Am J Physiol Regul Integr Comp Physiol 303: R48–R56, 2012.
First published May 9, 2012; doi:10.1152/ajpregu.00002.2012.
Growth and regression of vasculature in healthy and diabetic mice after
hindlimb ischemia
Natalia Landázuri,1 Giji Joseph,1 Robert E. Guldberg,2,3 and W. Robert Taylor1,4,5
1
Division of Cardiology, Department of Medicine, Emory University, Atlanta, Georgia; 2Woodruff School of Mechanical
Engineering Georgia Institute of Technology, Georgia Institute of Technology and Emory University, Atlanta, Georgia;
3
Institute for Bioengineering and Bioscience, Georgia Institute of Technology, Atlanta, Georgia; 4Wallace H. Coulter
Department of Biomedical Engineering, Georgia Institute of Technology and Emory University, Atlanta, Georgia;
and 5The Veterans Affairs Medical Center, Decatur, Georgia
Submitted 4 January 2012; accepted in final form 8 May 2012
perfusion; angiogenesis; arteriogenesis
THE FORMATION of the vascular network during embryogenesis
encompasses complex and tightly regulated growth of blood
vessels, followed by maturation of some vessels and disconnection and pruning of others (14, 24, 26, 27). Vascularization
of the retina in neonate mice follows a similar pattern. Pups are
born with an immature retinal vasculature. Shortly after birth,
vessels rapidly grow and give rise to a dense capillary network.
Then, in a temporally and spatially controlled manner, some
blood vessels regress while others mature and give rise to a
functional and stable vascular network (14, 36). These scenarios indicate that controlled vessel growth and rarefaction are
integral parts of vascularization during early stages of devel-
Address for reprint requests and other correspondence: W. R. Taylor, Div.
of Cardiology, Emory Univ. School of Medicine, 101 Woodruff Circle, Suite
319 WMB, Atlanta, GA 30322 (e-mail: [email protected]).
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opment. The adult vasculature, while more quiescent, is also
capable of adapting to changing physiological conditions by
remodeling blood vessels (9, 11, 29 –31). This ability can be
compromised in the presence of cardiovascular risk factors
such as diabetes (1, 20).
One of the animal models most extensively used to study
adult revascularization under physiological and pathological
conditions is hindlimb ischemia (HLI). In this model, blood
flow through the femoral artery of one leg is interrupted by
ligation and/or excision of the artery (8), leading to an endogenous response to revascularize the ischemic tissue. Researchers have been able to identify some of the factors that lead to
formation of new vessels and to growth of preexisting vessels,
namely 1) the mechanical environment, where increased shear
stress leads to collateral growth; 2) upregulation of pro-angiogenic growth factors and cytokines; and 3) mobilization of
progenitor and inflammatory cells from the bone marrow, the
peripheral circulation, or even the local tissue (5, 7, 15, 16, 23,
31, 32, 35, 37, 39). However, the extent to which vessel
rarefaction occurs after the initial phase of vessel growth
during vascular recovery in the adult is unknown. Given that
regression of newly formed or remodeled vessels can dictate
the final outcome of adult vascular repair, characterizing this
process is key to designing therapies for ischemic cardiovascular disease.
In this study, we propose to characterize the phases of vascular recovery after an ischemic insult in a mouse model of
HLI. Also, we propose to compare how a cardiovascular risk
factor may alter this process by comparing the kinetics of
recovery between control and diabetic mice. In particular, we
based our analysis on three main parameters: morphology of
the vasculature measured by microcomputed tomography (micro-CT), recovery of blood flow quantified by LASER Doppler
perfusion imaging (LDPI), and functionality of the ischemic
limb by voluntary running in a mouse activity wheel system.
METHODS
Animals. Male 129 mice were purchased from Charles River
Laboratories (Wilmington, MA), and male C57Bl6/J mice were purchased from The Jackson Laboratory (Bar Harbor, ME). The animals
were fed a standard chow diet ad libitum and had free access to water.
All protocols were approved by the Institutional Animal Care and Use
Committee of Emory University and done in accordance with the
National Institutes of Health Guide for the Care and Use of Laboratory Animals.
Hindlimb ischemia model. At 9 to 13 wk of age, the animals were
anesthetized with intraperitoneal injections of xylazine (10 mg/kg)
and ketamine (80 mg/kg). A unilateral incision was made over the left
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Landázuri N, Joseph G, Guldberg RE, Taylor WR. Growth and
regression of vasculature in healthy and diabetic mice after hindlimb
ischemia. Am J Physiol Regul Integr Comp Physiol 303: R48 –R56, 2012.
First published May 9, 2012; doi:10.1152/ajpregu.00002.2012.—The
formation of vascular networks during embryogenesis and early
stages of development encompasses complex and tightly regulated
growth of blood vessels, followed by maturation of some vessels, and
spatially controlled disconnection and pruning of others. The adult
vasculature, while more quiescent, is also capable of adapting to
changing physiological conditions by remodeling blood vessels. Numerous studies have focused on understanding key factors that drive
vessel growth in the adult in response to ischemic injury. However,
little is known about the extent of vessel rarefaction and its potential
contribution to the final outcome of vascular recovery. We addressed
this topic by characterizing the endogenous phases of vascular repair
in a mouse model of hindlimb ischemia. We showed that this process
is biphasic. It encompasses an initial rapid phase of vessel growth,
followed by a later phase of vessel rarefaction. In healthy mice, this
process resulted in partial recovery of perfusion and completely
restored the ability of mice to run voluntarily. Given that the ability to
revascularize can be compromised by a cardiovascular risk factor such
as diabetes, we also examined vascular repair in diabetic mice. We
found that paradoxically both the initial growth and subsequent
regression of collateral vessels were more pronounced in the setting of
diabetes and resulted in impaired recovery of perfusion and impaired
functional status. In conclusion, our findings demonstrate that the
formation of functional collateral vessels in the hindlimb requires
vessel growth and subsequent vessel rarefaction. In the setting of
diabetes, the physiological defect was not in the initial formation of
vessels but rather in the inability to sustain newly formed vessels.
BIPHASIC VASCULAR RECOVERY AFTER HINDLIMB ISCHEMIA
as ratios of surgery to control leg for each animal to account for
potential changes in vessel volume after injection of microfil.
Measurements of perfusion by LASER doppler perfusion imaging.
LDPI with a LASER of 810 nm (MoorLDI, Moor Instruments,
Wilmington, DE) was used to evaluate the perfusion in the ischemic
and nonischemic legs after surgery. The distance between the source
of the laser and the leg was 21 cm, the scan speed was 4 ms/pixel, and
the resolution was 256 ⫻ 256 pixels. Mean perfusion was estimated
in the foot and in the ischemic portion of the leg, extending from the
thigh to the ankle. The nonischemic legs and feet were used as
controls. The results for mean perfusion were reported as ratios of the
surgery to nonsurgery leg or foot for each animal.
Voluntary running wheel activity system. Immediately after HLI,
animals were individually housed in cages with a single activity wheel
(Lafayette Instrument, Lafayette, IN). The animals had free access to
an activity wheel. The distance run in the wheels was recorded daily.
Quantification of microvasculature by lectin staining. Eight and
twenty three days after HLI, paraffin sections of hindlimb tissue were
stained with FITC fluorescein-labeled GSL I-isolectin B4 (Vector
Laboratories, Burlingame, CA). Sections were imaged by fluorescent
microscopy using a ⫻20 objective. The number of microvessels from
five animals per group and four fields of view per hindlimb was
counted. The results for density of microvessels were reported as
ratios of the surgery to nonsurgery limb.
Data analysis. The data were summarized as means ⫾ SE. Statistical analysis was performed using Prism software Version 4.01. A
t-test was used to compare the means between two groups. A two-way
analysis of variance (ANOVA) was used to compare measurements of
various parameters over time. The Bonferroni comparison test was
used to conduct pairwise comparisons between means. To compare
the microvasculature density, a general linear model using day,
diabetic versus nondiabetic and surgery leg versus nonsurgery leg as
factor variables was used. The least square means was calculated and
Fig. 1. Morphological assessment of vascular
development after hindlimb ischemia surgery
in 129 mice. Representative images of microcomputed tomography (micro-CT) scans of
the surgery leg at various days after surgery,
and of the control leg immediately after surgery. Images of the vasculature in the control
leg were similar over time.
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medial thigh of the mouse. The superficial femoral artery and vein
were ligated proximal to the caudally branching deep femoral artery
and proximal to the branching of the tibial arteries. The portion of the
artery and vein between the ligation points was excised. The skin was
closed with interrupted silk sutures.
Mouse model of diabetes. Eight-week-old C57Bl6/J mice received
a daily intraperitoneal injection of 60 mg/kg of streptozotocin for 5
consecutive days after overnight fasting. Glucose levels were measured 3 to 4 wk after induction of diabetes (immediately before
hindlimb ischemia) and every other week thereafter. Mice with
glucose levels above 300 mg/dl were considered diabetic.
Measurement of morphological parameters by micro-CT. After the
animals were euthanized, the thoracic cavity was opened and the
inferior vena cava was severed. The vasculature was flushed with
0.9% normal saline containing 4 mg/ml papaverine at a pressure of
⬇100 mmHg via a needle inserted into the left ventricle.The excess
papaverine was flushed with saline and the vasculature fixed with 10%
neutral buffered Formalin. Formalin was flushed from the vessels
using saline. The vasculature was injected with undiluted Microfil
(MV-122, Flow Tech; Carver, MA) as previously described (12).
Samples were stored at 4°C overnight for contrast agent polymerization. Mouse legs (not including the feet) were dissected from the
specimens and soaked in 10% neutral buffered Formalin to ensure
complete tissue fixation. Tissues were subsequently treated for 48 h in
Cal Ex II (Fisher Scientific; Pittsburgh, PA) and then stored in 10%
neutral buffered Formalin. The hindlimb vasculature was imaged with
a micro-CT imaging system (␮CT 40, Scanco Medical; Bassersdorf,
Switzerland). The scanner was set to a voltage of 55 kVp and a current
of 145 ␮A. Resolution was set to medium, and the limbs were scanned
at a 30-␮m voxel size. A threshold of 110 was chosen based on visual
interpretation of thresholded two-dimensional tomograms. Surgery
and control legs were evaluated individually to quantify the threedimensional histomorphometric values vessel volume, connectivity,
number, thickness distribution, and spacing. The results were reported
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BIPHASIC VASCULAR RECOVERY AFTER HINDLIMB ISCHEMIA
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Fig. 2. Quantification of morphological parameters based on microCT scans of the surgery leg () and the nonsurgery leg (}) in 129 mice. For each parameter,
a ratio between the surgery and the nonsurgery leg was calculated (Œ). The dotted lines indicate a ratio of 1. The quantified parameters were the following:
vascular volume normalized for tissue volume (A), density (B), connectivity (C), spacing (D). Each point represents the mean ⫹ SE, n ⫽ 6 to 8.
AJP-Regul Integr Comp Physiol • doi:10.1152/ajpregu.00002.2012 • www.ajpregu.org
BIPHASIC VASCULAR RECOVERY AFTER HINDLIMB ISCHEMIA
the significance of the interactions was assessed. Differences at P ⬍
0.5 were considered statistically significant.
RESULTS
vessels of diameter larger than 420 ␮m remained lower in
the surgery at all time points (Fig. 3, C and D).
Taken together, our results showed that vascular volume,
density, and connectivity in the surgery leg exceeded or were
equal to these same parameters in the nonsurgery leg. Interestingly, we noticed that interconnected collaterals extensively
developed for 1 wk after surgery but were gradually reduced at
later time points.
To assess the extent to which each of these two phases of
collateral remodeling, namely, growth and rarefaction, contributed to recovery of perfusion, we scanned both limbs by LDPI.
We first focused on the area we analyzed by micro-CT; that is,
the region extending from the ankle to the thigh. We observed
that perfusion increased until day 7 post-HLI and then reached
a plateau at a ratio between 0.5 and 0.6 (Fig. 4, A and C). This
indicated that perfusion to the surgery leg increased during the
phase of vascular growth but remained unchanged during the
phase of vascular pruning.
While most studies quantify the number of vessels in the
areas we used for micro-CT analysis (thigh or calf), LDPI is
usually targeted to the feet. When measuring the perfusion
ratio between the feet, we found that it increased linearly with
time for 3 wk and then reached a plateau at a ratio of 0.5 on day
21 (Fig. 4, B and C), thus demonstrating that distal perfusion
continued to increase during growth and pruning of blood
vessels in the proximal area.
Finally, to evaluate the extent to which vascular remodeling
and recovery of perfusion related to functional recovery of
Fig. 3. Thickness distribution of developing blood vessels after hindlimb ischemia (HLI) surgery in 129 mice. Based on micro-CT scans, the thickness distribution
of blood vessels was assessed at various days after HLI in the surgery leg (A) and the control leg (B). The vascular volume including only vessels of a given
diameter (C, D) was calculated as a ratio of surgery leg to the nonsurgery leg. Each point represents the mean ⫹ SE, n ⫽ 6 to 8.
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To obtain a morphological assessment of the time course of
collateral development after induction of HLI, at various days
after surgery we prepared the legs from 129 mice for scanning
by microCT (Fig. 1). We assessed the following quantitative
parameters provided by these scans: vascular volume normalized for tissue volume, connectivity, mean vessel density (i.e.,
number), spacing, and vessel thickness distribution. Vascular
volume, density, and connectivity were lower in the surgery leg
than in the nonsurgery leg immediately after surgery. At day 7,
their values peaked and exceeded those in the nonsurgery leg.
The ratios of surgery to control leg reached 1.25 ⫾ 0.08 for
volume, 1.13 ⫾ 0.02 for density, and 2 ⫾ 0.34 for connectivity,
and then slowly declined over several weeks. Forty days
post-HLI, the ratios for density and volume were 1, whereas
the ratio for connectivity was 1.5 (Fig. 2, A–C). Consistent
with these findings, we observed that vascular spacing
decreased in the surgery leg over 7 days postsurgery and
then increased slowly (Fig. 2D). While analyzing the distribution of vessels of various thicknesses as a function of
time, we observed that vessels of small diameter substantially developed only in the surgery leg (Fig. 3, A–D). The
volume occupied by vessels of diameter smaller than 330
␮m was up to 1.5 times higher in the surgery leg compared
with the control leg. In contrast, the volume occupied by
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BIPHASIC VASCULAR RECOVERY AFTER HINDLIMB ISCHEMIA
limb usage, we placed the animals in a voluntary running
wheel activity system the day of HLI and recorded the distance
mice run on a daily basis. We found that within 4 wk after HLI,
mice could run the same distance as control mice (Fig. 4D).
To determine whether these findings could be generalized to
mouse strains other than 129, we conducted a similar experiment using C57Bl6/J mice. In addition, to assess how a
cardiovascular risk factor would alter vessel remodeling and
recovery of perfusion, we also used diabetic mice on a
C57Bl6/J background. Our micro-CT results indicated that the
biphasic pattern of vascular remodeling in control and diabetic
mice was similar to that observed in 129 mice. The volume of
blood vessels in the surgery leg rapidly increased during the
first week after HLI, reached values higher than those in the
control leg, and then decreased (Fig. 5A). Interestingly, we
observed that diabetic mice demonstrated a robust increase in
parameters vascularity after HLI. We found that the vascular
volume of size-delineated vessels and microvessel density
were higher at day 8 in the diabetic mice (Fig. 5, B–F).
However, we also observed a striking reduction in vascular
volume and microvessel density in diabetic mice at 23 days
post-HLI, suggesting that in the diabetic mice, the newly
formed vasculature was not sustained (Fig. 5, A–F). Taken
together, these data indicated that small vessels in diabetics
paradoxically underwent more pronounced growth at early
time points after induction of ischemia and then subsequently
did not sustain this higher level of vascularization with resultant pruning of the neovasculature.
To investigate how these differences in vascular remodeling
affected recovery of perfusion, we scanned the feet of the mice
at various times after HLI. The perfusion ratio was equivalent
between control and diabetic mice up to 14 days post-HLI, but
control mice demonstrated significantly improved recovery
over the diabetic mice beginning at day 23 post-HLI (Fig. 6, A
and B). These findings suggested that excessive pruning of
vessels hindered distal recovery of perfusion.
Finally, we assessed the distance that mice run on a daily
basis after HLI. The distance that control mice run increased
over time and reached a plateau level 9 to 11 days post-HLI,
when the distance run by mice with and without HLI was
similar (Fig. 6C). In contrast, the ability of diabetic mice to run
did not improve over the same period of time (Fig. 6D).
DISCUSSION
In the present study, we showed that the endogenous process
of vascular repair after an ischemic insult in the adult mouse is
biphasic. It encompasses an initial phase of vessel growth
followed by a later phase of vessel rarefaction. In healthy 129
and C57Bl6/J mice, this process resulted in partial recovery of
perfusion and completely restored the ability of mice to run
voluntarily. In diabetic mice, these two phases were more
pronounced than in healthy mice, with a paradoxical exaggerated increase in vascularity at early time points followed by an
enhanced vascular rarefaction at later time points. These discrepant responses resulted in impaired recovery of perfusion,
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Fig. 4. Measurement of recovery in 129 mice based on perfusion and ability to run. The perfusion ratio between the surgery and the control limb was calculated
selecting only the feet (A) or the portion of the legs extending from the hip to the ankle (B) (n ⫽ 10). Representative LASER Doppler perfusion imaging (LDPI)
images at days 4, 21, and 42 after HLI (C). Mice with or without HLI were placed in a voluntary running wheel activity system. The distance run after HLI was
recorded on a daily basis (D) (n ⫽ 6). Each point represents the mean ⫹ SE.
BIPHASIC VASCULAR RECOVERY AFTER HINDLIMB ISCHEMIA
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as measured by LDPI, and impaired functionality of the ischemic limb, as measured by running distances.
The initial phase of rapid vessel growth started shortly after
induction of HLI. Interconnected blood vessels developed by
day 7. Total vascular volume, mean density, and mean connectivity in the surgery leg peaked and exceeded the values
from the control leg. This phase appears to temporally coincide
with the upregulation of angiogenic and arteriogenic key factors reported in the literature and with the mobilization of cells
to the site of injury (8, 19, 28, 33).
During the second phase, which lasted several weeks, vessels gradually regressed. By day 40 postsurgery, vascular
volume and density in the surgery leg had decreased and were
similar in both legs. Connectivity still remained higher in the
surgery limb but maintained a decreasing trend. This general
process recapitulates other scenarios of vascular development,
including early postnatal adaptations to the environment and
the control of arterial branching in embryogenesis. Postnatal
vessel maturation and rarefaction are tightly regulated by
signaling molecules that participate in both arteriogenesis and
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Fig. 5. Quantification of morphological parameters of the vasculature in control and diabetic C57Bl6/J mice. At various days after HLI, the total vascular volume
(normalized for tissue volume) was measured based on micro-CT scans, and the density of microvessels (⬍30 ␮m) was quantified by histology. The vascular
volume ratio between the surgery and the nonsurgery leg was calculated based on micro-CT parameters (A). The vascular volume including only vessels of a
given diameter was calculated as a ratio of surgery leg to the nonsurgery leg (B--E). The dotted lines indicate a ratio of 1. *Statistically significant differences
between control and diabetic mice (P ⬍ 0.05), n ⫽ 5– 8. The density of microvessels was calculated as the ratio of the surgery leg to the nonsurgery leg (F).
*Statistically significant differences between surgery and nonsurgery legs (P ⬍ 0.05). Each point represents the means ⫹ SE.
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BIPHASIC VASCULAR RECOVERY AFTER HINDLIMB ISCHEMIA
angiogenesis. For example, upregulation of vascular endothelial growth factor sustains immature vessels and facilitates
vessel maturation, whereas downregulation of vascular endothelial growth factor results in the selective pruning of immature vessels that have not yet acquired a periendothelial coating
(3, 4). This type of signaling to prune vessels responds to the
surrounding environment, in particular, to the metabolic requirements of the tissue. For example, infants treated with
hyperoxia can suffer from retinopathy of prematurity, because
their retinal vessels regress to match lower oxygen requirements (2, 4, 22). Another factor that determines the fate of
vessels is hemodynamics. In fact, high shear stress prevents
apoptosis of endothelial cells, thus inhibiting regression of
highly perfused vessels (10). Also, during embryogenesis, the
flow velocity difference between a branch and the main artery,
as well as the branching angle, appear to regulate the rate of
disconnection of side branches. This disconnection ultimately
permits progression of blood flow into more distal areas (27).
In our model, growth of vessels during the first week
postsurgery coincided with an increase in perfusion. Perfusion
continued to increase for an additional week and then remained
unchanged while vessels regressed. This suggests that pruning
was selective for inefficient or nonfunctional vessels. As recognized by others, rarefaction of the unnecessary vasculature
can decrease the tortuosity of the vessels, as well as the
resistance to blood flow, thereby enhancing perfusion (32, 34).
This biphasic behavior affected vessels of all sizes, yet the
volume occupied by vessels of diameter smaller than 330 ␮m
remained higher in the surgery leg than in the control leg, while
the volume of vessels larger than 420 ␮m remained higher in
the control leg (Fig. 3). Most likely, the transient thickening of
large, mature vessels increased the blood flow to the small
branching collaterals, thus temporarily stimulating them to
remodel and grow. Of importance, we observed this biphasic
behavior in two strains of mice that have been shown to differ
in their response to ischemia, namely 129 and C57Bl6/J (18),
suggesting that growth and regression of the vasculature could
be generalized to various genetic backgrounds.
In healthy mice, this endogenous vascular repair process
resulted in only 50% to 60% recovery in perfusion. The
persistent alteration of vascular morphology, with increased
distribution of vascular volume to smaller vessels, may help
explain the continued perfusion deficit. In addition, we should
note that femoral artery occlusion can lead to partial atrophy of
the affected lower leg (6), thereby decreasing its blood flow
requirements compared with the control leg. Interestingly,
mice completely recovered their ability to run voluntarily,
8,000 to 10,000 meters per day, when recovery of perfusion
was around 25% for C57Bl6/J mice and 55% for 129 mice.
This indicates that incomplete perfusion fulfills the metabolic
requirements for intense physical activity.
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Fig. 6. Recovery of perfusion and functional use of hindlimbs in nondiabetic and diabetic C57Bl6/J mice. Representative images obtained by LDPI 3 wk after
HLI (A). Perfusion ratio between the surgery and the control foot was measured at various time points after HLI (B). *Statistically significant difference between
nondiabetic and diabetic mice (P ⬍ 0.05). Mice with or without HLI were placed in a voluntary running wheel activity system. The distance run after HLI was
recorded on a daily basis (C, D). *Statistically significant differences between animals with HLI and animals without HLI (P ⬍ 0.05). Each point represents the
mean ⫹ SE, n ⫽ 6.
BIPHASIC VASCULAR RECOVERY AFTER HINDLIMB ISCHEMIA
Perspectives and Significance
The findings from this study emphasize the contribution of
vessel rarefaction to vascular repair in the adult. Our results
also point to the prospect that poor revascularization in the
presence of a cardiovascular risk factor, such as diabetes, can
be in part due to excessive vessel regression. Most current
preclinical therapies for vascular repair consist of delivering a
therapeutic agent shortly after HLI and have had limited
success (13, 15, 21, 25, 32–34, 38). Also, some of these
therapies have led to an initial acceleration of revascularization
but not to a long-term benefit (17). Thus our results raise the
possibility that the disparity observed between promising preclinical angiogenic therapies and clinical outcomes in humans
may be related to the point in time when results are evaluated.
Moreover, our findings highlight the importance of designing
therapies to sustain arteriogenesis, as opposed to angiogenesis
alone.
Based on our observations, we propose that therapies should
not only focus on the early stage of vessel growth but also on
the second stage of vessel rarefaction.
ACKNOWLEDGMENTS
This work was supported by National Heart, Lung, and Blood Institute
Grants PO1 HL-095070 and RO1HL-062820.
DISCLOSURES
No conflicts of interest, financial or otherwise, are declared by the author(s).
AUTHOR CONTRIBUTIONS
Author contributions: N.L., R.E.G., and W.R.T. conception and design of
research; N.L. and G.J. performed experiments; N.L. analyzed data; N.L. and
W.R.T. interpreted results of experiments; N.L. prepared figures; N.L. drafted
manuscript; N.L. and W.R.T. edited and revised manuscript; N.L., R.E.G., and
W.R.T. approved final version of manuscript.
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