2006 The Journal of Clinical Investigation http://www.jci.org Volume 122 Number 6 June 2012
Blood flow reprograms lymphatic vessels
to blood vessels
Chiu-Yu Chen,1 Cara Bertozzi,1 Zhiying Zou,1 Lijun Yuan,1 John S. Lee,1 MinMin Lu,1
Stan J. Stachelek,2 Sathish Srinivasan,3 Lili Guo,1 Andres Vincente,4 Patricia Mericko,1
Robert J. Levy,2 Taija Makinen,4 Guillermo Oliver,3 and Mark L. Kahn1
1Department of Medicine and Division of Cardiology, University of Pennsylvania, Philadelphia, Pennsylvania, USA. 2Department of Cardiology,
The Children’s Hospital of Philadelphia, Philadelphia, Pennsylvania, USA. 3Department of Genetics, St. Jude Children’s Research Hospital,
Memphis, Tennessee, USA. 4Lymphatic Development Laboratory, Cancer Research UK, London, United Kingdom.
Human vascular malformations cause disease as a result of changes in blood flow and vascular hemodynamic
forces. Although the genetic mutations that underlie the formation of many human vascular malformations
are known, the extent to which abnormal blood flow can subsequently influence the vascular genetic program
and natural history is not. Loss of the SH2 domain–containing leukocyte protein of 76 kDa (SLP76) resulted
in a vascular malformation that directed blood flow through mesenteric lymphatic vessels after birth in mice.
Mesenteric vessels in the position of the congenital lymphatic in mature Slp76-null mice lacked lymphatic iden-
tity and expressed a marker of blood vessel identity. Genetic lineage tracing demonstrated that this change in
vessel identity was the result of lymphatic endothelial cell reprogramming rather than replacement by blood
endothelial cells. Exposure of lymphatic vessels to blood in the absence of significant flow did not alter ves-
sel identity in vivo, but lymphatic endothelial cells exposed to similar levels of shear stress ex vivo rapidly
lost expression of PROX1, a lymphatic fate–specifying transcription factor. These findings reveal that blood
flow can convert lymphatic vessels to blood vessels, demonstrating that hemodynamic forces may reprogram
endothelial and vessel identity in cardiovascular diseases associated with abnormal flow.
Human vascular malformations are common congenital dis-
eases that can result in a variety of clinical disorders later in life.
Patients may present with stroke and neurologic impairment
from lesions in the central nervous system (1), high-output heart
failure from arterio-venous shunting through large malforma-
tions (2), and disfigurement due to large or numerous cutaneous
lesions (3). In the past two decades, significant progress has been
made in identifying the genetic basis for many common human
vascular malformations (4). These studies have revealed defects
in many of the pathways known to regulate endothelial and ves-
sel identity (e.g., NOTCH signaling in Alagille syndrome, ref. 5;
TGF-β signaling in hereditary hemorrhagic telangiectasia, ref. 6)
and function (e.g., endothelial junction formation in cerebral
cavernous malformation, ref. 7), and some human vascular mal-
formations have been successfully modeled in genetically altered
mice deficient in the same pathways (8, 9).
Despite progress in determining the genetic origins of human
vascular malformation, the treatment of these lesions remains
primarily mechanical and is limited by a lack of molecular under-
standing of the natural history of vascular malformations. A hall-
mark of human vascular malformations, especially those with
significant clinical consequences, is the shunting of blood away
from natural, hierarchical vascular circuits consisting of arteries,
arterioles, capillaries, venules, and veins and into abnormal cir-
cuits that lack the organization and mechanisms normally used
to control blood flow and hemodynamic forces. In the case of
large vascular malformations, such shunts frequently result in
hemorrhage or high-output heart failure many years after they
first form. Understanding whether and to what extent hemody-
namic forces shape the molecular and genetic landscape of these
vascular structures would provide much needed insight into how
the pathologic syndromes associated with human vascular mal-
formations arise and may be treated.
Mice lacking the SLP76 adaptor protein experience abnormal
lymphatic vascular development, in which blood-lymphatic vas-
cular connections form during embryonic life (10, 11). A fraction
of such mice survive to adulthood, at which time they exhibit
large arterio-venous malformations that form as a consequence
of these connections (10). In contrast to experimental studies of
hemodynamic effects on vessel identity and function that utilize
surgical interventions, alterations in blood flow in Slp76–/– animals
arise gradually as a consequence of vascular remodeling in much
the same way as in human vascular malformations. In the present
study, we determined the effect of blood flow and hemodynamic
forces on the identity of the lymphatic vessels that constitute part
of the efferent loop of the vascular malformation in Slp76–/– ani-
mals. Our studies demonstrate that lymphatic endothelial and
vessel identity are negatively regulated by blood flow and that
changes in hemodynamic forces can completely reprogram vessel
identity in postnatal life. These findings suggest that the genetic
basis for human vascular malformations is only the first half of a
story that is also written by the molecular and genetic responses
to hemodynamic forces.
SLP76-deficient mice undergo late separation of the blood and lymphatic
circulation. The hematopoietic signaling proteins SLP76 and SYK
have been shown to regulate blood and lymphatic vascular sepa-
Authorship note: Chiu-Yu Chen, Cara Bertozzi, and Zhiying Zou contributed equally
to this work.
Conflict of interest: The authors have declared that no conflict of interest exists.
Citation for this article: J Clin Invest. 2012;122(6):2006–2017. doi:10.1172/JCI57513.
The Journal of Clinical Investigation http://www.jci.org Volume 122 Number 6 June 2012
ration during development (10). Animals lacking these proteins
develop mixing of blood and lymphatic circulation in the skin
and intestine, defects that result in embryonic edema and neo-
natal chylous ascites (10, 12, 13). Despite these defects, approxi-
mately one-third of SLP76-deficient mice survive to adulthood,
when they no longer appear edematous but instead exhibit a high-
cardiac-output state due to the presence of an intestinal vascular
shunt (discussed below and in ref. 10). These findings suggested
that the blood-lymphatic connections that form in SLP76-defi-
cient animals might be transient and that vascular remodeling
might result in both the resolution of blood-lymphatic mixing
and formation of the vascular shunt. To better understand the
natural history of the vascular defects in these animals, we ana-
lyzed the blood and lymphatic vessels in the skin and intestine of
deficient animals and wild-type littermates at various time points
during development and postnatal life.
Between E12 and E15, lymphatic vessels invade the skin as they
grow by angiogenic sprouting from the central lymph sacs adjacent
to the cardinal veins (14, 15). At this time point, Slp76–/– embryos
were identifiable by marked cutaneous edema and the presence of
blood-filled cutaneous lymphatics (Figure 1A). By E18, however,
Slp76–/– animals exhibited normal abdominal skin folds, indicating
resolution of the edema, and blood-filled lymphatics appeared less
visible or completely resolved (Figure 1A). Resolution of blood-filled
lymphatics in the skin of late-gestation Slp76–/– animals was also
detectable histologically by the absence of red blood cells in cutane-
ous LYVE1+ lymphatic vessels (Figure 1B). A similar rapid resolu-
tion of the cutaneous vascular phenotype has also been reported for
SYK-deficient embryos between E16.5 and E18.5, although the basis
for this observation was not investigated in detail (12). Lymphatic
vessels first appear in the mouse intestine after E15, a time point
considerably later than their appearance in the skin (16). Signifi-
cantly, in SLP76-deficient mice, blood-filled intestinal lymphatics
and intestinal edema were observed after E18, a time point at which
the skin phenotype was noted to have resolved (Figure 1B).
These findings suggested that blood-lymphatic connections in
SLP76-deficient mice are transient and resolve at one site even as
they form at another site in the same animal. To map the formation
and resolution of connections between the blood and lymphatic
circulations of these animals, we injected biotin-conjugated lectin
into the blood to label endothelial cells in contact with circulat-
ing blood. Subsequent staining with FITC-streptavidin and anti-
LYVE1 antibodies was used to functionally identify lymphatic
endothelium exposed to circulating blood. LYVE1– blood vessels
but not LYVE1+ lymphatic vessels in the skin, intestine, and mesen-
tery of wild-type neonates were positive for lectin staining (Figure
1C), confirming that circulating blood does not normally come
into contact with lymphatic vessels. In contrast, in neonatal SLP76-
deficient animals, the LYVE1+ lymphatic vessels of the intestinal
wall and mesentery were positive for lectin staining even though
LYVE1+ lymphatic vessels in the skin were lectin negative (Figure
1C). Thus, blood-lymphatic mixing was present in the intestine but
not the skin of neonatal SLP76-deficient mice. When blood-lym-
phatic mixing was studied using this approach in mature, 12-week-
old littermates, however, the LYVE1+ intestinal vessels of SLP76-
deficient mice were no longer lectin+ (Figure 1D). These findings
demonstrate that blood-lymphatic connections in SLP76-deficient
mice ultimately resolve and suggest that the intestinal shunts pre-
viously described in mature animals form as a consequence of the
vascular remodeling that resolves those connections.
Mesenteric vessels in the anatomic position of lymphatics lack lymphatic
identity after exposure to blood flow in Slp76–/– mice. Mature SLP76-
deficient mice develop a high-cardiac-output state due to the pres-
ence of intestinal shunts in which afferent arterial blood is directly
returned to the heart through efferent mesenteric vessels, bypass-
ing the intestinal microvasculature (10). Previous angiographic
studies have shown that the efferent shunt vessels (SVs) corre-
spond anatomically to the mesenteric veins and collecting lym-
phatic vessels (10), suggesting that mesenteric lymphatics become
annexed to the blood vascular system during the remodeling pro-
cess that achieves vascular separation in the intestine. The results
of the studies of blood-lymphatic mixing shown in Figure 1 were
notable for indicating an absence of large LYVE1+ lymphatic ves-
sels in the mesentery of mature SLP76-deficient mice, even though
these animals had characteristic mesenteric vascular bundles con-
taining a thick-walled artery and two thin-walled vessels consistent
with the congenital vein and lymphatic vessels (Figure 1D). This
finding suggested that the mesenteric lymphatic vessels exposed
to flowing blood due to blood-lymphatic vascular mixing in the
intestine of SLP76-deficient animals lose lymphatic identity.
To better define the identity of the endothelial cells lining the effer-
ent SVs, we examined expression of prospero homeobox 1 (PROX1),
a transcription factor that specifies and maintains lymphatic
endothelial identity (17, 18); podoplanin (PDPN), a glycoprotein
expressed by lymphatic but not blood endothelial cells (19, 20); the
lymphatic marker LYVE1 (21); and vWF, a procoagulant protein syn-
thesized by blood but not by lymphatic endothelial cells (LECs) (22,
23); in addition to the pan-endothelial protein PECAM. Mesentery
from wild-type and SLP76-deficient neonates and wild-type 12-week-
old animals contained thick-walled, PECAM1+vWF+PROX1–
LYVE1– arteries, thin-walled PECAM1+vWF+PROX1–LYVE1– veins,
and thin-walled PECAM1+PROX1+PDPN+LYVE1+vWF– lym-
phatic vessels (Figure 2, A and B). At age 12 weeks, the artery and
vein were visible as two adjacent blood-filled vessels in the mes-
entery of wild-type mature animals. In contrast, the mesentery
of 12-week-old SLP76-deficient mice contained a third blood-
containing vessel in the anatomic position of the congenital lym-
phatic that participates in the intestinal shunt (Figure 2B and see
below; and ref. 10). These vascular bundles contained thick-walled,
PECAM1+vWF+PROX1–PDPN–LYVE1– arteries and large, thin-
walled PECAM1+vWF+PROX1–PDPN–LYVE1– SVs (Figure 2B).
Previous studies have shown that SLP76 is required in platelets
to prevent the formation of blood-lymphatic connections in the
intestine (10) and that these connections send blood flow through
mesenteric collecting lymphatic vessels that ultimately carry effer-
ent blood from a vascular shunt that forms in the intestine proper
(11). To test whether the observed loss of lymphatic mesenteric col-
lecting vessels in SLP76-deficient animals could result from loss of
SLP76 in endothelial cells themselves, we next assessed the natural
history of these vessels in Vav-Cre;Slp76fl/– mice. Previous lineage
tracing experiments performed using both Vav-Cre;R26RYFP and
Vav-Cre;R26RYFP;Slp76fl/– mice have revealed high-level Cre-medi-
ated recombination in hematopoietic cells, with no detectable
recombination in either blood or LECs of the intestine or else-
where (11, 24). Mature Vav-Cre;Slp76fl/– mice developed vascular
shunts identical to those described in SLP76-deficient animals,
and these vessels lacked lymphatic identity (Figure 3A). Thus, the
loss of endothelial and vessel identity in mesenteric collecting
lymphatics is a secondary event and not due to endothelial cell–
autonomous loss of SLP76 signaling.
2008 The Journal of Clinical Investigation http://www.jci.org Volume 122 Number 6 June 2012
Finally, to determine whether the SVs that arise in Slp76–/–
and Vav-Cre;Slp76fl/– mice acquire arterial or venous identity,
we stained for EPHB4, a venous marker, and CX40, an arterial
marker. SV endothelium was EPHB4+ and CX40–, while that
of mesenteric arteries was EPHB4– and CX40+, a result consis-
tent with venous identity (Figure 3A). These findings suggested
that the mesenteric lymphatics of SLP76-deficient animals lose
lymphatic vessel identity and acquire venous identity as they
Late separation of the blood and lymphatic circulations in SLP76-deficient mice. (A) Cutaneous edema resolves in Slp76–/– embryos between E15
and E18. Arrows indicate sites of raised skin indicative of edema at E15 (top) and the presence of normal skin folds indicating a lack of edema at
E18 (bottom). (B) Blood-filled lymphatics resolve in the skin of Slp76–/– embryos at the same time that they appear in the intestine. LYVE1 stain-
ing (brown) identifies lymphatic vessels in the skin and intestine of E15 and neonatal Slp76–/– animals. Arrows indicate blood-filled lymphatics in
E15 skin and in neonatal intestine. (C) Functional identification of blood-filled lymphatics in neonatal Slp76–/– animals using intravenous injection
of biotinylated lectin. Blood-perfused vessels are identified by FITC-streptavidin binding to biotin-lectin after intravenous injection (green), and
lymphatic vessels are visualized by LYVE1 immunostaining (red). Co-stained vessels are present in the intestine and mesentery but not skin of
Slp76–/– neonates (arrows). (D) Late resolution of mesenteric and intestinal blood-lymphatic mixing in Slp76–/– mice demonstrated using staining
for injected biotin-lectin and LYVE1. Scale bars: 50 μm.
The Journal of Clinical Investigation http://www.jci.org Volume 122 Number 6 June 2012
become exposed to blood flow from blood-lymphatic connec-
tions in the intestine (Figure 3B).
Prox1CreERT2 lineage tracing reveals that LECs give rise to venous endo-
thelial cells in the mesenteric vessels of Slp76–/– mice. The hypothesis
that mesenteric lymphatic vessels in SLP76-deficient mice alter
identity in response to blood flow is based on their characteris-
tic anatomical position within the mesenteric vascular bundle.
It is possible, however, that SVs do not derive from the congeni-
tal lymphatic due to a complex remodeling process. In addition,
even if correctly identified, the endothelial cells lining this vessel
Mesenteric SVs in adult Slp76–/– mice lose lymphatic identity. (A) Neonatal mesenteric lymphatics in Slp76–/– mice express the lymphatic
endothelial molecular markers LYVE1 and PROX1, but not the blood endothelial marker vWF. Shown is antibody staining of serial sections. (B)
The mesenteric SVs in adult Slp76–/– mice express PECAM but not LYVE1, PDPN, or PROX1. A, artery; V, vein; L, lymphatic. Scale bars: 20 μm.
2010 The Journal of Clinical Investigation http://www.jci.org Volume 122 Number 6 June 2012
may have been molecularly reprogrammed or replaced to confer
blood vessel identity. To determine whether LECs give rise to the
blood endothelial cells that line efferent SVs, we bred SLP76-
deficient mice onto a Prox1CreERT2;Rosa26RYFP background in
which tamoxifen induction of CRE activity in Prox1-expressing
cells activates permanent expression of YFP in LECs (15). Slp76–/–;
Prox1CreERT2;Rosa26RYFP animals and control littermates were
treated with tamoxifen from P1 to P5, a time point prior to intesti-
nal vascular separation and the appearance of the intestinal shunt,
and YFP was detected by immunohistochemistry at 12 weeks of
age. Neonatal tamoxifen induction conferred mosaic YFP expres-
sion in the endothelial cells of lymphatic but not blood vessels
in the mesentery of 12 week-old Slp76+/–;Prox1CreERT2;Rosa26RYFP
control animals (Figure 4A). YFP+ endothelial cells were identified
in the large mesenteric SVs that lack lymphatic identity in Slp76–/–;
Prox1CreERT2;Rosa26RYFP littermates (Figure 4B). YFP+ endothelial
cells in these vessels were negative for PROX1, PDPN, and LYVE1
but expressed vWF (Figure 4B). These results suggested that
Mesenteric SVs in adult Vav-Cre;Slp76fl/– mice lose lymphatic identity and acquire venous identity. (A) Mesenteric SVs that form in Vav-Cre;Slp76fl/–
mice lose expression of lymphatic molecular markers and acquire expression of the blood vessel marker vWF and venous marker EPHB4 but not
the arterial marker CX40. Scale bars: 50 μm. (B) Model of vascular remodeling in SLP76-deficient mice. Shown are the vascular anatomy and
flow through the intestinal and mesenteric vessels of neonatal wild-type, neonatal Slp76–/– (KO neonate), and mature Slp76–/– (KO adult) animals.
SLP76-deficient radiation chimeras (KO chimera) develop a vascular phenotype like that observed in KO neonates. In the wild-type animal, affer-
ent mesenteric blood flow is carried by the mesenteric artery (A, red), while efferent blood and lymph are carried by the mesenteric vein (V, blue)
and lymphatic (L, green), respectively. In the KO neonate or KO chimera, blood-lymphatic mixing allows blood to enter the mesenteric lymphatics,
but flow is minimal, and lymphatic identity is preserved. In the KO adult, mesenteric lymphatics become incorporated into an arterio-venous shunt
that directs efferent blood flow through them (right), a process that produces an SV (blue) with blood vessel identity. Histologic studies reveal the
presence of small lymphatic vessels that retain lymphatic identity in mature KO animals.
The Journal of Clinical Investigation http://www.jci.org Volume 122 Number 6 June 2012
LECs were reprogrammed from blood endothelial cells in vessels
exposed to flowing blood, but only a small number of such cells
could be identified using this Prox1CreERT2 allele.
The small number of YFP+ endothelial cells detected in the
mesenteric SVs of Slp76–/–;Prox1CreERT2;Rosa26RYFP could indicate
that most LECs are simply replaced by blood endothelial cells, or
it could reflect inefficient labeling due to reduced levels of Prox1
expression in postnatal animals compared with embryos (25) and/
or low levels of CreERT2 expressed behind Prox1 using an IRES in
the knock-in Prox1CreERT2 allele. To distinguish between these possi-
Genetic lineage tracing demonstrates that the blood endothelial cells lining mesenteric SVs in Slp76–/– mice arise from LECs. (A) Lineage tracing
studies performed using a Prox1CreERT2 knock-in line. Antibody staining of mesentery from 12-week-old Slp76+/–;Prox1CreERT2;Rosa26RYFP animals
exposed to tamoxifen as neonates reveals YFP only in PROX1+LYVE1+PDPN+vWF–LECs (top). Antibody staining of mesentery from 12-week-old
Slp76–/–;Prox1CreERT2;Rosa26RYFP animals exposed to tamoxifen as neonates reveals YFP in PROX1–LYVE1–PDPN–vWF+ blood endothelial cells
that line large SVs (bottom). Black scale bars: 50 μm; white scale bars: 10 μm. (B) Lineage tracing studies using a Prox1CreERT2 BAC transgenic line.
Studies were performed as described in A using a single tamoxifen injection at P14. Note that with this Prox1CreERT2 line, virtually all the endothelial
cells of the SVs shown are YFP+ but PROX1–LYVE1–PDPN–vWF+EPHB4+. Scale bar: 50 μm; applies to all panels in B.
2012 The Journal of Clinical Investigation http://www.jci.org Volume 122 Number 6 June 2012
bilities, we repeated this experiment using a recently described BAC
Prox1CreERT2 transgene that labels postnatal LECs highly efficiently
(26). In contrast to lineage tracing studies using the Prox1CreERT2
knock-in animals, a single tamoxifen injection at P14 resulted in
virtually 100% labeling of LECs in Prox1CreERT2;Rosa26RYFP BAC
transgenic mice, although sparse labeling of venous endothelial
cells could also be detected with this regimen (Figure 4B). Analysis
of Slp76–/–;Prox1CreERT2;Rosa26RYFP BAC transgenic mice revealed
that in approximately half of SVs, virtually all endothelial cells
were YFP+, indicating that these vessels were of lymphatic origin
(Figure 4B). The YFP+ endothelium of these vessels was uniformly
PROX1–LYVE1–vWF+EPHB4+, consistent with a venous blood ves-
sel identity (Figure 4B). These genetic studies demonstrate that the
LECs of mesenteric lymphatic vessels of SLP76-deficient mice are
neither lost nor replaced during exposure to blood flow, but are
instead reprogrammed to venous blood endothelial cells.
Lymphatic endothelial identity is maintained in the presence of blood.
Molecular reprogramming of the LECs lining SLP76-deficient mes-
enteric lymphatic vessels may be a response to factors in the blood
that are not present in lymph, to hemodynamic shear forces present
in blood vessels but not in lymphatic vessels, or to both. Lethally
irradiated wild-type mice that are reconstituted with SLP76-defi-
cient hematopoietic cells develop blood-filled mesenteric lymphat-
ics (10, 27), but these mature animals do not undergo subsequent
vascular separation and fail to develop the shunts or cardiomegaly
observed in Slp76–/– animals (10, 27). Previous angiographic studies
of SLP76-deficient mice revealed that paired mesenteric SVs in the
anatomic position of the congenital vein and lymphatic are exposed
to high levels of blood flow (10). In contrast, angiographic studies
performed in SLP76-deficient radiation chimeras revealed minimal
blood flow in blood-filled mesenteric lymphatics compared with
adjacent veins (Supplemental Videos 1 and 2; supplemental materi-
al available online with this article; doi:10.1172/JCI57513DS1), and
Doppler ultrasound confirmed that there was minimal blood flow
in these vessels (Figure 5, A–D). Unlike the endothelial cells lining
Slp76–/– SVs, the endothelial cells lining blood-filled lymphatic ves-
sels in Slp76–/– radiation chimeras exhibited persistent expression of
PROX1, PDPN, and LYVE1 and no detectable vWF 16 weeks after
reconstitution (Figure 5E). These studies demonstrate that expo-
sure to blood is not sufficient to convert lymphatic vessels to blood
vessels in vivo and suggest that fluid flow is necessary.
The fluid shear forces experienced by Slp76–/– mesenteric lymphatics are
sufficient to downregulate PROX1 expression in LECs ex vivo. To define
a potential link between blood flow and the loss of lymphatic
vessel identity in vivo, we calculated the hemodynamic forces in
SLP76-deficient mesenteric SVs using high-frequency ultrasound
to measure vessel size and blood flow (Figure 6). Two-dimensional
(2D) ultrasound of wild-type mesentery revealed a small-caliber
artery and a large-caliber vein (Figure 6A). The inability to visual-
ize the wild-type lymphatic vessel was likely due to compression
of this very low-pressure vessel by the ultrasound transducer. In
contrast, the 3 blood-filled vessels that constitute a typical Slp76–/–
mesenteric vascular bundle were easily visualized using 2D ultra-
sound, including a small-caliber artery and two large-caliber SVs
(Supplemental Videos 1 and 2, and Figure 6B). Flow profiles in the
vessels visualized by 2D ultrasound were obtained using pulsed-
wave Doppler ultrasound (Figure 6, C and D). The small-caliber
arteries of both wild-type and SLP76-deficient mice displayed
high-velocity pulsatile flow in the direction of the intestine (i.e.,
afferent blood flow) (Figure 6C). The larger-caliber veins in wild-
type animals and SVs in SLP76-deficient animals were character-
ized by non-pulsatile, lower-velocity flow away from the intestine
(i.e., efferent blood flow) (Figure 6D). The wall shear stress in each
vessel was next calculated using the measured mean flow veloc-
ity and vessel diameter, assuming a constant blood viscosity η of
7 mPa*s (28–30). Shear stresses in wild-type veins and the SVs
of SLP76-deficient animals were similar in magnitude, although
SVs exhibited greater heterogeneity (Figure 6E and Supplemental
Table 1). These studies suggest that mesenteric lymphatic vessels
in SLP76-deficient animals become exposed to fluid shear stresses
similar to those experienced by mesenteric veins, a result consis-
tent with their molecular venous identity (Figure 3A).
To determine whether fluid shear stress in this range might be
sufficient to negatively regulate lymphatic endothelial identity, we
subjected primary human LECs to either pulsatile or steady flow
with a shear stress of 20 dynes/cm2. Following 8 hours of shear
stress, LECs exhibited a greater than 6-fold reduction in the level
of PROX1 mRNA expression compared with static cultured cells
(Figure 6F). Exposure to fluid shear forces did not alter expression
of the pan-endothelial gene PECAM1 (Figure 6F and Supplemental
Figure 1). Expression of the secondary lymphatic markers LYVE1
and PDPN was not significantly reduced in response to shear
forces (Figure 6F). Real-time PCR assessment of other endothelial
identity markers and known shear-responsive genes revealed little
change in the venous markers COUP-TFII and EPHB4, and increas-
es in the arterial markers HEY1, HEY2, and EFNB2, as well as the
shear-responsive gene KLF2 (Supplemental Figure 2). The gener-
ally arterial shift in gene expression observed in these cells is in
contrast to the venous identity observed in vivo and may reflect
the difference in the way these forces are exerted, i.e., rapid and
full onset in vitro versus slow and gradual onset in vivo. Consis-
tent with the drop in PROX1 mRNA levels, LECs demonstrated
dramatically reduced PROX1 protein levels after exposure to shear
stress (Figure 6G). Finally, loss of PROX1 mRNA was transient in
this system, and levels recovered after 24 hours without shear (Fig-
ure 6H). These studies confirm that fluid shear forces like those
experienced by lymphatic vessels in postnatal SLP76-deficient
animals are sufficient to negatively regulate the expression of the
lymphatic endothelial fate regulator PROX1.
Hemodynamic forces have long been postulated to regulate vessel
identity, but testing this hypothesis has not been straightforward.
The fact that vessel identity is established by a genetic program
prior to the presence of significant and diverse hemodynamic
forces has made it particularly difficult to discern later contribu-
tions of fluid forces on vessel identity, particularly in the case of
pathologic flow states such as those in vascular malformations.
Most experimental approaches have utilized exogenous, surgical
manipulations to dramatically alter blood flow in the developing
or mature cardiovascular system to test the effect of hemodynam-
ic forces on vessel identity and cardiovascular organ formation
(31–33). While these approaches have provided valuable insights,
whether they accurately reflect the more gradual responses that
occur in the context of congenital or acquired vascular diseases
is unclear. In addition, whether hemodynamic forces can perma-
nently reprogram endothelial and vessel identity is not known.
In the present study, we show that postnatal vascular remodel-
ing in SLP76-deficient mice exposes normally formed mesenteric
lymphatic vessels to flowing blood that reprograms the LECs lin-
The Journal of Clinical Investigation http://www.jci.org Volume 122 Number 6 June 2012
ing these vessels as blood endothelial cells. Similar changes are
observed in LECs exposed to fluid shear ex vivo, suggesting that
hemodynamic forces can reprogram lymphatic vessels to blood
vessels through transcriptional pathways that establish and main-
tain endothelial identity.
The key finding in this study is the demonstration that lym-
phatic vessels exposed to blood flow in vivo after birth are repro-
grammed to acquire blood vessel identity. Studies performed more
than 30 years ago revealed that endothelial cell turnover in large
vessels such as the aorta is low but non-uniform and higher in
areas of turbulent blood flow where hemodynamic forces are more
varied (34, 35). Thus, the onset of significant fluid shear forces
in the lymphatic vessels of SLP76-deficient mice could stimulate
endothelial turnover and the replacement of LECs by either circu-
lating blood endothelial cells or circulating blood endothelial pre-
cursor cells believed to contribute to the endothelium of injured or
new vessels (36–39). Alternatively, the gradual rise in hemodynam-
ic shear forces may alter the gene expression of the LECs lining
these vessels and reprogram them to a blood endothelial identity.
Our genetic lineage tracing experiments demonstrate that virtu-
ally all of the PROX1-negative endothelial cells that line the SVs of
surviving SLP76-deficient animals derive from PROX1-expressing
cells. These studies therefore provide definitive evidence of molec-
ular reprogramming of endothelial and vessel identity in response
to blood flow in vivo.
Our findings provide strong evidence that hemodynamic forces
underlie the reprogramming of lymphatic vessels to blood vessels
in response to blood flow. Studies using Vav-Cre;Slp76fl/– mice dem-
Lymphatic endothelial identity is maintained in the presence of blood in Slp76–/– radiation chimeras. (A) The mesenteric vessels of Slp76–/– radia-
tion chimeras were visualized using 2D ultrasound. (B–D) Pulsed-wave Doppler signals indicative of blood flow detected in the mesenteric artery,
vein, and lymphatic are shown. Note that arterial flow is directed opposite to that of venous flow and that arterial flow is pulsatile (B, corresponding
to heart rate), while venous flow is phasic (C, corresponding to respiration). No significant signal in either direction was obtained from the lym-
phatic (D). (E) Blood-filled mesenteric lymphatics retain lymphatic identity in Slp76–/– radiation chimeras. Mesenteric vessels in wild-type lethally
irradiated mice reconstituted with Slp76+/+ (+/+) or Slp76–/– (–/–) bone marrow are shown 16 weeks after reconstitution (left). Analysis of serial
sections reveals that mesenteric lymphatics in Slp76–/– radiation chimeras that are exposed to blood but not flow retain expression of LYVE1,
PROX1, and PDPN and do not express vWF (right). Scale bars: 50 μm.
2014 The Journal of Clinical Investigation http://www.jci.org Volume 122 Number 6 June 2012
onstrate that the endothelial and vessel identity changes observed
do not reflect an unexpected role for SLP76 in endothelial cells,
but instead arise due to changes in the vascular environment that
result from blood-lymphatic vascular connections in the intestine.
Radiation chimeras reconstituted with SLP76-deficient hemato-
poietic cells develop blood-filled mesenteric lymphatics but have
little or no flow in those vessels, most likely because these mature,
irradiated animals do not remodel their vasculature to create
arterio-venous-lymphatic shunts like neonatal animals. These ani-
mals demonstrate that the formation of blood-lymphatic connec-
tions and contact with blood is not sufficient to alter lymphatic
endothelial and vessel identity. In contrast, LECs exposed to fluid
Fluid shear forces equivalent to those in Slp76–/– mesenteric lymphatics drive loss of PROX1 expression in LECs. (A) Representative mesenteric
vessels in 12-week-old Slp76+/+ and Slp76–/– animals that were studied using Doppler ultrasound. The dilated, blood-containing vein and congeni-
tal lymphatic are denoted as SV1 and SV2. (B) The vessels shown in A were visualized using 2D ultrasound. (C and D) The pulsed-wave Doppler
signals and measured flow velocity of blood in the vessels indicated in A and B are shown. Note the difference in direction of flow between the
arteries and veins or efferent SVs. (E) Calculated shear stresses for Slp76+/+ veins and Slp76–/– SVs. Slp76–/– data points in the same color indicate
values of paired SVs from the same mesenteric bundle. (F) LECs exposed to flow downregulate PROX1. LECs were subjected to a shear stress
of 20 dynes/cm2 for 8 hours, and the expression of the indicated mRNAs measured using qPCR. n = 7. **P < 0.01. (G) Loss of PROX1 protein in
LECs exposed to shear. Anti-PROX1 immunostaining in primary LECs is shown compared with DAPI staining of cell nuclei. (H) Shear-mediated
downregulation of PROX1 is transient in cultured endothelial cells. PROX1 levels were measured after 8 hours of static culture, 8 hours of flow, or
8 hours of flow followed by 24 hours of static culture. *P < 0.05, **P < 0.01. Scale bars: 25 μm.
The Journal of Clinical Investigation http://www.jci.org Volume 122 Number 6 June 2012
shear forces in the absence of blood ex vivo exhibit rapid downregu-
lation of PROX1. Loss of PROX1 in LECs has been shown to result
in the loss of other lymphatic identity markers such as LYVE1 and
PDPN (17, 20), and genetic deletion of Prox1 has recently been
shown to be sufficient to convert LECs to blood endothelial cells in
vivo (18). Thus, our studies support the concept that expression of
the PROX1 transcription factor is required to maintain lymphatic
endothelial and vessel identity (18) and that loss of PROX1 expres-
sion in LECs exposed to fluid shear forces associated with blood
flow is a likely mechanism by which lymphatic vessels are repro-
grammed to blood vessels in SLP76-deficient mice.
What are the molecular pathways through which hemody-
namic forces negatively regulate PROX1 and endothelial lym-
phatic identity? We found that HEY1 and HEY2 are strongly
upregulated in coordination with downregulation of PROX1
in LECs exposed to fluid shear forces (Supplemental Figure 2),
and HEY1/2 or NOTCH expression was recently demonstrated
to negatively regulate lymphatic endothelial identity in cultured
LECs by reducing expression of PROX1 (40). To test the role of
HEY1/2 expression and loss of lymphatic endothelial identity in
SLP76-deficient mice in vivo, we have examined Slp76–/–;Hey2LacZ/+
mice, in which LacZ is expressed in place of Hey2 (41). However,
we did not detect LacZ expression in the endothelium of mesen-
teric arteries or SVs, suggesting that the NOTCH/HEY signaling
is not the basis for this endothelial reprogramming event in vivo.
It is possible that flow-dependent activation of HEY1/2 signaling
plays a transient role in this process that was not detected by these
studies, but it seems more likely that ex vivo studies of cultured
endothelial cells performed over hours do not fully model molec-
ular changes that take place over weeks in vivo. Flow chamber
experiments have identified large numbers of endothelial genes
that are upregulated and downregulated by fluid flow, includ-
ing many involved in endothelial identity (31, 42, 43), but there
is no definitive means of testing whether and to what extent these
genes mediate endothelial responses to hemodynamic forces in
vivo. Transiently blocking blood flow in very young embryos
can distinguish between programmed endothelial identity gene
expression and gene expression driven by fluid forces (44, 45), but
this approach is not feasible when hemodynamic changes arise
more gradually, as they do in patients with congenital vascular
and cardiac defects. Thus, the identity of the molecular signals
that downregulate PROX1 expression in response to fluid flow
in vivo is not yet defined.
An important implication of this study is that vessel identity
remains plastic after vascular development is complete and may be
radically altered by hemodynamic forces later in life. The healthy
mature vasculature is thought to be very quiescent, but many con-
genital and acquired human cardiovascular diseases are associated
with persistent changes in blood flow and fluid shear forces, e.g.,
the left-to-right shunting of blood to the low-pressure pulmonary
vasculature from the high-pressure arterial system in congenital
heart disease. Molecular changes in endothelial and vessel identity
are very likely to accompany the hemodynamic alterations in these
diseases. Defining these molecular changes is expected to provide
new insight into the pathogenesis and treatment of human cardio-
vascular diseases such as vascular malformations.
Histology and immunohistochemistry. Intestine and mesentery were dissected
from murine neonates and adults and fixed in 4% paraformaldehyde
for 24–48 hours, dehydrated in 100% ethanol, and embedded in paraf-
fin. Serial 8-μm-thick sections were subjected to hematoxylin and eosin
and/or immuno histo chemical staining as detailed by the University of
Pennsylvania Molecular Cardiology Research Center Histology and Gene
Expression Core (http://www.med.upenn.edu/mcrc/histology_core/).
Antibodies used were MEC13.3 rat anti–mouse CD31 (BD Biosciences
— Pharmingen) at 1:500, rabbit anti-LYVE1 at 1:1,000, rabbit anti-Prox1
(Abcam) at 1:100, anti-CX40 (Alpha Diagnostics) at 1:100, and anti-
EPHB4 (Cell Sciences) at 1:50.
Injection of biotinylated lectin. Intracardiac injection of 50 μg of biotinylated
Lycopersicon esculentum (tomato) lectin (Vector Laboratories) was performed
in neonatal Slp76–/– mice and wild-type littermate controls. Co-injection
with FITC-dextran (Sigma-Aldrich) was used to determine successful
perfusion. Biotinylated tomato lectin (300 μg; Vector Laboratories) was
injected intravenously into 10- to 12-week-old Slp76–/– mice and wild-type
littermate controls via tail vein. The mice were then euthanized and fixed
by perfusion with 4% paraformaldehyde. Tissues were then processed for
immunohistochemical analysis as described above.
Hemodynamic studies. A Visual Sonics Vevo 770 Imaging System with
single-element mechanical transducers was used to image the mesenteric
vascular structures and blood flow spectra. The center frequency was set
at 40 MHz (for lateral and axial resolutions of 68 and 38 nm, respectively).
Twelve-week-old mice were anesthetized with Avertin, and their intestine
and mesentery were dissected and exposed. Body temperature was moni-
tored via a rectal thermometer and maintained between 36°C and 37°C
using a heating pad and lamp, and heart rate and electrocardiogram were
also continuously monitored. To eliminate interference from structures
underlying the mesentery, the mesenteric and intestinal areas of interest
were spread on a slide glass. A thin layer of prewarmed ultrasound gel was
applied to the slide glass and covered with a plastic membrane before tissue
exposure. A second membrane was placed on the tissue surface to keep it
clean for later dissection. Another layer of prewarmed thick ultrasound gel
was placed on this membrane to provide a coupling medium for the trans-
ducer. Two mesenteric vascular bundles from each mouse were selected
for imaging. Cross-sectional images of the vessels were recorded and the
lumen diameters measured by 2D ultrasonography. The transducer was
then rotated 90° to obtain a longitudinal view of the vessels. For Doppler
spectral analysis, the transducer was adjusted for an angle of insonation
less than 60°. The mean blood flow velocities were measured and used to
calculate wall shear stress for the vessels of interest.
The wall shear stress in each vessel was next calculated using the mea-
sured mean flow velocity Vmean and vessel diameter ID, assuming a constant
blood viscosity η of 7 mPa*s (28–30): τ (shear stress) = γ • η = (4Vmeanη)/ID.
Although the wall shear rate γ was not directly measured, it was calculated
from the measured parameters by using Poiseuille’s parabolic model of
LEC isolation. Human microvascular LECs were harvested according to
previously published methods (46, 47). In brief, microvascular cells were iso-
lated from human foreskins, and endothelial cell colonies were allowed to
expand on gelatin-coated tissue culture dishes in EGM-MV growth medium
(Lonza). At confluence, cells were detached using Cell Dissociation Solu-
tion (Sigma-Aldrich) and trypsin (Lonza). Immunomagnetic purification
was performed by incubating the resuspended cells with monoclonal mouse
anti–human podoplanin antibody (clone 18H5, Research Diagnostics Inc.)
for 30 minutes at 4°C, followed by incubation with pan-mouse IgG mag-
netic beads (Invitrogen) for 15 minutes at 4°C. LECs attached to beads were
magnetically separated and plated on gelatin-coated dishes. LECs were sub-
sequently cultured and passaged using EGM-2 medium supplemented with
VEGF or EGM2-MV medium (Lonza). Purity of greater than 95% was veri-
fied using flow cytometry for CD31 and podoplanin.
2016 The Journal of Clinical Investigation http://www.jci.org Volume 122 Number 6 June 2012
Cell culture under shear stress. Passage 4 LECs were cultured to confluence
on gelatin-coated glass slides for use in a parallel plate flow chamber. Pul-
satile flow with shear stress of 20 dynes/cm2 was achieved using a Master-
flex peristaltic pump and a parallel plate flow chamber provided by J.A.
Frangos (CytoDyne, San Diego, California, USA). Non-pulsatile flow with
shear stress of 20 dynes/cm2 was achieved using a 6-slide parallel plate flow
chamber designed by Flexcell International Corp. The medium reservoir
was maintained at 37°C using an incubator or water bath. Cells were incu-
bated in the flow chamber for 8 hours. Static control cells were fed with
fresh medium at the beginning of the experiment and maintained in the
incubator for the duration of the experiment.
Real-time PCR analysis. Total RNA from LECs (primary isolation described
above) and human umbilical vein endothelial cells (Lonza) was isolated
using an RNeasy Mini Kit (QIAGEN) according to the manufacturer’s
protocols. For reverse transcriptase reactions, 1 μg of total RNA and 100
ng of random hexamers was used to generate cDNA with the First-Strand
cDNA Synthesis Kit (Invitrogen) according to the manufacturer’s proto-
cols. Real-time semi-quantitative PCR and relative quantitation normal-
ized to GAPDH was performed using SYBR Green Master Mix reagent
(Applied Biosystems) on the ABI Prism 7900HT Sequence Detection
System (Applied Biosystems). Real-time PCR primers were designed and
obtained from IDT. Primer sequences are listed in Supplemental Methods.
Immunocytochemistry. Cells were fixed in 4% paraformaldehyde for 15 min-
utes at room temperature and permeabilized with 0.2% Triton X-100/PBS.
Samples were blocked with 2% bovine serum albumin in PBS and stained with
rabbit anti-Prox1 antibody (Abcam), followed by Alexa Fluor 488 secondary
antibody (Molecular Probes, Invitrogen), and mounted with DAPI-containing
Vectashield mounting medium (Vector Laboratories) to visualize cell nuclei.
Genetic reconstitution with SLP76-deficient bone marrow and angiography stud-
ies. Wild-type mice were lethally irradiated and reconstituted with SLP76-
deficient bone marrow according to published methods (10, 27). Recipient
mice were sacrificed for study 8–10 weeks after transplantation. FITC-
dextran angiography with real-time video microscopy was performed on
anesthetized animals as previously described (10). Tissues were harvested,
fixed, and processed for immunohistochemical analysis.
Statistics. P values were calculated using an unpaired, 1-tailed Student’s
t test. A P value less than 0.05 was considered significant. All bar graphs
and error bars represent mean values and SEM.
Study approval. Animal studies were approved by the Institutional Animal
Care and Use Committee at the University of Pennsylvania, Philadelphia,
This work was supported by an American Heart Association (AHA)
award (Scientist Development Award 0730286N to S.J. Stachelek),
Cancer Research UK (to T. Makinen), and the National Heart,
Lung, and Blood Institute (NHLBI; HL073402 to G. Oliver and
HL072798 to M.L. Kahn).
Received for publication February 20, 2012, and accepted in
revised form April 5, 2012.
Address correspondence to: Mark L. Kahn, University of Pennsyl-
vania, 952 BRB II/III, 421 Curie Blvd., Philadelphia, Pennsylvania
19104, USA. Phone: 215.898.9007; Fax: 215.573.2094; E-mail:
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