Endothelial Nitric Oxide Synthase Regulates Microlymphatic
Flow via Collecting Lymphatics
Jeroen Hagendoorn, Timothy P. Padera, Satoshi Kashiwagi, Naohide Isaka, Fatima Noda,
Michelle I. Lin, Paul L. Huang, William C. Sessa, Dai Fukumura, Rakesh K. Jain
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Abstract—Functional interactions between the initial and collecting lymphatics, as well as the molecular players involved,
remain elusive. In this study, we assessed the influence of nitric oxide (NO) on lymphatic fluid velocity and flow, using
a mouse tail model that permits intravital microscopy and microlymphangiography. We found that NO synthase (NOS)
inhibition decreased lymphatic fluid velocity in the initial lymphatics, without any effect on their morphology. Using
the same model, we found a similar effect in eNOS⫺/⫺ mice and in mice treated with a selective endothelial NOS (eNOS)
inhibitor. Next, we uncoupled the superficial initial lymphatics from the deeper collecting lymphatics by ligating the
latter and found that lymphatic fluid velocity in NOS-inhibited mice became equal to that in control animals.
Surprisingly, lymphatic fluid velocity was significantly increased after ligating the collecting lymphatics, and there was
a concomitant increase in injection rate and mean lymphatic vessel diameter. Our results provide the first in vivo
evidence that eNOS affects function of the whole microlymphatic system and that it is regulated via the collecting
lymphatics. (Circ Res. 2004;95:204-209.)
Key Words: microlymphatics 䡲 eNOS 䡲 lymphatic function 䡲 edema 䡲 lymphangiography
T
and right lymphatic duct and, eventually, drained into the
jugular and subclavian veins.
Functionally, determinants of lymph flow are extrinsic
propulsive forces such as the lymph formation rate, respiration, and skeletal muscle movement, and the intrinsic contractility of the smooth muscle layer of the collecting lymphatics.5 Although there is a positive pressure difference
between the thoracic duct and dorsal foot lymphatics in
humans in upright position, lymph flow is present during
basal physiological conditions in caudocranial direction.6 It is
speculated, therefore, that the contractile collecting lymphatics act as a primary driving force for lymph propulsion. A
number of studies have confirmed systematic contractions of
the collecting lymphatics in various ex vivo preparations.7–10
Moreover, oxygen tension is lower in mesenteric collecting
lymphatics than in the surrounding interstitial fluid, implying
in vivo energy consuming contractile processes of the lymphatic vessel wall.11 Thus, the transient contraction of each
lymphangion forces fluid into the proximal lymphangion and,
because one-way valves prevent backflow, this would result
in net fluid flow toward the heart. The relative importance of
collecting lymphatic vessel contractility on overall lymph
flow and the interactions of initial and collecting lymphatics
have, however, not been described.
he lymphatic network plays a physiologically crucial,
dual role in both immune function and the maintenance
of tissue interstitial fluid balance through the transport of
interstitial fluid back to the blood circulation. Despite its
importance in pathologies such as edema and cancer, mechanistic insights into lymphatic function have been hampered
by a lack of quantitative research models. In addition,
molecular factors that may affect these mechanisms have
remained elusive.
Structurally, the microlymphatic system has been well
characterized.1 First, interstitial fluid is taken up by blindended, capillary structures (⬇60 ␮m in diameter) known as
the initial lymphatics. These consist of adjacent lymphatic
endothelial cells, which lack a continuous basement membrane and possess overlaps that act as primary valves.2 The
initial lymphatics are dynamically coupled to collagen fibers
of the interstitium via anchoring filaments,3 so that increased
interstitial volume and resultant radial tension on the lymphatics leads to increased convective interstitial-lymphatic
fluid transport. Then, fluid is transported to larger lymphatic
structures (100 to 150 ␮m in diameter) that have a smooth
muscle layer4 and intraluminal valves, which divide the
lymph vessels into functional units called lymphangions.
From these collecting lymphatics, lymph fluid is transported,
via lymph nodes and lymphatic trunks, to the thoracic duct
Original received October 17, 2003; resubmission received February 5, 2004; revised resubmission received May 26, 2004; accepted June 3, 2004.
From the Department of Radiation Oncology (J.H., T.P.P., S.K., N.I., D.F., R.K.J.), E.L. Steele Laboratory for Tumor Biology, Massachusetts General
Hospital and Harvard Medical School, Boston, Mass; Division of Cardiology (F.N., P.L.H.), Cardiovascular Research Center, Massachusetts General
Hospital and Harvard Medical School, Charlestown, Mass; and the Department of Pharmacology (M.I.L., W.C.S.), Boyer Center for Molecular Medicine,
Yale University School of Medicine, New Haven, Conn.
Correspondence to Rakesh K. Jain, E.L. Steele Laboratory for Tumor Biology, Department of Radiation Oncology, Massachusetts General Hospital
and Harvard Medical School, 100 Blossom St, Cox 7, Boston, MA 02114. E-mail [email protected]
© 2004 American Heart Association, Inc.
Circulation Research is available at http://www.circresaha.org
DOI: 10.1161/01.RES.0000135549.72828.24
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eNOS and Microlymphatics
205
procedures were performed following the guidelines of the Institutional Animal Care and Use Committee of the Massachusetts
General Hospital.
Experimental Design
Figure 1. Schematic overview of mouse tail lymphangiography.
Fluorescent dye is injected with constant pressure in the interstitium of the tail-tip for lymphatic uptake. Inset, Intravital
microscopy reveals a hexagonal network of initial lymphatics.
RTD indicates region of intravital microscopy of lymphatic transport and residence time distribution analysis. Arrow indicates
location of ligation of collecting lymphatic vessels.
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Nitric oxide (NO), a major regulator of (micro)vascular
function,12–14 was found to be generated by lymphatic endothelial cells.8 Lymphatic endothelial cells express nitric oxide
synthase (NOS).15 Exogenous NO inhibits the pacemaking
activity of lymphatic smooth muscle cells by activating
protein kinases via the cyclic GMP pathway.7 Applied NO
was shown to resemble flow induced inhibition of contraction
frequency of mesenteric lymphatics, whereas N-monomethylL-arginine (L-NMMA), a nitric oxide synthase inhibitor,16
could partially attenuate this effect.9 Nevertheless, data on the
differential role of the NOS isoforms on the components of
the microlymphatic system in vivo are needed.
In this study, we adapted a previously described model of
the microlymphatic circulation17 for the combined and separate assessment of initial and collecting lymphatic function in
the mouse tail. This model uses constant pressure infusion of
a fluorescent dye in the interstitium, which is taken up only
by the lymphatics (Figure 1). Intravital microscopy is then
combined with residence time distribution (RTD) analysis to
assess lymphatic fluid velocity and mean lymphatic vessel
(LV) diameter, thereby coupling dynamic and ultrastructural
data. The effect of NO on lymphatic function was assessed in
mice treated with L-NMMA or the recently described eNOS
inhibitor cavtratin18 and in eNOS⫺/⫺ mice. Our results provide
the first in vivo evidence that (1) eNOS affects lymphatic
fluid flow, (2) this is mediated via the collecting lymphatics,
(3) eNOS inhibition does not affect the diameter of the initial
lymphatics, and (4) the collecting lymphatics provide outflow
resistance and have a regulatory role in microlymphatic
function.
Materials and Methods
Animals
Studies were performed in 7- to 10-week-old female C57BL/6 and
nude mice. In addition, male and female eNOS⫺/⫺ mice backcrossed
for 10 generations to C57BL/6 background were compared with ageand gender-matched wild-type animals from the same heterozygoteheterozygote breeding pair. All animals were bred and maintained in
animal facilities at Massachusetts General Hospital. Fifty-six mice
were used for these experiments: 24 for the inhibition experiments,
15 for the ligation experiments, and 17 additional controls. All
Mice received a subcutaneous osmotic pump (ALZET Model
1003D, Durect Corp) 3 days before the lymphatic function measurements for continuous infusion of L-NMMA or N-monomethyl-Darginine (D-NMMA) (controls) at 350 mg/kg daily, as described.19
For selective eNOS inhibition, mice received a daily intraperitoneal
injection of cavtratin, a cell-permeable peptide derived from
caveolin-1,18 at 2.5 mg/kg or the control peptide AP at 1.2 mg/kg,
during 3 days. The following groups were studied: group 1 (n⫽4),
L-NMMA administration; group 2 (n⫽4), D-NMMA administration;
group 3 (n⫽4), eNOS⫺/⫺ mice; group 4 (n⫽4), wild-type mice; group
5 (n⫽4), cavtratin administration; group 6 (n⫽4), control peptide
(AP)18 administration; group 7 (n⫽8), L-NMMA administration plus
bilateral collecting lymph vessel ligation; group 8 (n⫽7), D-NMMA
administration plus bilateral collecting lymph vessel ligation. Additional control groups consisted of: nude mice (n⫽3) to confirm that
lymphatic fluid velocities were consistent with our previous data17
and C57BL/6 mice (n⫽4) without pump implantation.
Surgical Procedure
Mice in the experimental groups 7 and 8 underwent ligation of the
deep collecting lymphatic vessels of the tail immediately before the
microlymphangiography, so that development of edema was avoided
(Figure 1). Mice were anesthetized intramuscularly (90 mg/kg
ketamine and 9 mg/kg xylazine) and placed on a heated surgical
microscopy table. The translucent collecting lymphatic vessels were
separated from the tail veins with microsurgical forceps through
small, bilateral incisions in the axial direction, and ligated with a
10-O nonabsorbable suture (Prolene, Ethicon). The incision site was
closed with surgical glue, taking care to avoid circumferential
tension on the tail that could interfere with superficial lymphatic
function.
Quantitative Lymph Flow Measurements Using
Residence Time Distribution Analysis
Fluorescence intensity measurements were performed using residence time distribution (RTD) analysis as described previously.17
Briefly, mice were anesthetized and placed on a small plate.
FITC-dextran (2.5%) (MW⫽2 million; Sigma) in PBS was infused
into the interstitial tissue of the tail tip, with a constant pressure of 40
cm H2O via a 30-gauge needle. Thus, changes in blood vessel
permeability would not affect RTD measurements of initial lymphatic fluid velocity. The mouse was transferred to an epifluorescence microscopy setup as described previously.20 Eight adjacent
fluorescent images of the tail, with a field dimension of 3.5⫻2.5 mm,
were obtained from distal to proximal, every 10 minutes until
saturation was reached in the most proximal region. The temporally
consecutive fluorescent images were analyzed offline using NIH
Image Analysis software. The average fluorescence intensity was
determined for each image and used to calculate the mean residence
time for each region, the lymphatic fluid velocity in the tail
lymphatic network, and the mean LV diameter.
Immunohistochemistry
Lymphatic vessels of the tail were histologically identified using
ferritin lymphangiography (type I ferritin, Mr 480 000; Sigma Chemical Co) as described before.21 Distribution of the NOS isoforms on
lymphatic vessel walls was examined immunohistochemically using
monoclonal antibodies against eNOS, inducible NOS (iNOS), and
neuronal NOS (nNOS) (BD Transduction Laboratory, Inc) as described before.22
Mean Arterial Blood Pressure
Eight-week-old female C57BL/6 mice were weighed and anesthetized. Mean arterial pressure (MAP) was measured by cannulation of
the exposed left carotid artery with a PE-10 intravascular polyeth-
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respectively; P⫽0.14) (Figure 2B). In addition, there was no
difference in mean lymphatic vessel diameter (61.5⫾0.7
versus 61.6⫾1.6 ␮m, respectively; NS) (Figure 2C). To
exclude a confounding effect of blood pressure at the time
point studied, we measured MAP in a separate group of mice,
and found no difference between mice that had received
L-NMMA and controls (71.7⫾1.4 versus 72.7⫾3.1 mm Hg,
respectively; NS). These data show that NOS inhibition
decreases initial lymphatic fluid velocity without affecting
mean lymphatic vessel diameter in the superficial network.
The absence of a significant effect on injection rate should be
interpreted with caution, because this is only an indirect
indicator of lymphatic uptake. Although the collecting lymphatics are not directly functionally evaluated in this experiment, the regular connections between the initial and collecting lymphatics make it reasonable to assume that the time
course of lymphatic filling in the deep collecting lymphatics
mirrors that in the superficial initial network.17 Taken together, these data suggest that NOS inhibition decreases
overall lymph flow.
eNOS Inhibition Decreases Initial Lymphatic
Fluid Flow
Figure 2. Effects of eNOS deficiency on lymphatic function parameters measured in L-NMMA–treated mice, eNOS⫺/⫺ mice,
and cavtratin-treated mice. A, Lymphatic fluid velocity (␮m/s) is
decreased in eNOS⫺/⫺ mice compared with wild-type mice and
after treatment with L-NMMA or cavtratin compared with mice
that received D-NMMA or the control peptide, AP.18 After ligation of the collecting lymphatics, this effect is eliminated.
*P⬍0.05. B, Injection flow rate (nL/min) is significantly different
between unligated and ligated controls. *P⬍0.05. C, Mean lymphatic vessel diameter (␮m) is not different between L-NMMA–
treated animals and controls, but there is a significant difference
between unligated and ligated controls. *P⬍0.05.
ylene catheter, connected to a pressure transducer (Gould Inc). MAP
was measured for 15 minutes, after 3 days of L-NMMA administration19 (n⫽5) and compared with PBS controls (n⫽3).
Statistics
Results are presented as mean⫾SE. Student t test (equal variances
not assumed) was used to evaluate statistical significance (defined
as P⬍0.05).
Results
NOS Inhibition Decreases Initial Lymphatic
Fluid Flow
We performed lymphatic function measurements in mice that
had received 3 days of L-NMMA treatment for NOS inhibition, and found that overall lymphatic fluid velocity in the
dermal lymphatic network was decreased by 42% compared
with controls that had received D-NMMA (5.1⫾0.6 versus
8.7⫾0.4 ␮m/s, respectively; P⬍0.05) (Figure 2A). The lymphatic fluid velocity in the control group was comparable to
that in mice without an infusion pump (8.7⫾0.4 versus
8.6⫾1.2 ␮m/s, respectively) as well as to that in humans.23
Injection rate of the fluorescent tracer into the interstitium
was not significantly different between L-NMMA–treated
animals and controls (11.3⫾1.2 versus 15.7⫾1.5 nL/min,
We performed immunohistochemistry for eNOS, iNOS, and
nNOS on tail sections, after ferritin lymphangiography to
identify the lymphatic vessels. eNOS protein was localized to
the walls of the collecting lymphatic vessels of the mouse tail
(Figure 3). There was no discernible staining of iNOS or
nNOS in the lymphatics (data not shown). Next, we repeated
the lymphatic function measurements in eNOS⫺/⫺ mice and
wild-type controls and found, consistent with the L-NMMA
treated animals, that lymphatic fluid velocity was decreased
(5.9⫾0.6 versus 8.5⫾0.7 ␮m/s, respectively; P⬍0.05), without a significant difference in injection rate (23.5⫾4.4 versus
20.7⫾3.2 nL/min, respectively; NS), or in mean lymphatic
vessel diameter (66.4⫾2.6 versus 66.7⫾1.6 ␮m, respectively;
NS). On microlymphangiography, there were no evident
morphological abnormalities in the initial lymphatics of
eNOS⫺/⫺ mice compared with wild-type mice. We then
performed lymphatic function measurements in mice that had
received 3 days of cavtratin, an eNOS inhibitor that does not
have any effect on iNOS.24 The used dose of cavtratin caused
a decrease in lymphatic fluid velocity (6.6⫾0.3 versus
8.8⫾0.2 ␮m/s, respectively; P⬍0.05) (Figure 2A), without a
significant difference in injection rate (14.9⫾0.7 versus
17.2⫾1.5 nL/min, respectively; NS) (Figure 2B), or in mean
lymphatic vessel diameter (60.7⫾2.3 versus 62.4⫾1.9 ␮m,
respectively; NS) (Figure 2C). These data delineate eNOSderived NO in the regulation of lymphatic function. With the
given dose of cavtratin, previously shown to have no effect on
blood pressure,18 lymphatic fluid velocity appeared less
decreased compared with L-NMMA–treated mice. Possibly,
the relatively large molecular size of Cavtratin prevented an
optimally effective concentration from reaching the lymphatic system. Taken together, these data show that eNOS
inhibition decreases lymphatic fluid flow.
eNOS Inhibition Does Not Affect Structure or
Function of Uncoupled Initial Lymphatics
We hypothesized that eNOS inhibition affected lymphatic
function via the collecting lymphatics. Therefore, we uncou-
Hagendoorn et al
eNOS and Microlymphatics
207
lymphatic function of the nonligated and ligated control
groups. In the ligated mice, the lymphatic fluid velocity was
significantly higher than in non-ligated mice (11.2⫾0.5
versus 8.7⫾0.4 ␮m/s, respectively; P⬍0.05) (Figure 2A). In
addition, the injection rate was increased (21.8⫾1.9 versus
15.7⫾1.5 nL/min, respectively; P⬍0.05) (Figure 2B), as was
the mean lymphatic diameter (78.1⫾2.3 versus
61.6⫾1.5 ␮m, respectively; P⬍0.05) (Figure 2C). The mean
lymphatic vessel diameter is inversely proportional to initial
lymphatic network resistance, which is thus decreased in the
ligated group. These data indicate that, in an intact microlymphatic network, the collecting lymphatics provide outflow
resistance to the initial network and regulate overall lymph
flow.
Discussion
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Figure 3. eNOS is expressed in collecting lymphatics. Crosssections through mouse tail prepared after ferritin lymphangiography. A, Functional initial lymphatic vessels (arrows) containing
ferritin are highlighted green. A collecting lymphatic vessel
(asterisk) can be identified as a larger ferritin containing structure adjacent to the tail vein (V). Scale bar⫽100 ␮m. B, eNOS is
expression (arrows) is localized to the wall of collecting lymphatics containing ferritin (asterisk). The expression pattern resembles that of the tail vein (V). Scale bar⫽15 ␮m.
pled the initial lymphatic network from the two deep, lateral
collecting lymphatics by ligating the latter near the tail-base
immediately before the experimental procedure (Figure 1).
After ligation, no significant difference in velocities was
found between L -NMMA–treated mice and controls
(10.5⫾0.6 versus 11.2⫾0.5 ␮m/s, respectively; NS) (Figure
2A). In addition, there was no significant difference in
injection rate (25.0⫾1.3 versus 21.8⫾1.9 nL/min, respectively; NS) (Figure 2B), or in mean lymphatic vessel diameter
(77.2⫾2.1 versus 78.1⫾2.3 ␮m, respectively; NS) (Figure
2C). Thus, after functionally removing the collecting lymphatics, the impairment of lymphatic fluid transport during
NOS inhibition was eliminated. These data imply that blocking eNOS-derived NO decreases lymphatic fluid velocity in
the whole microlymphatic network, but that this effect is
mediated via the collecting, and not the initial lymphatics.
Initial Lymphatic Resistance Is Decreased After
Ligation of the Collecting Lymphatics
To further examine the functional interaction between the
initial and collecting lymphatic networks, we compared the
We have shown that pharmacological blockade or genetic
deletion of eNOS decreases lymphatic fluid velocity in the
skin microlymphatic network and that this is mediated via the
collecting lymphatics. These data provide new mechanistic
and molecular insights into the regulation of microlymphatic
function.
How do the flexible initial lymphatic vessels and the
contractile collecting lymphatics act in concert to induce and
sustain overall lymph fluid flow? If lymph flow were a
passive process, ie, governed by Starling forces and driven
purely by lymph formation rate, the microlymphatic system
could be described in terms of an electrical circuit, where the
relative resistances of the initial and collecting lymphatics
determine the actual flow in both compartments.25 This
circumstance may be true in states of high lymph formation
rate, such as in our model, where the injection rate equals 10
to 20 times the baseline physiological lymph formation rate.17
The collecting lymphatics can constrict, resulting in increased
outflow resistance for the entire network (Figure 4). Removing the collecting lymphatics functionally by ligation, removes the control of lymph flow. This leads to lower
resistance in the initial lymphatics, an increased interstitiallymphatic pressure gradient, and thus to increased lymph
flow. In addition to the increased infusion rate, the amount of
fluorescent solution near the interstitial injection site appeared less in the ligated animals. This suggests that, in
conjunction with higher lymph fluid velocity and wider
lymphatic vessel diameter, overall lymph formation must be
augmented. Although establishing the exact mechanistic relationship between collecting and initial lymphatics warrants
further investigation and may be revealed by mathematical
modeling, these data represent strong evidence that collecting
lymphatics act as regulators of microlymphatic lymph flow.
What is the role of eNOS expression and NO in these
mechanic processes? Our data show that lymph flow is
decreased in the initial lymphatic network under eNOS
inhibition and in eNOS⫺/⫺ mice. Because the collecting
lymphatics are not visualized in this model, we cannot
exclude shunting of flow to the deeper network. If, however,
the collecting lymphatics are calculated to equal 35% of the
cross-sectional area of the total lymphatic network, a simple
mass balance implies that a ⬇75% increase in velocity would
be necessary in the collecting lymphatics for fully compen-
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Figure 4. Schematic representation of
the effects of eNOS blockade and ligation on the mouse tail lymphatic network.
A, Microlymphatic network consists of
hexagonal initial lymphatics and two
deep collecting lymphatic vessels. Fluorescent tracer is injected with constant
pressure in the interstitium of the distal
end of the mouse tail. B, In the physiological situation, the tracer is transported
through the initial and collecting lymphatics. The latter have a muscular wall
and intraluminal valves. C, A constricted
state of the collecting lymphatics under
eNOS inhibition increases resistance and
decreases fluid velocity in the lymphatic
network. D, Proximal ligation of the collecting lymphatics leaves the initial lymphatics as the only route for fluid flow.
Loss of control of lymph fluid transport
and decrease in total resistance, to
which lymph vessel diameter is inversely
proportional, leads to increased fluid
velocity and injection flow rate.
satory flow in the L-NMMA–treated animals. Moreover, we
did not observe a reciprocal increase in relative resistance of
the superficial network in terms of smaller mean lymphatic
vessel diameter or incomplete network staining. Our data
imply that an overall more-constricted state of the collecting
lymphatics under eNOS inhibition leads to decreased total
lymph flow. Whereas baseline NO production is needed for
active lymph propulsion via collecting lymphatic smooth
muscle contractility,7 NO may act, in states of high passive
interstitial fluid drainage, to lower overall lymphatic
resistance.
We found that eNOS is insignificant in affecting initial
lymphatic function. This is in agreement with the structural
properties and draining role of the initial lymphatics, because
these are mechanically coupled to the interstitial space, which
intrinsically regulates initial lymphatic permeability.1,2
Clinically, these findings raise the question of the role of
eNOS in lymphatic pathologies, such as edema. Whether NO
donors could be used to specifically target the collecting
lymphatics for the treatment of (noninflammatory) edema or
influencing the function of epicardial lymphatics,26 warrants
further investigation. In cancer, it has not been elucidated if
tumors, shown to exhibit an abnormal lymphatic drainage
pattern,27,28 can influence peritumor lymphatic function via
eNOS.
In conclusion, we show that mice treated with eNOS
inhibitors and eNOS⫺/⫺ mice exhibit decreased lymphatic
fluid velocity in the microlymphatic network and that this
effect can be eliminated by functionally removing the collecting lymphatics. As a mechanism for the effect of eNOS
inhibition on overall lymph flow, we show that the collecting
lymphatics respond to NO and provide outflow resistance to
the initial lymphatics.
Acknowledgments
This study was supported by the NIH (Bioengineering Research
Partnership Grant R24-CA85140). Dr Jeroen Hagendoorn is a
Fulbright Foundation fellow. The authors thank Dr Lance Munn for
assistance with illustrations; Michele Riley, Sylvie Roberge, and
Carolyn Smith for excellent technical assistance; and Prof Dr Inne
Borel Rinkes and Dr Theo van Vroonhoven (Utrecht University)
for support.
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Downloaded from http://circres.ahajournals.org/ by guest on June 15, 2017
Endothelial Nitric Oxide Synthase Regulates Microlymphatic Flow via Collecting
Lymphatics
Jeroen Hagendoorn, Timothy P. Padera, Satoshi Kashiwagi, Naohide Isaka, Fatima Noda,
Michelle I. Lin, Paul L. Huang, William C. Sessa, Dai Fukumura and Rakesh K. Jain
Circ Res. 2004;95:204-209; originally published online June 10, 2004;
doi: 10.1161/01.RES.0000135549.72828.24
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