valve segments during slow, ramp-wise
pressure elevation, either from the input or output side of the valve.
Tests were conducted over a wide range of baseline pressures (and
thus diameters) in passive vessels as well as in vessels with two levels
of imposed tone. Surprisingly, the pressure gradient required for valve
closure varied ⬎20-fold (0.1–2.2 cmH2O) as a passive vessel progressively distended. Similarly, the pressure gradient required for
valve opening varied sixfold with vessel distention. Finally, our
functional evidence supports the concept that lymphatic muscle tone
exerts an indirect effect on valve gating.
LYMPHATIC VALVE GATING
METHODS
Vessel isolation. All animal protocols were approved by the University of Missouri Animal Care and Use Committee and conformed
to the Public Health Service Policy for the Humane Care and Use of
Laboratory Animals (PHS Policy, 1996).
Male Sprague-Dawley rats (150 –300 g) were anesthetized with
sodium pentobarbital (60 mg/kg ip), and a loop of intestine from each
animal was exteriorized. Lymphatic vessels (80 –180 ␮m inner diameter) were dissected from mesenteric arcades and placed in physiological saline solution (PSS) with albumin (APSS) at room temperature. The animal was subsequently euthanized with Nembutal (120
mg/kg ic). APSS contained the following (in mM): 145.0 NaCl, 4.7
KCl, 2.0 CaCl2, 1.2 MgSO4, 1.2 NaH2PO4, 0.02 EDTA, 5.0 glucose,
2.0 sodium pyruvate, 3.0 3-(N-morpholino) propanesulfonic acid, and
0.5 g/100 ml purified BSA (pH ⫽ 7.4 at 37°C). APSS was used for
initial dissection and to fill the cannulation pipettes, whereas PSS was
used for the regular bath solution in protocols studying spontaneous
contractions. High K⫹-PSS (KPSS) was used in some protocols to
impose a higher level of tone and was made with the same composition as PSS except for equimolar substitution of K⫹ for Na⫹. The
passive properties of the vessel, including most valve gating tests and
passive pressure-diameter curves, were assessed in Ca2⫹-free PSS,
which was identical to PSS except that 3.0 mM EDTA was substituted
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for CaCl2. All chemicals were obtained from Sigma (St. Louis, MO)
except albumin (no. 10856; U.S. Biochemicals, Cleveland, OH).
Cannulation, pressurization, and imaging methods. After removal
of fat and loose adventitia, a segment of lymphatic vessel with one or
two valves was transferred to a 3-ml chamber, filled with PSS, and
cannulated at each end with a glass micropipette held in a Lucite
holder mounted on a Burg-style V-track system (17). The entire
apparatus was transferred to the stage of an inverted microscope (an
inverted Zeiss ACM equipped with Zeiss ⫻3.2 and ⫻6.3 Neofluor
objectives). Input and output pressures (Pin and Pout, respectively)
were initially set from standing reservoirs, and the axial length of the
vessel was adjusted so that there was no slack in the vessel with Pin
and Pout temporarily set to ⬃13 cmH2O; this was necessary to prevent
axial buckling of the vessel at high pressures, which otherwise
interfered with accurate diameter tracking. A continuous flow of
heated PSS (2 ml/min) prevented changes in osmolarity associated
with evaporation.
The vessel image was digitized using a fire-wire CCD camera
(model A641FM; Basler, Ahrensburg, Germany). Camera resolution
was 1,624 ⫻ 1,236 pixels (14 frames per second, monochrome, 12
bits/pixel), but the progressive scan settings were adjusted to acquire
a custom video format of 1,624 ⫻ 510 pixels at 30 Hz. The effective
resolution was 0.53 and 0.99 ␮m/pixel using Zeiss 6.3 ⫻ and 3.2 ⫻
objectives, respectively. Inner diameter was continuously tracked
using a custom computer algorithm (10). Pin and Pout were measured
using low-pressure transducers (CyberSense model 104; Nicholasville, KY) and digitized in synchrony with diameter data at 60 Hz
using an A-D card (model PCI 6030e; National Instruments, Austin
TX). Video images were captured in AVI files along with embedded
pressure and diameter data, so that all the measured parameters could
be replayed offline (using LabVIEW; National Instruments); this
method also permitted diameter tracking to be performed at additional
sites along the vessel, if needed. The video was compressed (5:1)
without degrading the image quality, using a lossless video codec
(Lagarith).
After initial equilibration, pressure control was switched from
standing reservoirs to a custom-made, analog servo-control system
(Cardiovascular Research Institute, Texas A&M University) in which
servo-null style pumps (model V102; Ling Dynamic Systems,
Royston, UK) were connected to the pipettes and powered by a
hardware-based servo-controller through unitary-gain power amplifiers. The controller compared the output signal from the pressure
transducers to reference voltages specified by a computer (running
LabVIEW) and adjusted the pump voltages accordingly (57). Pressure
steps between 0 and 50 cmH2O could be achieved within 50 ms, and
combinations of step- or ramp-pressure waveforms could be applied
to the input and/or output ends of the isolated lymphatic segment.
Servo-null pressure recording. In segments with two valves, the
servo-null method was used to directly measure intraluminal pressure
(PL). Servo-null micropipettes were pulled from borosilicate glass
(1.0/0.5 mm outer diameter/inner diameter, Omega-dot; Frederick
Haer, Bowdoin, ME) using a Sutter P-97 puller (Sutter Instrument,
Novato, CA). The tips were either gently broken or beveled to an
outer diameter of ⬃2 ␮m. The pipettes were back-filled with 2 M
NaCl and connected to a servo-null micropressure system (model 4A;
IPM, La Mesa, CA) to monitor intraluminal pressure in the central
lymphangion. Positioning was achieved using a hydraulic micromanipulator (model MO-102; Narishige, East Meadow, NY). The servonull pump incorporated the same type of low-pressure transducer used
in the servo-controller. The system was calibrated using a water
manometer along with the other pressure transducers before each
experiment. The output signal was filtered at 50 Hz (model LPBF01G; NPI Electronic; Tamm, Germany).
After cannulation, but before the bath temperature was raised to
37°C, the vessel was passive; micropuncture was typically performed
during this time to minimize trauma to the vessel. The servo-null
system was zeroed with the micropipette positioned just outside the
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access resistance created by the cannulation pipettes. Second,
for strongly pumping segments with two valves, in which
intraluminal pressure in the lymphangion is measured with a
servo-null system, the output valve is often observed to open at
the peak of systole even when peak systolic pressure does not
reach or exceed the pressure in the output pipette (unpublished
observations), as would be predicted if only the pressure
gradient determined valve opening; moreover, the discrepancy
between the two pressures appears to depend on the degree to
which the vessel is distended.
For these reasons, we hypothesized that the gating of secondary lymphatic valves is governed primarily by the passive,
trans-valve pressure gradient but is also influenced by leaflet
stiffness or other mechanical factors related to vessel distention. To test this hypothesis, we devised protocols to measure
the small pressure gradients required to open or close lymphatic valves under carefully controlled conditions and to
determine whether those pressure gradients vary as a function
of vessel diameter.
An additional goal of our study was to determine the valve
gating patterns during the active, lymphatic contraction cycle.
Surprisingly, these patterns have not been documented in detail
previously but are assumed to be similar to those observed for
heart valves during the cardiac cycle. However, at least two
studies have noted the apparent absence of a period of isovolumic systole in spontaneously contracting lymphatic segments
(2, 18), when closure of both valves of a lymphangion should
be required for intraluminal pressure development (38). Thus
we sought to measure valve gating patterns during active
lymphatic contractions in relation to pressure and diameter. We
predicted that the patterns would be more complex than those
of cardiac valves because 1) lymphatic preload and afterload
can vary independently or simultaneously depending on the
prevailing local pressure conditions, and 2) there are differences in the flow regimes in lymphatic vessels (low Reynolds
numbers and highly viscous flow) vs. the heart (large Reynolds
numbers and important inertial effects).
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LYMPHATIC VALVE GATING
Valve timing protocols. The movements of the input and output
valve leaflets relative to changes in diameter and intraluminal pressure
were determined at various levels of Pin and Pout. Initially, Pout was set
to the same level as Pin and several spontaneous contractions were
recorded. Subsequently, a slow, ramp-wise elevation in Pout was
imposed, with the final pressure being just beyond the level at which
the vessel failed to eject during systole. The Pout level at which this
occurred was quite variable (4 –20 cmH2O), as detailed in a separate
study (unpublished observations). During these protocols, the video,
along with associated analog data, was saved periodically into an AVI
file at 28 –30 Hz. Movements of the lymphatic valves were detected
upon replay of the AVI files, in synchrony with the extracted diameter
and pressure data, using a custom computer algorithm (in LabVIEW)
to measure the change in image intensity in a region of interest
overlaying the base and/or midpoint of a valve leaflet.
The most accurate recordings of valve gating were made in vessels
in which closed valve leaflets were oriented perpendicular to the
image plane; most output valves were intentionally positioned in this
way when the vessel was cannulated to optimize valve tracking. Two
densitometric windows were positioned along the midline of the
vessel, with the mean pixel intensity of one window used to detect the
Fig. 1. A: video montage of the isolated lymphatic vessel preparation, showing the positions of the input, output, and servo-null pipettes relative to the 2 valves.
Black and red rectangles indicate the approximate positions of the diameter tracking windows in the central and output segments, respectively. Shadow at bottom
left is caused by the servo-null pipette. Pin, input pipette pressure; Pout, output pipette pressure; PL, intraluminal pressure in the central segment; arb., arbitrary
units. Calibration bar ⫽ 100 ␮m. B: representative record of the lymphatic response to ramp-wise elevation in Pout. Initially, Pin and Pout were both set to 1 cmH2O
and, after 7 spontaneous contractions, a Pout ramp to 8 cmH2O (at 4 cmH2O/min) was imposed. Bottom trace: time course of the inner diameter changes in the
central segment (black) and the output segment (red). Pressure traces show Pin (blue), Pout (red), and PL (black) superimposed on a common scale. As the Pout
ramp progressed, greater intraluminal systolic pressures were developed by the central segment during each contraction cycle but diastolic pressure returned to
1 cmH2O each cycle. Top two traces: net densitometer signals recorded from pairs of windows positioned over the midlines of each valve region (see METHODS
for details and representative window positions in Supplemental Fig. S1), where blue is position of the input valve leaflets and red is position of the output valve
leaflets. Top traces: open and closed positions of the respective valves obtained after applying a threshold window to the densitometer traces (any signal below
the lower edge of the window corresponds to a closed valve). *Contraction cycle in which the output valve did not transiently close. This experiment represents
the response of 9 different vessels.
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vessel wall. Successful micropuncture left the pipette tip free from
obstruction in the vessel lumen. Servo-null measurements of PL were
made immediately downstream from the input valve (see image
montage in Fig. 1A) to protect the central lymphangion from changes
in axial length that occurred upon penetration of the micropipette
through the vessel wall. The success rate, without subsequent impairment of contractions after warming, was ⬃80%. The criteria used to
assess valid servo-null pressure recordings were the same as described
previously (33). The calibration of each micropipette was adjusted
after bath temperature reached 37°C and checked periodically during
the experiment if pipette plugging was suspected; the test involved
simultaneously raising Pin and Pout and adjusting the calibration gain
and offset as appropriate to match the pipette pressures.
After micropuncture, the vessel was equilibrated for 30 – 60 min at
37°C and 3 cmH2O until a stable pattern of spontaneous contractions
developed. Subsequently, Pin and Pout were lowered to a baseline
pressure of 1 cmH2O for an additional 5- to 10-min equilibration
period before pressure ramps were imposed. All vessels, even those
with two valves (consisting of 1 complete lymphangion surrounded by
2 partial lymphangions), showed nearly synchronized contractions of
the entire cannulated segment.
LYMPHATIC VALVE GATING
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gradient by tracking particles that flowed backwards through the
valve. For particle tracking, the cannulation pipettes were filled either
with unfiltered APSS containing some particulate matter (2 experiments) or with a dilute suspension of red blood cells (1:8 in APSS; 2
experiments) obtained by cardiac puncture of the same rat. When
particles or cells exited the output pipette at defined pressure gradients, sequences of AVI images were captured showing the particles
moving retrograde through the valve toward the input pipette
(Supplemental Fig. S3). Frame subtraction was used to generate a
32-bit image in Image J (National Institutes of Health; http://
rsb.info.nih.gov/ij) to give a middle-gray image with bright spots
indicating the position to which the particle had moved and dark
spots to indicate the position from which the particle had moved.
This subtracted image was then overlaid onto the average of the
two images used for the subtraction and the particle positions were
pseudocolored.
Confocal microscopy. Vessel segments were observed at ⫻40 –50
magnification using a Leica AOBS SP2 Confocal Multiphoton Microscope System. The cells were loaded with CellTracker Green (5
␮M in PSS; Molecular Probes). Images were acquired at 0.3-␮m
intervals in the z-axis (vertically through the lumen) using 489 nm
excitation and 508 nm emission wavelengths. The image stacks were
reconstructed using Leica confocal software to produce various threedimensional projections.
Data analysis. Custom analysis programs written in LabVIEW
were used to extract embedded data from the AVI files, detect valve
leaflet movements, and detect diameter and pressure changes off-line.
Data were compiled and plotted in Excel (Microsoft, Redmond, WA)
and Igor (Wavemetrics, Lake Oswego, OR) and/or Photoshop (Adobe,
Mountain View, CA). Typically, one vessel was used for data analysis
from each animal. Curve fitting was performed using Igor. Supplemental video files were made using custom LabView programs to
crop the video sequences, overlay the embedded data traces, and add
appropriate labels. The files were converted from AVI to MP4 format
using Aiseesoft Converter Suite (Beijing, China).
RESULTS
Valve gating during the lymphatic contraction cycle. To
determine the sequence of changes in valve position relative to
changes in inner diameter and intraluminal pressure, two-valve
lymphatic segments were subjected to different combinations
of Pin and Pout. A typical recording is shown in Fig. 1B. With
Pin and Pout initially set to equal levels, Pout was slowly
elevated from 1 to 8 cmH2O at a standard ramp rate of 4
cmH2O/min. Pin was held constant as Pout was elevated. Typical diameter and pressure responses to a Pout ramp are represented by the two bottom traces. The second trace from the top
is the net densitometer signal (red is output valve, axis at left;
blue is input valve, axis at right) with the shaded area over the
densitometer trace representing the range of that signal corresponding to an open valve. The top traces show the binary
valve positions (using the same color scheme). Progressive
elevation of Pout was associated with a progressive decrease in
the open times of both valves and a progressive increase in the
closed time of the input valve; in this example, the output valve
stopped opening halfway through the ramp, as noted below.
Vessel diameter was measured on both sides of the output
valve to illustrate the effect of increasing Pout on the diameters
of the respective segments. As Pout increased, the vessel distended progressively on the output (downstream) side of the
valve (red trace), as reflected by increases in both end diastolic
diameter and end systolic diameter (ESD). In contrast, end
diastolic diameter in the central chamber, on the upstream side
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amount of overlap between the leaflets when the valve was closed or
partially closed (Supplemental Fig. S1, window 4; Supplemental
Material for this article is available online at the Am J Physiol Heart
Circ Physiol website); this signal was highest when the valve was
completely open (Supplemental Fig. S1A) and lowest when it was
completely closed. However, pixel intensity within the vessel lumen
was also influenced by vessel constriction and/or lateral movements
associated with constriction. Therefore, a second window was positioned in the vessel midline (Supplemental Fig. S1, window 3) and
used to monitor the change in lumen intensity associated with contraction. The difference between the mean pixel intensities of the two
windows (after both were normalized to their control values in
diastole) yielded a net densitometer signal that more closely reflected
only the movements of the valve leaflets. The baseline level of the net
densitometer trace also reflected the backward bulging of the output
valve as Pout was progressively elevated (e.g., Supplemental Fig. S1,
B–E, red trace). The degree to which the densitometer output accurately reflected the valve positions was confirmed by replaying the
video (at ⬃1/3 real time) and manually toggling a button to denote
whether the valve was open or closed in each frame; the resulting
manual and automated traces were virtually identical. A thresholding
method was then applied to the net densitometer trace to convert it to
a digital recording of valve position, where 1 was open and 0 was
closed.
Valve closure and opening tests. To determine the trans-valve
pressure gradient required to open or close a valve, single valves were
studied in isolation (see Fig. 4B). Initial valve gating tests were
performed in Ca2⫹-free solution to prevent interference from intraluminal pressure spikes associated with spontaneous contractions. Only
one densitometer window was needed under these conditions, with the
window positioned along the midline of the closed leaflets.
For valve closure tests, pressures were initially set to equal “baseline” values (typically 3 cmH2O), and the leaflet positions were
monitored as Pout was slowly ramped (4 cmH2O/min) to a higher
value, with Pin held constant, until the valve closed. The point of valve
closure was evident by a rapid decline in the densitometer signal and
also by a smaller but simultaneous drop in upstream diameter. This
procedure was repeated three times at each baseline pressure while the
video was saved to an AVI file. The baseline pressure was then set to
another level, either 0.2, 0.5, 1, 2, 3, 5, 7, 11, 15, or 20 cmH2O for 1–2
min, and the ramp test was repeated in triplicate. A complete set of
measurements at all of the above pressures was typically obtained.
For valve opening tests, Pout was set to a level higher than the
baseline Pin required to close the valve; Pin was then ramped up to a
higher level, while Pout was held constant, until the valve opened.
Valve opening was evident by an abrupt increase in the densitometer
signal. This procedure was repeated in triplicate for each of the
baseline pressures while the video was recorded.
Because the valve tests under passive conditions might not accurately reflect the behavior of the valves under physiological conditions, the closure test was repeated in the presence of imposed basal
tone. A number of different agonists potentially could have been used
to impose tone (13, 39), but accurate measurement of the small
pressure gradients required to test valve gating also required elimination of spontaneous contractions. After several different methods were
attempted for simultaneously imposing tone and inhibiting contractions, the one that worked most consistently was to exchange the bath
for KPSS ⫹ substance P (SP). Elevated [K⫹] led to cessation of
spontaneous contractions and produced a transient constriction, while
the simultaneous addition of SP led to the development of stable tone,
at least at low pressures (see Supplemental Fig. S2). The SP dose was
adjusted to induce various levels of tone, from ⬃10% tone at 5 ⫻
10⫺8 M to ⬃80% tone at 1 ⫻ 10⫺6 M. Tone was calculated as the
percent diameter decrease at 3 cmH2O compared with that of the
passive vessel.
Particle tracking. In a few vessels, we sought to confirm that a
valve indeed remained open in the face of an adverse pressure
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LYMPHATIC VALVE GATING
open position at the end of systole. In early diastole, when the
rate of the diameter change was relatively high, PL dipped
transiently below Pin (and Pout). This negative pressure, relative
to Pin, observed previously in isolated bovine mesenteric lymphangions under similar conditions (24) and that we hereafter
call the “suction effect,” pulled the output valve leaflets backward into the closed position, as indicated in the densitometer trace and the valve position trace. This phenomenon
was observed in ⬃75% of vessels when Pin and Pout were
equal and at a low level (ⱕ3 cmH2O); diameter typically
decreased by ⱖ50% in systole under these conditions, as
evident in this example (see also the examples in Supplemental Figs. S4 and S5). The suction effect was diminished
somewhat during the second contraction, was only present in
four of the seven contractions before the ramp (missing in
contractions marked by asterisk in Fig. 1B), and was essentially
absent in Fig. 2B when the systolic periods were characterized
by smaller amplitude changes.
When Pout was higher than Pin, the valve gating pattern was
much more consistent with the pattern observed for ventricular
valves during the cardiac cycle (56), as illustrated in Fig. 2B.
The output valve was closed throughout diastole. At the initiation of systole, the input valve closed, permitting a greater rise
in PL than could be recorded when Pin and Pout were equal (due
to partial shunting of the PL spike through an open output valve
into the output cannula). When PL exceeded Pout, the output
valve opened. Pressure development in the central lymphangion during the time that both valves were closed implies that
a brief period of isovolumic systole occurred (Fig. 2B, inset
with expanded time scale at right). During this time, diameter
was decreasing at the site immediately upstream from the
output valve but the effect on volume was apparently balanced
by an increase in diameter at the more proximal end (near the
sinus) of the central lymphangion (data not shown); indeed,
diameter increases near the sinus were typical during systole
when Pout was elevated (data not shown). In midsystole, peak
Fig. 2. Expanded views of selected contractions from the recording in Fig. 1 show the
timing of the input and output valves at
different levels of Pout. Traces are the same
as described in Fig. 1. A: lymphatic contraction cycle when Pin ⫽ Pout (1 cmH2O). Vertical lines denote the periods of systole, as
determined from the diameter recordings.
B: lymphatic contraction cycle when Pout ⬎ Pin.
Vertical lines denote the periods of systole,
as determined from the diameter recordings.
B, inset: first contraction with an expanded
time scale; here the dotted vertical lines denote a period of isovolumic systole. ESD,
end systolic diameter; EDD, end diastolic
diameter.
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of the output valve (black trace), decreased slightly as Pout
increased, reflecting a modest myogenic constriction. Myogenic constriction was a very typical response during Pout ramp
protocols and will be described in more detail a separate study.
In Fig. 1B, bottom, pressures in the input pipette (blue trace),
output pipette (red trace), and central lymphangion (black
trace) are overlaid on a common y-axis scale. As Pout was
raised from 1 to 3 cmH2O, PL transiently increased during each
contraction such that peak systolic PL exceeded Pout; when this
occurred, the output valve opened, as indicated on the “valve
position” trace, and partial emptying of the luminal contents
resulted. At Pout levels from 4 – 6 cmH2O (denoted by vertical
dotted lines), the output valve opened during each spontaneous
contraction and some ejection occurred, even though peak PL
did not clearly exceed Pout. This phenomenon is discussed in
subsequent figures and was not due to unequal calibrations of
the two transducers. At Pout levels ⬎6 cmH2O, the lymphangion continued to contract spontaneously and generated a rise
in PL sufficient to close the input valve, but the peak systolic
pressure was insufficient to open the output valve (as was
evident by the closed state of the output valve in the valve
position trace). Contraction frequency increased by ⬃70%
during this particular Pout ramp, followed by rate-sensitive
inhibition (12) that was evident by a long delay before contractions resumed after termination of the ramp. The video
sequence is available as Supplemental Video S1.
Expanded views of the timing of valve leaflet positions
relative to diameter and intraluminal pressure are shown in Fig. 2
when Pin ⫽ Pout (A) and later in the ramp when Pout ⬎ Pin (B).
There were significant differences in the patterns of valve
gating under these two conditions. In Fig. 2A, both valves were
open before the first period of systole began (systole and
diastole are denoted by vertical dotted lines). When systole was
initiated, the fall in diameter and rise in PL essentially coincided. Closure of the input valve coincided with the period in
which PL exceeded Pin. The input valve then returned to the
LYMPHATIC VALVE GATING
areas calculated from this image, the ratio of the valve opening
to the lumen is 1:7.
A typical valve used for a closure test is shown under
brightfield illumination in Fig. 4B, with the valve viewed from
the side so that the insertion points lie at the top and bottom
surfaces of the vessel. Figure 4, C and D, shows the data
obtained during the closure test. Initially, pressures in the
cannulation pipettes were equal (in Fig. 4, C and D, middle
traces) and the valves were open (densitometer signals were
high). As Pout slowly increased, the valve closed (denoted by a
vertical dotted line). At this time, diameter decreased because
pressure on the upstream side of the valve dropped to Pin (from
approximately half-way between Pout and Pin) at the moment of
closure. The magnitude of the diameter change depended on
the degree to which the vessel was distended initially.
At the low baseline pressure used for the test in Fig. 4C, the
minimal pressure gradient (Pout ⫺ Pin) required to close the
valve was 0.17 cmH2O. In contrast, when the closure test was
repeated on the same valve, at a higher baseline pressure (Fig.
4D), a minimal pressure gradient of 1.27 cmH2O was required
for closure. The set of three closure tests for this vessel at the
lower pressure level are available as Supplemental Video S3.
Particle tracking during the imposition of an adverse pressure gradient confirmed that valves with the morphology
shown in Fig. 4B, top, were indeed open. Supplemental Fig.
S3A shows a vessel segment with an apparently open valve
when Pin ⫽ Pout ⫽ 5 cmH2O. As Pout was elevated ramp wise
from 5 to ⬃6.7 cmH2O, with Pin held constant, a single particle
emerged from the “output” pipette (point #1) and moved
backwards through the valve, down the prevailing pressure
gradient, until it exited the “input” pipette at point #17. The
positions of the particle at sequential times in the AVI file are
shown by the yellow dots (1–17). Supplemental Fig. S3B
shows particle velocity as a function of the relative position,
and Supplemental Fig. 3C shows the diameter, pressure and
densitometer data during the Pout ramps associated with this
test. The times at which the movements of this particle were
Fig. 3. Example recording from a vessel that was able to eject
for every contraction during a Pout ramp. Horizontal dotted line
corresponds to contractions that resulted in opening of the
output valve without peak systolic PL equaling or exceeding
Pout. At the end of the ramp, Pin and Pout were elevated
simultaneously to 10 cmH2O to check the calibration of the
pipette used to measure PL.
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systolic PL clearly exceeded Pout, but this was not the case at
higher levels of Pout (see Fig. 3). Systolic PL peaked before the
point in time when ESD was reached. At ESD, PL was
essentially equal to Pout, at which point the output valve began
to close. The input valve remained open until PL fell below Pin.
Note the slightly longer open time for the input valve and its
correspondence with the PL trace in the second contraction of
Fig. 2B, presumably because Pout and thus peak PL were higher
so that the fall to Pin required more time.
Discrepancy between the level of peak systolic PL and Pout
during ejection. A common observation during Pout ramps is
illustrated in Fig. 3. Peak systolic PL clearly did not exceed or
even match Pout after Pout was greater than ⬃3 cmH2O;
nonetheless, the output valve opened transiently during each
contraction (red valve position trace), as indicated by the
horizontal dotted line. At first we suspected transducer drift or
partial plugging of the servo-null pipette, which would have
altered the PL calibration. However, a quick calibration test
following the Pout ramp (at time ⫽ 148.1 min) verified that
neither of these problems occurred. It is still possible that the
frequency response of the servo-null pipette was not sufficient
to accurately detect the PL peak; however, this behavior was
observed in a number of vessels with varying degrees of
discrepancy between peak systolic PL and Pout. This difference
in the two pressures in the face of continued ejection, along
with the “bias” of the valves to be open in the absence of a
pressure gradient, led us to more carefully examine the pressure gradients required to close or open a valve at different
levels of vessel distention.
Valve closure test using a Pout ramp. A three-dimensional
reconstruction of a valve in a rat mesenteric lymphatic vessel,
obtained using confocal microscopy, is shown in Fig. 4A. The
view is taken from downstream, looking back against the
normal flow direction into the valve. Both the valve leaflets and
their downstream insertion points are visible, and the bicuspid
appearance of the valve is evident. A video that rotates through
a 360° view is available as Supplemental Video S2. Based on
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LYMPHATIC VALVE GATING
recorded correspond to AVI frame numbers (1,156 –1,295,
with total duration ⫽ 4.8 s) in the second ramp; the pressure
gradient was ⬃1 cmH2O when this particle moved across the
field of view. In other experiments (see Supplemental Video
S4), red blood cells introduced into the pipette system were
observed to move in the same direction through an open valve
and to stop abruptly the instant the valve closed.
Valve opening test using a Pin ramp. An example of the test
devised to measure the minimal pressure gradient required to
open a valve is shown in Fig. 5. With Pout held constant, Pin
was raised ramp wise until the valve opened and the difference
between Pin and Pout at the point of opening was recorded as
the minimal pressure gradient required to open the valve.
Figure 5A shows a recording of the valve opening test at a low
baseline pressure, where Pin ⫺ Pout was ⫺0.09 cmH2O at the
moment the valve opened, indicating that the valve opened
before the pressures were equal. The red and blue diameter
traces are the diameter changes on the output side and input
sides of the valve, respectively. The test was repeated two more
times at the same baseline pressure (the full sequence is
available as Supplemental Video S5). At a higher baseline
pressure and diameter (Fig. 5B), the same valve opened when
Pin was 0.32 cmH2O below Pout. These tests suggest that vessel
AJP-Heart Circ Physiol • VOL
distention has an effect on the pressure gradient required for
valve opening but that the influence of vessel distention on the
trans-valve pressure gradient is smaller than that for valve
closure at a comparable baseline pressure.
Summary analyses for valve gating tests. The data sets from
the valve closure and opening tests, performed at 10 different
baseline pressures, were analyzed in two different ways. In
Fig. 6A, the pressure gradient required for valve closure (filled
circles, axis at left) is plotted as a function of baseline pressure.
The reference pressure was designated as the side on which the
pressure ramp was imposed (output); thus ⌬P was calculated as
Pout ⫺ Pin for the valve closure test. Over the lower end of the
baseline pressure range from 0.1 to 3 cmH2O, the pressure
gradient required for valve closure increased from 0.1 to 1.4
cmH2O. However, as baseline pressure increased from 3 to 20
cmH2O, the pressure gradient required for valve closure increased only an additional 30% (1.4 to 2.1 cmH2O). Interestingly, the change in slope of the curve fitting these data roughly
corresponded to the “elbow” in the passive pressure-diameter
relationship for the same vessel (red), which is plotted on the
axis at right in Fig. 6A. The data from the valve opening
tests on the same vessel are also plotted in Fig. 6A (open
circles). The reference pressure was designated as the side
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Fig. 4. Valve closure tests performed on a
single-valve vessel in Ca2⫹-free solution.
A: confocal reconstruction of a secondary valve in
a rat mesenteric lymphatic obtained in Ca2⫹free solution to prevent contractions. View
is taken from downstream, looking back
(against normal flow) into the valve. Structures at 1 o’clock and 7 o’clock at the inner
edge of the lumen are the valve insertion
points, or buttresses. Calibration bar ⫽ 40
␮m. B: images of a representative vessel with
the valve in the open (top) and closed (bottom) positions. Single densitometer window
used to detect valve leaflet position is shown
in yellow. Calibration bar ⫽ 120 ␮m. C and
D: densitometer, pressure, and diameter
traces are similar to those described in previous figures (no PL recordings were necessary under these conditions because all pressures could be controlled). Tests begin with
Pin (red trace) and Pout (blue trace) equal in
which case the valve is open. A ramp-wise
increase in Pout is imposed until the valve
closes. Pout and Pin levels at the instant of
closure are recorded and the difference (Pout ⫺
Pin) is the minimal pressure gradient required
for closure. Diameter traces on both sides
of the valve are recorded at sites corresponding to the colored windows in B. Test
in C starts at the lowest baseline pressure
level used (0.2 cmH2O), whereas the test in
D starts at the highest level of baseline
pressure used (20 cmH2O). Both recordings are from the same vessel. Note the
difference in the diameter and pressure
scales between C and D.
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LYMPHATIC VALVE GATING
on which the pressure ramp was imposed (input); thus ⌬P
was calculated as Pin ⫺ Pout for the valve opening test.
Although the general shapes of ⌬P-baseline pressure curves
were fairly representative of those for most vessels, plotting
data sets from multiple vessels in this manner resulted in
substantial scatter in the data along the baseline pressure axis.
Because baseline pressure determined the passive vessel diameter, the data were expressed as a function of vessel diameter.
In Fig. 6B, the pressure gradient required for valve closure
(closed circles) and valve opening (open circles) for the same
vessel are plotted as a function of the normalized passive
diameter (D/Dmax), where Dmax is the maximum passive diameter in Ca2⫹-free PSS at 20 cmH2O. Here the ⌬P for each test
is defined as in Fig. 6A. It is evident that the pressure gradient
required for valve closure increased 23-fold (0.1 to 2.3
cmH2O) as the diameter approached the maximal passive
diameter. The data set for the valve opening tests on the same
vessel show a similar trend in that the passive vessel diameter
altered the pressure gradient required for valve opening; however, the magnitude of the effect was much smaller (4-fold)
over the baseline pressure range of 0.2 to 20 cmH2O.
The results of valve closure tests performed on 17 vessels
and valve opening tests performed on 10 vessels are summarized in Fig. 7. All tests were performed in Ca2⫹-free solution
with the x-axis representing normalized diameter. Higher baseline pressures are associated with progressive distention of the
vessel up to the maximum diameter recorded at 20 cmH2O, and
it is apparent that a valve in a highly distended vessel can
remain open in the face of a substantial adverse pressure
gradient (on average 2.2 cmH2O, but as high as 5 cmH2O; Fig.
7A). Likewise, the opening tests in Fig. 7B show that valve
opening occurs at a progressively more negative pressure
gradient (Pin ⫺ Pout) as a vessel distends. However, the range
AJP-Heart Circ Physiol • VOL
of the pressure gradient required for valve opening changes
only approximately sixfold with vessel diameter compared
with the ⬎20-fold range of the pressure gradient required for
valve closing (only one vessel required a pressure gradient
more negative than ⫺1 cmH2O for valve opening). The data
sets in Fig. 7 were fit with the power functions listed in
Supplemental Table S1. We impute no physiological or mathematical significance to the power functions; they merely
provided better fits of the data sets than exponential or third
order polynomial functions.
Effect of vessel tone on valve closure. We were concerned
that valve gating tests under passive conditions might not
accurately reflect the behavior of the valves under physiological conditions; e.g., that the relationships in Fig. 7 might be
altered by the presence of spontaneous vessel tone. An effect of
tone on gating would be predicted if smooth muscle cells
directly or indirectly controlled tension of the valve leaflets.
The graph in Fig. 8A shows the results of valve closure tests in
four vessels using KPSS ⫹ 1 ⫻ 10⫺7 M SP to impose tone
(open circles), along with tests performed subsequently on
the same valves in Ca2⫹-free solution (closed circles). The tone
induced by this dose of SP (20.4 ⫾ 2.2%) slightly exceeded the
upper limit of basal tone observed under several physiological
conditions (11, 13, 22, 23). Tone was relatively stable at low
pressures but did not completely recover after tests at baseline
pressures ⬎10 cmH2O. Thus, at some pressures, tone changed
during the set of three closure tests at each baseline pressure.
For this reason, all of the data points (rather than only the
average of 3 trials at each baseline pressure) were plotted in
Fig. 8. There appeared to be slight difference in the curves
fitting these data sets, with the curve for the SP data shifted
slightly to the left of the Ca2⫹-free curve. Curve fits of the
individual (paired) data sets (not shown) confirmed that the SP
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Fig. 5. Valve opening tests performed on a singlevalve vessel in Ca2⫹-free solution. A: valve opening
test at a low baseline pressure. Densitometer, pressure, and diameter traces are similar to those described in previous figures. Blue traces are the respective recordings on the input side of the valve,
and red traces are the respective recordings on the
output side of the valve. Test begins with Pout
sufficiently higher than Pin to maintain a closed
valve. A ramp-wise increase in Pin is imposed until
the valve opens. Pout and Pin levels at that instant are
recorded and the difference (Pin ⫺ Pout) is the
minimal pressure gradient required for opening.
This value was typically negative as shown here.
B: valve opening test is repeated at the highest level
of Pout used (20 cmH2O). Both A and B are from the
same vessel. Note the difference in the diameter and
pressure scales between the 2 panels.
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LYMPHATIC VALVE GATING
DISCUSSION
Fig. 6. Summary of valve closure and opening tests for one lymphatic vessel.
A, left axis: pressure gradient required for valve closure (Pout ⫺ Pin; ) or
opening (Pin ⫺ Pout; Œ) plotted as a function of baseline pressure. Each point
is the average ⌬P (Pout ⫺ Pin) for 3 trials. Right axis: continuous pressure
diameter relationship for the same vessel determined from a simultaneous
Pin ⫹ Pout ramp from 0.2 to 20 cmH2O in Ca2⫹-free PSS after valve gating
tests were completed; data were normalized to the passive diameter at 20
cmH2O. B: pressure gradient required for valve closure (Pout ⫺ Pin; ) or opening
(Pin ⫺ Pout; Œ) plotted as a function of normalized diameter. All measurements
were made in Ca2⫹-free PSS. All curve fits are power functions. D/Dmax,
passive diameter/maximum passive diameter.
and Ca2⫹-free curves essentially overlapped in three of the four
vessels.
The valve closure tests were repeated on four additional
vessels in which the SP dose was adjusted (from 6 ⫻ 10⫺7 M
to 1 ⫻ 10⫺6 M) to impose ⬃50% tone (51.3 ⫾ 1.6%). In this
case, the curve that fit the aggregate closure test data for SP
was more noticeably shifted to the left compared with the curve
for the aggregate data in Ca2⫹-free solution (Fig. 8B). Curve
fits of the individual (paired) data sets (not shown) confirmed
that three of four vessels showed substantial leftward shifts in
the curves fitting the SP data relative to the curves fitting the
Ca2⫹-free data. The data sets in Fig. 8 were fit to power
functions, with coefficients listed in Supplemental Table S1. A
leftward shift in the ⌬P vs. D/Dmax relationship suggests that
the induction of tone increases the pressure gradient necessary
to close the valve at any given baseline pressure, compared
with a passive vessel.
AJP-Heart Circ Physiol • VOL
Fig. 7. Summary of measurements describing the pressure gradient required for
valve closure (A; n ⫽ 17) or valve opening (B; n ⫽ 10) as a function of
normalized diameter.
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Valve gating patterns during the contraction cycle. This
study is, to our knowledge, the first detailed description of the
timing of lymphatic valve movements relative to pressure and
diameter changes during the lymphatic contraction cycle. Thus
Fig. 2 is the equivalent of the “Wigger’s diagram” (32) for the
lymphangion. In the rat, the timing of lymphatic valve leaflet
movements during the contraction cycle closely resembles that
of cardiac valves if, and only if, Pout ⬎ Pin (Fig. 2B). At the
onset of systole, the input valve closes first, allowing pressure
in the chamber to rise during contraction, after which the
output valve opens when PL exceeds Pout (at low pressures). At
higher pressures, the output valve will typically open even
though PL does not reach Pout, according the relationship
defined in Fig. 7B. In either case, once open the output valve
closes at the end of systole when PL falls sufficiently below
Pout. Finally, the input valve reopens after diastolic PL has
returned to the baseline level. If diastolic PL does not return to
baseline before the next contraction is initiated, the input valve
remains closed and a slow rise in diastolic PL ensues, as can be
observed just before the onset of valve insufficiency (unpublished observations).
LYMPHATIC VALVE GATING
Flow measurements are usually included in cardiac cycle
diagrams (32). Unfortunately, we were not able to measure
lymphangion flow accurately under conditions in which diameter, pressure, and valve movements were simultaneously measured. Although centerline velocity can be measured using red
blood cells introduced into the input pipette (see Supplemental
Video S4), velocity during much of systole is often too high
(15, 16) to be accurately determined at ⬃30 frames per second
with the camera used in the present experiments. Flow measurements in conjunction with valve gating tests could increase
our understanding of some aspects of valve behavior but would
not likely result in highly detailed flow information such as the
presence of secondary flow structures near the valve leaflets.
This kind of information is currently better obtained from
carefully constructed computational flow simulations, such as
AJP-Heart Circ Physiol • VOL
we have performed for the straighter regions of lymphangions
(49). The outcomes of those simulations may then be used to
attempt to design better experimental approaches to measure
flow patterns around the valves.
Unlike the heart, where Pout always exceeds Pin, lymphatic
valves can exhibit multiple gating patterns depending on the
prevailing Pin and Pout (see Figs. 1–2). When Pin ⫽ Pout, the
input valve typically closes at the onset of systole and opens at
the end of systole. Because the output valve is open when the
trans-valve pressure gradient is zero (Fig. 7), it can occasionally remain open throughout the entire contraction cycle (e.g.,
contractions marked by asterisk in Fig. 1). More typically, the
output valve closes transiently in early diastole as PL dips
below the level of Pin and Pout (Fig. 2A). The dip in intraluminal pressure is associated only with contractions of large
amplitude that typically occur at low baseline pressure levels
(⬍3 cmH2O) and is the subject of another study (14). In the
heart, isovolumic systole is a brief period of time when both
input and output valves of the ventricle are closed, allowing
pressure development during ventricular contraction. This
phase may or may not occur in a lymphatic vessel. Benoit et al.
(2) noted, from in vivo observations of lymphatics in the rat
mesentery, that there was no discernable period of isovolumic
systole. The vessels in their experiments, which were performed in supine animals without an externally imposed afterload, most likely exhibited valve gating patterns resembling
those shown in Fig. 2A rather than Fig. 2B. Experiments with
single, isolated bovine mesenteric lymphangions studied at
equal diastolic Pin and Pout also demonstrated the absence of a
period of isovolumic systole (20). In contrast, our recordings
(Fig. 2B, inset) clearly show a brief period of isovolumic
systole when Pout ⬎ Pin. Differences in the level of afterload
possibly could reconcile the apparent discrepancy between the
observations of Benoit et al. (2) and Gashev (20) with those of
McHale and Roddie (38, 39) with respect to resolving a period
of isovolumic systole in the lymphangion contraction cycle.
Still other valve gating patterns can occur in lymphatic
vessels under conditions where Pin and Pout change, conditions
never experienced by the heart. For example, in edematous
tissues, lymphatic Pin may exceed Pout and the lymphangion
may behave more as a conduit, with continuous forward flow,
than as a pump (23, 48); similar behavior may occur normally
in intestinal lymphatics during high levels of lymph formation.
We are able to simulate this condition by setting Pin higher than
Pout and thereby imposing a favorable gradient for flow (23,
25). When Pin is increased ramp wise while Pout is held
constant, both valves typically remain open throughout the
ramp (see Supplemental Fig. S4). In contrast, when Pin and Pout
are simultaneously elevated, both valves will gate sequentially
during each contraction cycle until Pin and Pout exceed a certain
level (Supplemental Fig. S5). The simultaneous Pin ⫹ Pout
ramp might simulate a condition where a lymphatic vessel
would be pumping against a gravitational load in the face of
developing edema.
Effect of vessel distention on valve gating. Our measurements of valve gating in single-valve segments verify previous
predictions that the gating of lymphatic valves is primarily (or
solely) determined by the trans-valve pressure gradient. Predictions about passive valve gating have been made largely on
the basis of valve ultrastructure, which show that the leaflets
are composed of two thin endothelial cell sheets containing
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Fig. 8. Summary of valve closure measurements (Œ) after imposing a moderate
(A) or high (B) level of tone using high K⫹ physiological saline solution
(KPSS) ⫹ substance P (SP). Concentration of SP was 10⫺7 M for all vessels
in A, 6 ⫻ 10⫺7 M for 2 vessels in B and 1 ⫻ 10⫺6 M for the other 2 vessels
in B. Measurements in same vessels at same baseline pressures in Ca2⫹-free
PSS are shown (). Dotted lines are curve fit for SP data; solid lines are curve
fit for Ca2⫹-free data (n ⫽ 4 for A and B); curve fit parameters are listed in
Supplemental Table S1.
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While direct measurements of stiffness in this setting would be
extremely difficult to make, we can qualitatively visualize
leaflet behavior under various flow conditions. In some experiments, the leaflets exhibit measureable “flutter” in response to
rapid pressure oscillation at both low and high baseline transmural pressures (Supplemental Video S6), suggesting that they
remain reasonably compliant even when a vessel is near
maximally distended. A fuller assessment of this phenomenon
through high resolution three-dimensional imaging and computational modeling would be beneficial. In the meantime, the
findings of our study should provide valuable information for
models of lymphatic function such as the recent model developed by Bertram et al. (3).
Influence of vessel tone on valve gating. The idea that active
force generation by muscle cells in the lymphatic wall can
control valve gating is controversial. In bovine lymphatics,
some anatomical evidence exists for the presence of muscle
cells or muscle cell extensions into the valve leaflets (4, 5), yet
no functional data are available to support the concept that
muscle contraction per se can initiate valve closure/opening. In
this regard, we never observed valves to close without the
imposition of an appropriate trans-valve pressure gradient,
even in the presence of tone. However, the relationships
described in Fig. 8 and Supplemental Fig. S6 clearly demonstrate that tone has an influence, albeit an indirect one, on valve
closure. The reciprocal implication is that high levels of tone
will lead to enhanced valve opening when Pin ⬍ Pout (Fig. 7B),
i.e., in the presence of high tone an output valve will open well
before PL reaches Pout, meaning that active force developed by
lymphatic muscle during systole would facilitate valve opening. It should be noted that “tone” in this context reflects the
effect of steady-state stimulation of the vessel with KPSS ⫹ SP
in our protocols. Whether the same principle also applies to the
transient, active force development by lymphatic muscle during the contraction cycle will be difficult to determine using
methods similar to the ones we employed. Nevertheless, our
results (Fig. 8) are partially consistent with the idea of an active
Fig. 9. Plot of the trans-valve pressure gradient at the moment of valve closure
as a function of baseline (initial) pressure. Graph represents an alternative way
to express the closure test data for 1 of the 4 vessels used in Fig. 8B (⬃50%
tone group). Complete data sets for all 4 vessels are plotted in a similar manner
in Supplemental Fig. S6. Arrow indicates the increase in the adverse pressure
gradient required to close the valve if the vessel were to lose all tone at a
constant lumenal pressure of 2 cmH2O. Diameters indicated are the averages
calculated from the respective trials (with and without tone) at 2 cmH2O.
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elastin and collagen fibers (26). Schmid-Schönbein (50) noted
that the small size of the vessel and low Reynolds numbers for
lymph flow support the concept that valve leaflet movements
are dictated solely by trans-valve pressure gradients and viscous forces. Although such gradients are predicted to be small,
we could find no published measurements to validate this
assumption, apart from the work of Lobov (35) in an unpublished Russian thesis. Lobov reported that an adverse pressure
gradient of ⬃0.4 cmH2O was sufficient to close secondary
valves in bovine mesenteric lymphatic vessels at a baseline
pressure of 3 cmH2O; although a direct comparison with our
results is not possible without knowing D/Dmax for bovine
vessels in those particular experiments, the absolute value is
well within the range of the pressure gradients for closure of rat
mesenteric lymphatic valves shown in Fig. 7A.
A novel and important finding from our study is that the
trans-valve pressure gradient required for valve closure (and to
a lesser extent for valve opening) depends strongly on vessel
distension, the level of which is determined by the baseline
pressure at which the test is performed. At low pressures
(⬍2 cmH2O) that are within the physiological range of rat
mesenteric vessels (2), ⬃0.1 cmH2O pressure gradient
(Pout ⬎ Pin) is required for valve closure, whereas at higher pressures
(e.g., ⬎10 cmH2O), a trans-valve gradient of 2.2 cmH2O (and
in some cases ⬎5 cmH2O; Fig. 7A) is required. Thus there is
a ⬎20-fold range in the pressure gradient determining valve
closure over the range of pressures the vessel would experience
normally and under conditions of edema (44, 46). The baseline
pressure at which the ⌬P for closure substantially increases
(typically 2–3 cmH2O) corresponds approximately to the “elbow” in the passive pressure-diameter curve of the vessel (Fig.
6A) and suggests that the increase in the pressure gradients
required for opening/closing may be determined by tensioning
of the valve leaflets as the vessel progressively distends. The
⌬P-diameter relationship (Fig. 7A) explains why valves do not
always close when a vessel segment with a single-valve contracts under conditions where Pin ⫽ Pout (as occurs in Fig. 1
before the Pout ramp begins). At baseline pressures ⬎2 cmH2O,
the small fluctuations in intraluminal pressure associated with
contraction are insufficient to generate even a transient backpressure large enough to close the valve, since the pressure
spikes are largely shunted out the open cannulation pipettes.
The graph in Fig. 7A also explains why reverse flow (through
an open valve) can sometimes be observed in vivo (Supplemental Video S7). The graph in Fig. 7B explains why an output
valve can open even if internal (developed) pressure does not
exceed output pressure (Fig. 3).
There was a distinct lack of symmetry in the ranges of
pressure gradients necessary to open (6-fold) and close (20fold) the valve leaflets as transmural pressure increased (Fig.
6). This is perhaps related to the bias of the valves to remain in
the open position. Furthermore, increasing the transmural pressure likely results in less contact area between the leaflets and
makes coaptation more difficult. This would further bias the
valve to the open position. We occasionally noted that there
appeared to be less contact area between the leaflets at larger
vessel diameters, but the imaging techniques used here did not
allow for accurate assessment of contact area. It is also possible
that, like the vessels themselves (Fig. 6A), the valve leaflets
become stiffer with increasing diameter as the wall distends
and pulls radially on the base and/or buttress of the leaflets.
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AJP-Heart Circ Physiol • VOL
insufficient in distended vessels, because a greater ⌬P is
required for closure. Thus, if diastolic pressure rises, pressure
will be more likely to equalize through an open valve, rendering that valve insufficient. Indeed, we have observed this
sequence of events frequently under conditions that mimic
those seen in lymphedema (a simultaneous ramp increase in Pin
and Pout, with Pout ⬎ Pin; unpublished observations) and
insufficient valves have been reported to be associated with
distended lymphatic vessels in vivo, including vessels in patients with lymphedema (44, 51). A potential means to combat
this problem and minimize lymph reflux could be to increase
the resting tone of the collecting lymphatic vessels.
In summary, our findings provide several new insights into
the function of secondary lymphatic valves. 1) We have documented in detail for the first time the temporal sequence of
events among diameter, pressure, and valve gating during
the lymphatic contraction cycle. The timing of the lymphatic
valve movements is similar to that of cardiac valves only
under the specific condition where lymphatic afterload is
elevated. 2) We find that lymphatic vessel distention exerts a
profound influence on the pressure gradient required for valve
opening/closing. 3) We provide an explanation for why lymphatic valves in mesenteric vessels have a natural tendency to
remain open when the trans-valve pressure gradient is zero or
near zero. 4) Finally, we present functional evidence to support
the concept that lymphatic muscle tone exerts at least an
indirect effect on valve gating.
ACKNOWLEDGMENTS
We gratefully acknowledge the technical assistance of Shanyu Ho. Chris
Bertram provided valuable insights regarding the data analysis.
GRANTS
This work was supported by National Institutes of Health Grants HL089784 (to M. J. Davis), AG-030578 (to A. A. Gashev), HL-070308 (to D. C.
Zawieja), and HL-094269 (to J. E. Moore, Jr.).
DISCLOSURES
No conflicts of interest, financial or otherwise, are declared by the author(s).
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An extension of the idea described in the preceding paragraph is that valves will become more susceptible to becoming
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