Am J Physiol Regul Integr Comp Physiol 306: R901–R907, 2014.
First published March 26, 2014; doi:10.1152/ajpregu.00185.2013.
Functional adaptation of bovine mesenteric lymphatic vessels to mesenteric
venous hypertension
Christopher M. Quick,1,2 John C. Criscione,1,2 Akhilesh Kotiya,3 Ranjeet M. Dongaonkar,1
Joanne Hardy,1,4 Emily Wilson,1,5 Anatoliy A. Gashev,1,5 Glen A. Laine,1 and Randolph H. Stewart1
1
Michael E. DeBakey Institute, Texas A&M University, College Station, Texas; 2Department of Biomedical Engineering,
Texas A&M University, College Station, Texas; 3Department Biomedical Engineering, Dalhousie University, Halifax, Nova
Scotia, Canada; 4Large Animal Clinical Sciences, Texas A&M University, College Station, Texas; and 5Systems Biology and
Translational Medicine, Texas A&M Health Science Center, Temple, Texas
Submitted 15 April 2013; accepted in final form 24 March 2014
lymphangion; lymph flow; interstitial fluid; postnodal lymphatic vessels
THE LYMPHATIC SYSTEM drains fluid from the interstitial space
and transports it as lymph to the venous system. On the one
hand, lymphatic vessels have a very different structure than
blood vessels. Unlike blood vessels with primarily circumferential arrangement of smooth muscle, lymphatic vessels also
have smooth muscle layers that are oriented longitudinally
(38). On the other hand, lymphatic vessels are similar to veins.
Lymphatic vessels are relatively thin-walled and can collapse,
have muscle allowing phasic and tonic contraction, contain
unidirectional valves, and have a tone modulated by a number
of vasoactive mediators (14, 23, 46). Gaining far more attention in the last decade, however, is the observation that lymphangions, the segments of lymphatic vessels bound by valves,
form units that function like cardiac ventricles. Lymphangions
can act as chambers that cyclically contract, and are capable of
actively pumping lymph against an axial pressure gradient
(35). Similar to ventricles (21), increases in transmural pressure can cause lymphangions to increase stroke volume and
contraction frequency (22, 31). Not only does this property
allow lymph to be propelled from the low-pressure intersti-
Address for reprint requests and other correspondence: C. M. Quick,
Michael E. DeBakey Institute, TAMU 4466, Texas A&M Univ., College
Station, TX 77843-4466 (e-mail: [email protected]).
http://www.ajpregu.org
tial space to the higher-pressure venous system, it also
allows lymphatic vessels to respond to increases in interstitial fluid pressure by pumping more fluid out of the interstitium (3, 18, 43).
Mesenteric venous hypertension causes persistent intestinal
edema. Intestinal microvascular hypertension and resulting
interstitial edema may occur with portal venous thrombosis,
hepatic fibrosis, or mesenteric venous thrombosis. Acute venous hypertension leads sequentially to increased intestinal
microvascular pressure, increased microvascular filtration, increased interstitial fluid volume and pressure, and finally, a
several-fold increase in mesenteric lymph flow (13, 20, 27).
The increase in lymph flow is particularly pronounced when
hypertension is induced regionally by increased resistance of
regional veins (13, 36, 45), since, unlike central venous hypertension, regional venous hypertension does not raise the outlet
pressure of the lymphatic system (10, 28). Gashev et al. (17)
reported that increased luminal flow in vitro decreases the rate
and magnitude of lymphangion contraction in mesenteric lymphangions. Like blood vessels, this response is due to increased
endothelial shear stress and is mediated, in part, by the production of nitric oxide (17). Although regional venous hypertension has been used by numerous investigators to study
edema formation and lymphatic function (12, 13, 45), all
studies up to this point have been acute. Investigators have
yet to report that persistent intestinal edema secondary to
increased microvascular pressure involves adaptive responses
by lymphangions. Recent studies have reported that postnodal
bovine mesenteric lymphangions adapt to altered pressures in
vivo in as little as 3 days (8). Therefore, we used a mesenteric
venous hypertension model to test the hypothesis that postnodal bovine mesenteric lymphatic vessels adapt to become
weaker pumps within 3 days.
METHODS
Experimental groups. Experimental procedures and animal care
were performed in compliance with protocols approved by the Texas
A&M University Institutional Animal Care and Use Committee and
were consistent with the National Institutes of Health’s Guide for the
Care and Use of Laboratory Animals. Holstein cows used for this
study were randomly divided into two experimental groups: mesenteric venous hypertension (MVH) and sham surgery (Sham). Functional and histological analyses of lymphatic vessels were performed
to determine their response to MVH.
Surgical preparation. An intravenous catheter was placed in the
jugular vein for drug administration. Oxytetracycline (20 mg/kg) was
administered subcutaneously. Anesthesia was induced using xylazine
(0.1 mg/kg iv), followed by diazepam (0.01 mg/kg iv) and ketamine
0363-6119/14 Copyright © 2014 the American Physiological Society
R901
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Quick CM, Criscione JC, Kotiya A, Dongaonkar RM, Hardy J,
Wilson E, Gashev AA, Laine GA, Stewart RH. Functional adaptation of
bovine mesenteric lymphatic vessels to mesenteric venous hypertension. Am
J Physiol Regul Integr Comp Physiol 306: R901–R907, 2014. First published
March 26, 2014; doi:10.1152/ajpregu.00185.2013.—Lymph flow is the
primary mechanism for returning interstitial fluid to the blood circulation. Currently, the adaptive response of lymphatic vessels to mesenteric venous hypertension is not known. This study sought to
determine the functional responses of postnodal mesenteric lymphatic
vessels. We surgically occluded bovine mesenteric veins to create
mesenteric venous hypertension to elevate mesenteric lymph flow.
Three days after surgery, postnodal mesenteric lymphatic vessels from
mesenteric venous hypertension (MVH; n ⫽ 7) and sham surgery
(Sham; n ⫽ 6) group animals were evaluated and compared. Contraction frequency (MVH: 2.98 ⫾ 0.75 min⫺1; Sham: 5.42 ⫾ 0.81 min⫺1)
and fractional pump flow (MVH: 1.14 ⫾ 0.30 min⫺1; Sham: 2.39 ⫾
0.32 min⫺1) were significantly lower in the venous occlusion group.
These results indicate that postnodal mesenteric lymphatic vessels
adapt to mesenteric venous hypertension by reducing intrinsic contractile activity.
R902
LYMPHATIC ADAPTATION
Small intestine
LN
LN
MV
CL
MV
Fig. 1. Experimental induction of mesenteric venous hypertension. The veins
(solid lines) draining the small intestine are arranged in arcades so that
complete obstruction (Œ) of one mesenteric vein (MV) and constriction
(represented as circle with X) of an adjacent MV does not stop blood flow, but
rather redirects flow and increases local venous pressure. The resulting increase in intestine microvascular pressure causes microvascular filtration and
interstitial water volume to increase. As a result, lymph flow from the intestine
and through the lymph nodes (LN) and postnodal collecting lymphatic vessels
(CL) increases. Arrows indicate direction of flow for venous blood and lymph.
three times to ensure repeatability. If pressure in the MVH group was
less than 30 cmH2O, dextrose solution was injected into the vascular
occluder until that pressure was attained. This was done to ensure that
the elevated mesenteric venous pressure obtained during surgery was
maintained postoperatively. Venous pressure data were compared
using the Mann-Whitney rank sum test.
Sample collection. Three days following surgery, animals were
euthanized by captive bolt and exsanguination. Study samples were
collected immediately following euthanasia. The small intestine was
exposed for tissue sample collection. Postnodal mesenteric lymphatic
vessels draining the affected segment were ligated at the downstream
end. After carefully dissecting free from surrounding tissue, vessels
used for functional and structural analysis were placed in a thermos
containing warm (37°C) 1% albumin physiological saline solution
(APSS, pH 7.4) containing (in mM) 145 NaCl, 4.70 KCl, 2.00 CaCl2,
1.17 MgSO4, 1.20 NaH2PO4, 5.00 dextrose, 2.00 sodium pyruvate,
0.020 EDTA, and 3.00 MOPS. All the tissue samples were transported
a short distance to the laboratory.
Determination of intestinal water content. Intestinal water content
was measured to characterize the effects of the preparation on interstitial fluid balance. A jejunal segment was rinsed with saline to
remove the luminal contents, patted dry, and weighed. The segment
was placed in a drying oven and dried at 60°C to a constant weight.
Percent water content of the intestinal segment was calculated from
100 ⫻ (wet weight-dry weight)/wet weight. Data were compared
using a Student’s t-test.
Preparation of postnodal vessels. Experimental procedures were
similar to those reported previously (39, 48). Lymphatic vessels were
carefully cleared of all adipose tissue. Vessel segments ⬃3 cm in
length were isolated and instrumented in an isolated vessel bath in
which inlet and outlet pressures could be independently manipulated
and measured. Axial tension on the vessels was applied to a degree
just sufficient to raise the center portion of the vessel segment from
the bottom of the bath. Using vessel segments between valves, we
could ensure that there was no potential for valve closure to isolate the
pressure transducers from the lumen of the vessel. The vessels were
perfused and bathed with APSS with pH of 7.4 and warmed to 37°C.
Normal rectal temperature in beef cows is 36.7–39.1°C and in dairy
cows is 38.0 –39.3°C (1). Previous studies have set bath temperatures
between 35°C and 38°C (15, 29, 32, 33). We chose 37°C for the bath
temperature so that our results would be more comparable to those of
prior studies of bovine mesenteric lymphatic vessels (26, 30, 42, 51).
External pressure was set to 1 cmH2O by setting the bath solution
height 1 cm above the center line of the vessel. Transmural pressure
was determined as the difference between inlet and external pressures.
Flow through the vessel was minimized by maintaining inlet and
outlet pressures at equal levels throughout the measurement process.
Transmural pressure was adjusted by increasing or decreasing both
inlet and outlet pressures in tandem. Instantaneous vessel outer diameter was measured using custom video caliper software (IMAQ,
National Instruments, Austin, TX). Vessels were allowed to equilibrate for 15 min. Lymphatic vessel segments failing to exhibit stable
spontaneous contractions were discarded.
Functional analysis of postnodal lymphatic vessels. Postnodal
lymphatic vessels were exposed to five transmural pressures (3, 6, 9,
12, and 15 cmH2O). The instrumented vessel was allowed to equilibrate for 5 min at each transmural pressure setting followed by a
5-min recording period. Following analysis of the active vessel
properties, the bath and lumen were perfused with Ca2⫹-free APSS
for 30 min until the vessel was fully relaxed. Diameters of the passive
vessel were recorded for 1 min at each transmural pressure level (i.e.,
3, 6, 9, 12, and 15 cmH2O).
Analysis of passive properties. To detect changes in passive stiffness, passive pressure-diameter data were fit to an exponential function commonly used to characterize biomechanical properties (i.e.,
D ⫽ ␣e␤x) (34). The values of ␣ and ␤ were compared using the
Mann-Whitney rank sum test.
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(2 mg/kg). Animals were intubated, positioned in left lateral recumbency, and maintained using mechanical ventilation with 1–3% isoflurane in oxygen sufficient to maintain a surgical plane of anesthesia.
Arterial oxygen saturation was monitored (Cardell Veterinary Monitor-model MAX-12 HD; CAS Medical Systems, Branford, CT) and
maintained ⬎95% throughout the procedure. The right flank was
aseptically prepared and draped. A 25-cm incision was made midway
between the tuber coxae and the last rib. The small intestine was
exteriorized and kept moist using warm (37°C) 0.9% saline.
MVH. The veins draining the small intestine are arranged in
arcades, such that complete obstruction of one mesenteric vein does
not stop blood flow, but rather redirects flow, and increases local
venous pressure (Fig. 1). The proximal jejunal venous arcade was
identified. The vein draining one end of this arcade was carefully
isolated by blunt dissection and ligated. The vein draining the other
end of the arcade was isolated in a similar fashion and fitted with an
appropriately sized vascular occluder (Kent Scientific, Torrington,
CT). The occluder was fixed to the mesentery with a piece of suture
(2-0 polyglactin 910) and filled with 50% dextrose solution. To
measure pressure within the arcade, an upstream vein draining the
proximal jejunal venous arcade was isolated by blunt dissection, and
1/8⬙; O.D. polyvinyl chloride tubing (VWR Scientific, Suwanee, GA)
filled with heparinized saline was inserted into the vein and secured
with 2-0 polyglactin 910 suture. The PVC tubing and the tail of the
vascular occluder were exteriorized through a small skin incision
dorsal to the abdominal incision. The jejunum was replaced in the
abdomen, and the abdominal incision was closed in multiple layers.
The animals then recovered from anesthesia.
Sham surgery group. The same surgical procedures were performed in the sham-treated animals. Sham-treated animals, however,
were only instrumented with the intravenous tubing for measurement
of intestinal venous pressure. The mesenteric vein was not ligated, and
the vascular occluder was not applied.
Measurement of venous pressure. The day after surgery, mesenteric
venous pressure was measured by placing the cow in a restraining
chute. The venous catheter was flushed with heparinized saline to
ensure patency. A water manometer was used to measure venous
pressure in a fashion adapted from a common clinical technique for
measuring central venous pressure (49). The manometer was attached
to the venous catheter and fixed with the zero-point at the level of the
point of the shoulder. The equilibrium column height was recorded as
venous pressure. The system was zeroed, and pressures were recorded
R903
Transmural Pressure (cmH2O)
LYMPHATIC ADAPTATION
15
12
9
6
3
0
3
4
5
6
Passive Diameter (mm)
Functional data analysis. The functional data from postnodal
vessels were analyzed using two-way repeated-measures ANOVA
comparing the experimental groups (MVH vs. Sham) with transmural
pressure as the repeated measure. The Holm-Sidak method was used
for post hoc multiple-comparison tests. All volume measurements
were expressed in terms of cross-sectional area (i.e., volume normalized by length assuming uniform cylindrical contraction). The functional parameters compared were diastolic tone (100 ⫻ [passive
diameter ⫺ diastolic diameter]/passive diameter), maximum (diastolic) volume, minimum (systolic) volume, stroke volume, ejection
fraction (stroke volume/diastolic volume), contraction frequency, and
fractional pump flow (contraction frequency ⫻ stroke volume/diastolic volume). A calculated P value of less than 0.05 was considered
significant. In all cases, each cow contributed one vessel for analysis.
Histological analysis of postnodal lymphatic vessels. Changes in
the structural components of the postnodal lymphatic vessels were
determined using histological analysis. Each vessel was dehydrated
using a series of graded alcohol (70%, 80%, 90%, and 95% EtOH) and
then embedded with paraffin. Five-micrometer-thick cross sections of
the vessels were then obtained using a rotary microtome (model no.
RM2255, Leica Microsystems, Bannockburn, IL). The sections were
mounted on plus-coated slides and stained with Masson’s Trichrome
and hematoxylin and eosin (H&E). Images of each vessel cross
section were obtained, and their contrast and saturation increased
using image enhancement software (Picasa, Google, Mountain View,
CA). Images were then analyzed with a custom application developed
with MATLAB (R2008a; MathWorks, Natick, MA). Lymphatic muscle cells (stained pink) and collagen (stained blue) were identified
from Trichrome-stained images, and fibrous structures of the media
were identified from H&E-stained images. Using the filtered images,
we estimated the area occupied by each constituent.
Histological data analysis. Cross-sectional area occupied by
smooth muscle cells (SMCA), total cross-sectional area occupied by
collagen (TCA), and cross-sectional area occupied by medial collagen
were determined and normalized by total cross-sectional area (TA)
and medial area (MA) and were reported as means ⫾ SE. A calculated
P value less than 0.05 was considered significant. Each cow contributed one lymphatic vessel for analysis.
RESULTS
Fluid balance parameters. Mesenteric venous pressure measured one day after surgery was significantly higher in MVH
(31.4 ⫾ 2.2 cmH2O; n ⫽ 12) than Sham (11.6 ⫾ 2.8 cmH2O;
n ⫽ 5). Although the intestinal water content determined post
mortem 3 days after surgery in MVH (77.9 ⫾ 1.6%) was
elevated, it was not significantly different from the Sham group
(73.3 ⫾ 2.6%).
DISCUSSION
Decreased lymphatic pump function in mesenteric venous
hypertension. Within 3 days of induction of mesenteric venous
hypertension, postnodal vessels demonstrated decreased contraction frequency and fractional pump flow. The fact that
MVH vessels were weaker pumps was due primarily to decreases in contraction frequency. Contraction frequency in our
study may not have increased with increases in transmural
pressure as much as previously reported. We believe that
apparent differences between our study and previous studies in
contraction frequency data can be explained by differences in
preparation. The classic study demonstrating the effect of
Table 1. Comparison of passive, diastolic, and systolic
diameters of bovine mesenteric postnodal lymphatic vessels
in Sham and MVH groups
Transmural
Pressure, cmH2O
Passive
Diameter, mm
3
6
9
12
15
3.64 ⫾ 0.37
3.74 ⫾ 0.37
3.79 ⫾ 0.38
3.80 ⫾ 0.39
3.84 ⫾ 0.40
3
6
9
12
15
4.23 ⫾ 0.32
4.30 ⫾ 0.32
4.34 ⫾ 0.34
4.36 ⫾ 0.35
4.39 ⫾ 0.34
Diastolic
Diameter, mm
Systolic
Diameter, mm
Sham Group
3.38 ⫾ 0.39
3.51 ⫾ 0.39
3.59 ⫾ 0.39
3.66 ⫾ 0.38
3.71 ⫾ 0.36
1.87 ⫾ 0.27
2.15 ⫾ 0.29
2.49 ⫾ 0.29
2.89 ⫾ 0.30
3.14 ⫾ 0.30
MVH Group
4.06 ⫾ 0.31
4.21 ⫾ 0.31
4.27 ⫾ 0.33
4.30 ⫾ 0.35
4.31 ⫾ 0.35
2.15 ⫾ 0.26
2.93 ⫾ 0.30
3.40 ⫾ 0.35
3.77 ⫾ 0.35
4.00 ⫾ 0.34
Data are presented as means ⫾ SE. MVH, microvascular hypertension.
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Fig. 2. Passive pressure-diameter relationship of MVH (solid symbols) and
Sham (open symbols). Passive stiffness for each vessel was estimated from the
parameters estimated using nonlinear regression (D ⫽ ␣e␤x). There was no
significant change in passive stiffness in the pressure range of interest.
Functional analysis. There was no difference in passive
stiffness between the MVH and Sham group in the pressure
range of interest (Fig. 2). Passive diameter, diastolic diameter,
and systolic diameter at five transmural pressure levels (Sham,
n ⫽ 6; MVH, n ⫽ 7) are listed in Table 1. Diastolic tone,
normalized diastolic volume, normalized systolic volume, normalized stroke volume, ejection fraction, contraction frequency, and fractional pump flow for postnodal lymphatic
vessels (Sham, n ⫽ 6; MVH, n ⫽ 7) pooled over all transmural
pressure levels are listed in Table 2. Figure 3 depicts these
functional variables at each transmural pressure level. There
were no significant differences in normalized systolic volume,
normalized diastolic volume, or ejection fraction of the lymphatic vessels between the two groups (Table 2). However,
contraction frequency and fractional pump flow were significantly lower in the MVH group compared with the Sham group
(Table 2). Post hoc testing showed that contraction frequency
and fractional pump flow were significantly lower at particular
pressures (Fig. 3).
Histological analysis. There were no significant differences
detected in the SMCA/TA ratio (Sham: 0.33 ⫾ 0.070 and
MVH: 0.41 ⫾ 0.081), TCA/TA ratio (Sham: 0.69 ⫾ 0.069 and
MVH: 0.63 ⫾ 0.085), or MCA/TA ratio (Sham: 0.21 ⫾ 0.087
and MVH: 0.28 ⫾ 0.12). We also found no differences in
SMCA/MA ratio (Sham: 0.61 ⫾ 0.13 and MVH: 0.60 ⫾ 0.097)
and MCA/MA ratio (Sham: 0.39 ⫾ 0.11 and MVH: 0.40 ⫾
0.097) (Fig. 4).
R904
LYMPHATIC ADAPTATION
Table 2. Comparison of critical variables characterizing the response of bovine mesenteric postnodal lymphatic vessels
pooled over all transmural pressure levels
Group
Diastolic
Tone
Diastolic Volume,
% of PV
Systolic Volume,
% of PV
Stroke Volume,
% of PV
Ejection
Fraction
Frequency,
min⫺1
Fractional Pump
Flow, min⫺1
Sham
MVH
5.57 ⫾ 1.53
2.25 ⫾ 1.42
89.49 ⫾ 2.83
95.64 ⫾ 2.62
47.61 ⫾ 5.93
59.50 ⫾ 5.49
41.87 ⫾ 6.88
36.14 ⫾ 6.37
0.47 ⫾ 0.06
0.38 ⫾ 0.06
5.42 ⫾ 0.81
2.98 ⫾ 0.75*
2.39 ⫾ 0.32
1.14 ⫾ 0.30*
Data are presented as means ⫾ SE. Contraction frequency and fractional pump flow were significantly decreased in microvascular hypertension group (MVH;
n ⫽ 7) compared to Sham (n ⫽ 6). *P ⬍ 0.05 vs. Sham. PV, passive volume.
A
9
6
3
0
0
Ejection Fraction (%)
6
9
12
15
80
MVH
60
40
20
0
3
6
9
12
15
18
Fractional Pump Flow
4
SHAM Diastolic
MVH Diastolic
25
SHAM Systolic
MVH Systolic
0
0
3
*
6
9
12
15
18
MVH
4
2
0
0
3
0
6
15
SHAM
1
3
12
*
2
0
9
8
MVH
*
6
Transmural Pressure (cmH2O)
SHAM
*
3
50
6
9
12
15
Transmural Pressure (cmH2O)
Transmural Pressure (cmH2O)
E
75
D
SHAM
0
100
18
Transmural Pressure (cmH2O)
C
Fig. 3. Functional parameters for bovine mesenteric postnodal lymphatic vessels. A: diastolic tone. B: systolic and diastolic volumes
normalized by passive volume. C: ejection
fraction. D: contraction frequency. E: fractional pump flow. Contraction frequency and
fractional pump flow were significantly lower
in MVH (n ⫽ 7) compared with Sham (n ⫽
6). Data are presented as means ⫾ SE. *P ⬍
0.05 vs. Sham.
3
(%)
MVH
Contraction Frequency
(min-1)
Diastolic Tone
SHAM
Normalized Volume
B
12
18
Transmural Pressure (cmH2O)
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intervention that we used could potentially raise lymphatic
transmural pressure in MVH vessels in vivo above normal
values. Comparing Sham and MVH vessels in the same extended pressure range in vitro allowed us to detect any differences. Given the statistical difference between Sham and MVH
vessels at 9 and 12 cmH2O, this increased range is, in retrospect, justified. Because the passive properties of lymphangions have been reported to change significantly for the pressure range between 0 and 5 cmH2O (41), it may be interesting
in future biomechanical studies to explore changes in passive
stiffness in lower pressures than those illustrated in Fig. 2,
particularly in response to interventions that are less likely to
raise lymphatic transmural pressures than MVH.
Potential mechanical mechanisms for lymphatic pump inhibition.
The present work was primarily a functional study, and the
transmural pressure in bovine mesenteric lymphatic vessels by
McHale and Roddie (31) used transmural pressures of 0 to 6
cmH2O, while the current study used a pressure range of 3 to
15 cmH2O. If we compare the transmural pressures common
to both studies, the results for the Sham group appear similar to
McHale and Roddie’s study. In this study, the increase in mean
frequency from 3 to 6 cmH2O was ⬃17%. In the current study,
mean frequency in the Sham group at 6 cmH2O was 9% greater
than that at 3 cmH2O. However, mean frequency in the MVH
group in the present study was 5% lower at 6 cmH2O than at
3 cmH2O. This could be an aspect of the adaptive response to
venous hypertension. Because the frequency change from 3 to
6 cmH2O was not statistically significant, it is difficult to make
any claim as to its importance. We chose a much greater
pressure range than most in vitro studies, because the particular
LYMPHATIC ADAPTATION
A
TCA
TA
SMCA
Trichrome
H&E
Ratio of Total Medial Area
B
0.8
SHAM
MVH
0.6
0.4
0.2
0
SMCA/MA
C
MCA/MA
0.8
SHAM
Ratio of Total
Cross-section Area
MVH
0.6
0.4
0.2
0
SMCA/TA
TCA/TA
MCA/TA
Fig. 4. A: postnodal lymphatic vessel sections stained with Masson’s
Trichrome and hematoxylin and eosin (H&E) to estimate the percent area of
the constituents of interest: vessel total area (TA), medial area (MA), area
occupied by collagen (TCA), and smooth muscle cells (SMCA). B: between
the Sham (n ⫽ 8) and MVH (n ⫽ 8) groups, there were no significant
differences in the smooth muscle cell area/total area ratio (SMCA/TA), total
collagen area/total area ratio (TCA/TA), medial collagen area/total area ratio
(MCA/TA), smooth muscle cell area/medial area ratio (SMCA/MA), and
medial collagen area/medial area ratio (MC/MA). C: Ratio of total crosssectional area for SMC/TA, TCA/TA, and MCA/TA. Data are presented as
means ⫾ SE.
changes in function were not related to particular changes in
biomechanical properties. In particular, the decrease in pump
function with MVH may be related to changes in either
circumferential or longitudinal smooth muscle. It is possible
that changes in length, due to the longitudinal arrangement of
smooth muscle, leads to significant shortening in vivo. Because
our preparation set length, the difference in Sham and MVH
vessels manifested only as a change in radius. Similarly, the
mechanical stimuli in vivo that resulted in adaptation are
difficult to identify. Adaptive responses occurred as a result of
our hypertension model, but it is not clear whether this was due
primarily to enhanced lymph flow, elevated lymphatic pressure, or even tissue oncotic pressure changes. Some or all of
these mechanisms may be contributory, and their effects may
be interrelated. Changes in contractility can affect transmural
pressure and changes in transmural pressure can affect contractility (31). Elevated lymph flow increases shear stress, and
it is known to acutely inhibit lymphangion pumping (17, 34).
Since, in vivo, lymph flow is increased by increasing microvascular fluid filtration, it is not possible to increase lymph
flow without altering interstitial protein concentration (44) and
thus the content of lymph. Now that functional adaptations
have been identified, further biomechanical study is warranted,
including quantifying maximum tension generated (19, 52) or
alterations of sensitivity to shear stress (17).
Potential inflammatory mechanisms. The alterations in lymphatic function observed in this study may be the result of an
inflammatory response induced by the mesenteric venous hypertension. We did not seek to detect differences in immune
cell populations or indicators of inflammation within the sections of the vessel walls or surrounding tissue. Inflammatory
signaling molecules could originate in or near the intestinal
interstitium and travel via lymph to affect the mesenteric
lymphatic vessels. Using a model of bacterial tripeptide-induced ileitis in rats, Benoit and Zawieja (4) demonstrated that
mesenteric lymphatic vessels examined in vivo increased
stroke volume within the first hour. In that study, lymphatic
contraction frequency was not significantly affected. However,
Wu et al. (50) examined contractile behavior of mesenteric
lymphatic vessels in vivo and in vitro 3 and 6 days after
2,4,6-trinitrobenzenesulfonic acid (TNBS)-induced ileitis in
guinea pigs. They reported that intestinal inflammation was
associated with decreased contraction frequency, decreased
stroke volume, and increased diastolic diameter—results
broadly consistent with the current study. The inflammationrelated decrease in contraction frequency reported by Wu et al.
(50) was moderated by application of a nonselective COX
inhibitor (indomethacin), as well as by combined COX 1 and
COX 2 inhibitors. Numerous inflammatory mediators have
been demonstrated to affect lymphatic contractile behavior,
including prostaglandins, bradykinin, histamine, and substance
P (2, 6, 16, 23, 25). Recently, substance P has been shown to
activate both inflammatory signaling pathways and contractile
activity in lymphatic vessels (5). However, because substance
P acutely stimulates lymphatic contraction, it may not be a
good candidate for inducing the behavior observed in the
present study. The finding by Wu et al. that COX inhibition
partially reversed the effect of intestinal inflammation on
lymphatic pumping (50) provides support for the possible role
of prostacyclin.
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MA
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LYMPHATIC ADAPTATION
Perspectives and Significance
The results of the present study are the first to suggest that
postnodal lymphatic vessels can respond to edemagenic conditions by becoming weaker pumps. Given the great complexity of microvascular-interstitial-lymphatic interaction, mathematical models have been used to explore how changes in
lymphatic pumping affects interstitial fluid balance. Quick et
al. (40) developed a relatively simple model of a lymphangion
that suggests that decreases in lymphangion pumping frequency raises lymphangion effective lymphatic resistance, first
characterized by Drake et al. (11). Dongaonkar et al. (7)
illustrated with a system-level model that increased effective
lymphatic resistance will decrease lymph flow, increase interstitial volume, as well as “edemagenic gain” (i.e., the sensitivity of interstitial volume to increases in microvascular pressure) (9). These modeling insights suggest persistent adaptive
responses manifesting as “lymphatic pump failure,” therefore,
are either homeostatic, ameliorating elevated interstitial flow,
or maladaptive, exacerbating interstitial edema.
ACKNOWLEDGMENTS
The authors thank Dr. David C. Zawieja for careful review of the manuscript.
GRANTS
This work was supported by National Institutes of Health Grants R01
HL-092916 (E. Wilson and R. H. Stewart) and R01HL070308 (A. A. Gashev)
and National Science Foundation Grant CMMI-1063954 (C. M. Quick).
DISCLOSURES
No conflicts of interest, financial or otherwise, are declared by the authors.
AUTHOR CONTRIBUTIONS
Author contributions: C.M.Q., J.C., A.A.K., J.H., A.A.G., and R.H.S. conception
and design of research; C.M.Q., J.C., A.A.K., R.M.D., and R.H.S. analyzed
data; C.M.Q., J.C., R.M.D., J.H., E.W., A.A.G., G.A.L., and R.H.S. interpreted
results of experiments; C.M.Q. drafted manuscript; C.M.Q., R.M.D., E.W.,
A.A.G., G.A.L., and R.H.S. edited and revised manuscript; C.M.Q., J.C.,
A.A.K., R.M.D., J.H., E.W., A.A.G., G.A.L., and R.H.S. approved final
version of manuscript; A.A.K., J.H., and R.H.S. performed experiments;
R.M.D. and R.H.S. prepared figures.
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