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VASCULAR BIOLOGY
ALK1 signaling regulates early postnatal lymphatic vessel development
*Kyle Niessen,1 *Gu Zhang,1 John Brady Ridgway,1 Hao Chen,1 and Minhong Yan1
1Department
of Tumor Biology and Angiogenesis, Division of Research, Genentech Inc, South San Francisco, CA
In vertebrates, endothelial cells form 2 hierarchical tubular networks, the blood
vessels and the lymphatic vessels. Despite the difference in their structure and
function and genetic programs that dictate their morphogenesis, common signaling pathways have been recognized that
regulate both vascular systems. ALK1 is
a member of the transforming growth
factor-␤ type I family of receptors, and
compelling genetic evidence suggests its
essential role in regulating blood vascu-
lar development. Here we report that ALK1
signaling is intimately involved in lymphatic development. Lymphatic endothelial cells express key components of the
ALK1 pathway and respond robustly to
ALK1 ligand stimulation in vitro. Blockade of ALK1 signaling results in defective
lymphatic development in multiple organs of neonatal mice. We find that ALK1
signaling regulates the differentiation of
lymphatic endothelial cells to influence
the lymphatic vascular development and
remodeling. Furthermore, simultaneous
inhibition of ALK1 pathway increases apoptosis in lymphatic vessels caused by
blockade of VEGFR3 signaling. Thus, our
study reveals a novel aspect of ALK1
signaling in regulating lymphatic development and suggests that targeting ALK1
pathway might provide additional control
of lymphangiogenesis in human diseases. (Blood. 2010;115:1654-1661)
Introduction
Lymphatic vessels are a network of endothelial-lined vessels that
are critical for the collection and transport of interstitial fluid,
absorption of lipids in the intestine, and the transport of lymphocytes and antigen-presenting cells to the lymph nodes. Malformation or dysfunction of lymphatic vasculature contributes to the
pathogenesis of many human diseases. For instance, loss of
lymphatic function caused by genetic deficiency or damage of
lymphatic vessels causes lymphedema. However, solid tumors can
hijack the lymphatic vasculature to spread tumor cells to distant
sites. A better understanding of the signaling pathways that regulate the lymphangiogenesis will assist the design of novel therapeutics for human diseases that can better control the growth and
differentiation of lymphatic vessels.
Several important lymphatic endothelial regulators and markers
have been identified, such as Sox18, Prox1, VEGFR3, VEGFC/D,
podoplanin, and LYVE1. In the mouse, lymphatic development is
characterized by the induction of Sox18 expression at embryonic
day (E) 9.0, followed by Prox1 expression at E10.5 in a subset of
endothelial cells populating the dorsal cardinal vein.1,2 The Prox1expressing cells bud-off the cardinal vein and form the primary
lymphatic sacs and subsequently through the process of lymphangiogenesis, a functional lymphatic network is established.3 VEGFR3–
VEGFC/D signaling is essential for the lymphatic development. It
promotes migration, proliferation, and survival of lymphatic cells.
For instance, inactivation of VEGFR3 results in the failure of
Prox1-positive cells to bud-off from the cardinal vein.4 In addition,
sustained VEGFR3–VEGFC/D signaling is required for lymphatic
vessel stability as its inhibition results in the regression of
preexisting lymphatic vessels.5 Another critical regulator of lymphatic development is Prox1, a homeobox transcription factor that
regulates expression of the lymphatic phenotype genes podoplanin
and LYVE1 and is referred to as a master regulator of lymphatic
identity.6,7 Podoplanin is a membrane mucoprotein, and podoplanindeficient mice die at birth as the result of respiratory failure and
have defects in lymphatic development.8,9 LYVE1 is a hyaluronan
receptor, and LYVE1-deficient mice are viable but have defects in
lymphatic development.10
The blood vessels and the lymphatic vessels differ in their
functional and structural characteristics. During morphogenesis the
2 vascular systems are regulated by apparently distinct genetic
programs. Despite these differences, accumulating evidences also
support that common molecular pathways are used during angiogenesis and lymphangiogenesis. For instance, in addition to its
well-established role in the development of lymphatic vasculature,
VEGFR3 is also essential for the early blood vessel development in
embryos and contributes to tumor angiogenesis.11,12 Angiopoiteins
and their receptor Tie2 are known to be important for the
remodeling of endothelium and vascular stability during angiogenesis. The same signaling system also plays a role in lymphangiogenesis.13 EphrinB2 is essential for the angiogenic remodeling of blood
vessels in early embryo, and interestingly deletion mutation of
EphrinB2 causes abnormal development of lymphatic vessels.14,15
Activin receptor-like kinase 1 (ALK1) is a member of the
transforming growth factor-␤ (TGF-␤) type I family of receptors.
The TGF-␤ family of receptors elicits diverse tissue-specific
responses, including regulating proliferation, differentiation, migration, and survival.16-18 ALK1 is primarily expressed in the developing vascular system and plays a critical role in arteriogenesis and
arterial endothelial cell development.19,20 In addition, mutations in
ALK1 or its coreceptor endoglin are associated with the human
Submitted July 29, 2009; accepted October 20, 2009. Prepublished online as
Blood First Edition paper, November 10, 2009, DOI: 10.1182/blood-2009-07235655.
The online version of this article contains a data supplement.
*K.N. and G.Z. contributed equally to this work.
The publication costs of this article were defrayed in part by page charge
payment. Therefore, and solely to indicate this fact, this article is hereby
marked ‘‘advertisement’’ in accordance with 18 USC section 1734.
An Inside Blood analysis of this article appears at the front of this issue.
© 2010 by The American Society of Hematology
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BLOOD, 25 FEBRUARY 2010 䡠 VOLUME 115, NUMBER 8
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BLOOD, 25 FEBRUARY 2010 䡠 VOLUME 115, NUMBER 8
vascular disorder hereditary hemorrhagic telangiectasia,21,22 a
vascular disorder that leads to telangiectases and arteriovenous
malformations in skin, mucosa, and viscera. ALK1 signals through
a heteromeric complex that includes ALK1 and the TGF-␤ type II
receptors BMPR2, ACVR2A, or ACVR2B. Potential ALK1 ligands include bone morphogenic protein 9 (BMP9) and BMP10,
which directly bind and activate ALK1.23 Ligand binding results in
activation of ALK1 through transphosphorylation by the type II
receptor. Activated ALK1 in turn phosphorlyates Smad1 and
Smad5, which subsequently translocate to the nucleus to regulate
gene expression.24 In the mouse, ALK1 deficiency results in
embryonic lethality at E11.5 as the result of defects in the
remodeling of the primary capillary plexus into a functional
network of arteries, capillaries, and veins.19 In addition, ALK1deficient embryos have decreased smooth muscle cell recruitment
to the developing dorsal aorta.19 As ALK1-deficent embryos die at
the onset of lymphatic vessel development the role of ALK1 in
lymphatic development is unknown.
Here we show that ALK1 signaling is functionally active in
cultured lymphatic endothelial cells. In vivo, blockade of ALK1
signaling with the use of decoy receptors or ALK1-specific
antibody results in defective lymphatic development in early
postnatal mice. Our data demonstrate that, unlike VEGFR3 signaling, which is essential for the proliferation and survival of
lymphatic endothelial cells, ALK1 signaling regulates the differentiation of lymphatic endothelial cells to influence the lymphatic
vascular development and remodeling. Furthermore, we show that
inhibition of ALK1 signaling enhances the apoptosis of lymphatic
endothelial cells upon depletion of VEGFR3 ligands.
Methods
Reagents
hALK1Fc (aa. 1-118), hVEGFR3Fc (aa. 1-329), mALK1Fc (aa. 23-119),
mALK2 (aa. 1-123), and mALK7 (aa. 1-113) were purified from transient
transfection of 293T cells. mALK3Fc, mALK4Fc, mALK5Fc, and
mALK6Fc were purchased from R&D Systems. Goat anti-LYVE1 (AF2125)
and rabbit anti–active Caspase3 (AF835) were purchased from R&D
Systems. Cy3-anti–smooth muscle actin (C6198) and FITC-isolectin-B4
(L2895) were purchased from Sigma-Aldrich. Rat anti-CD31 (553370) was
purchased from BD Pharmingen. Hamster anti-podoplanin (CMV010) was
purchased from Cell Sciences. Secondary antibodies were all chicken
anti–(species on interest) Alexa-594 or Alexa-488, purchased from Invitrogen. Human umbilical vein endothelial cells (HUVECs; CC-2517) and
human microvascular dermal neonatal lymphatic endothelial cells (CC2505) and required media were purchased from Lonza. Detroit 551
fibroblast cells (CCL-110) were purchased from ATCC. The rabbit antiALK1 neutralizing antibody was generated by immunization with Histagged recombinant protein of the extracellular domain of murine ALK1.
Immune serum was purified by affinity chromatography by the use of a
mouse ALK1Fc.
ALK1 REGULATES LYMPHATIC DEVELOPMENT
1655
RNA extraction and real-time quantitative reverse-transcription
polymerase chain reaction
For stimulation of HUVEC and lymphatic endothelial cell assay, 13 000 cells
per well were plated into 96-well plates in growth media. Sixteen hours
later cells were serum starved (EGM2) for 3 hours and stimulated with
rhBMP9 or rhBMP10 for 3 hours. RNA isolation and cDNA synthesis was
performed by the use of TaqMan Gene Expression Cells-to-CT kit (Applied
Biosystems) according to manufacturer’s protocol. Gene expression was
analyzed by the use of TaqMan Gene Expression Master Mix (Applied
Biosystems), GAPDH (Hs00266705_g1)–, and Smad6 (Hs00178579_m1)–
specific TaqMan Gene Expression Assays (Applied Biosystems) and
analyzed on an Applied Biosystems 7500 reverse-transcription polymerase
chain reaction (RT-PCR) system. Primers used in RT-PCR include the
following: hAlk1 (L-AGCAGGCCTGTCTCAGGA, R-CCTGGGAGGTAGACGTTGC), hAlk2 (L-CCCAGCTGCCCACTAAAG, R-CGCCTTTTAAATTTTCGGAGA), hAlk3(L-GGACGAAAGCCTGAACAAAA, R-GCAATTGGTATTCTTCCACGA), hAlk4 (L-CTCAGGGTCTGGC-TCAGG,
R-ACATCACCACCCCTCCAG), hAlk5 (L-AACGTCAGGTTCTGGCTCA, R-GAATATCTTAACAGCAACTTCTTCTCC), hAlk6
(L-ATCAGGCCTCCCTCTGCT, R-CACTTTCACAGCTACCTTTTCG),
hAlk7 (L-GGAAGATGTGGCTGTGAAAAT, R-GTTGAGTCCAAGTTCCATTATCTTT), hAVCR2B (L-GAGGCCTCTCATACCTGCAT,
R-GGTCGCTCTTCAGCAATACA), hBMPR2 (L-GAAGCCTGGAAAGAAAATAGCC, R-AATCATCATAAGTTCAGCCATCC), hENG
(L-TCCCCAAGACCCAGATCC, R-GCATGCAGAAGGACAGTGAC),
hTGF␤RII (L-CCAATATCCTCGTGAAGAACG, R-GTATCTTGCAGTTCCCACCTG), and hGAPDH (L-GAGTCCACTGGCGTCTTCAC,
R-GTTCACACCCATGACGAACA).
Enzyme-linked immunosorbent assay
Mouse ALK1-7Fc binding assay. We coated 96-well plates with donkey
anti–human Fc (Jackson ImmunoResearch Laboratories). Plates were then
washed and blocked with phosphate-buffered saline (PBS), 0.5% BSA.
hIgG or Fc fusion proteins were captured in PBS, 0.5% BSA, and 0.05%
Tween-20. Plates were then washed 3 times with PBS, 0.5% BSA, and
0.05% Tween-20, and the rabbit anti-ALK1 neutralizing antibody was
added. Plates were washed 3 times with PBS, 0.5% BSA, and 0.05%
Tween-20 and then probed with anti–rabbit HRP (Jackson ImmunoResearch Laboratories). HRP activity was developed by the use of Sure
Blue Reserve TMP Microwell Peroxidase Substrate (Kirkegaard & Perry
Laboratories Inc), and absorbance was measured at 450 nm.
BMP9 binding assay. We coated 96-well plates with mouse Alk1Fc.
Plates were then washed and blocked with PBS and 0.5% BSA. Immobilized mALK1 was incubated with preimmune rabbit serum, ALK1immunized rabbit serum, or affinity-purified rabbit anti-ALK1. Plates were
then washed 3 times with PBS, 0.5% BSA, and 0.05% Tween-20, and
vehicle control or mouse BMP9-HIS was added to the plate. Plates were
washed 3 times with PBS, 0.5% BSA, and 0.05% Tween-20 and then
probed with mouse anti-HIS antibody (Roche). Plates were washed 3 times
with PBS, 0.5% BSA, and 0.05% Tween-20 and then probed with
anti–mouse AP (Jackson ImmunoResearch Laboratories). AP activity was
developed by the use of 1-Step PNPP substrate (Thermo Scientific), and
absorbance was measured at 405 nm.
Animal studies
All studies were conducted in accordance with the National Institutes of
Health Guide for the Care and Use of Laboratory Animals. An Institutional
Animal Care and Use Committee approved all animal protocols.
Tissue harvest
Tail dermis was collected by first removing the bone from the tail, followed
by incubation in 20mM ethylenediaminetetraacetic acid (EDTA), and
separation of the dermis and epidermis. Dermis was then fixed in 4%
paraformaldehyde and used in a standard immunofluorescence protocol.
Retina was isolated from the eye after overnight fixation in 4% paraformaldehyde. Retinas were then stained as previously described.25
Results
ALK1 signaling in lymphatic endothelial cells
Analysis of ALK1 mRNA expression in HUVECs and human
microvascular dermal neonatal lymphatic endothelial cells revealed that both cell types express ALK1 (Figure 1A). In addition
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1656
NIESSEN et al
BLOOD, 25 FEBRUARY 2010 䡠 VOLUME 115, NUMBER 8
lin, Smad1, Smad4, and Smad5, also reduced BMP9-induced
Smad6 up-regulation (Figure 1C). Target gene knockdown was
verified for each siRNA (supplemental Figure 1, available on the
Blood website; see the Supplemental Materials link at the top of the
online article). These data raise an interesting possibility that ALK1
signaling may also be involved in lymphatic endothelial cell
biology.
ALK1Fc disrupts both blood vessel and lymphatic vessel
development in vivo
Figure 1. Lymphatic endothelial cells express ALK1. (A) RT-PCR analysis of
gene expression in HUVECs and human microvascular dermal neonatal lymphatic
endothelial cells. RNA from a mixture of human cell lines with (⫹) and without (⫺)
reverse transcriptase enzyme was used as a positive and negative control. Sizes of
PCR products are indicated. (B) Expression level of Smad6 in HUVECs and
lymphatic endothelial cells stimulated with 50 pg/mL rhBMP9 or 500 pg/mL rhBMP10
for 3 hours. Inhibition of rhBMP9 and rhBMP10 induced up-regulation of Smad6
expression by pretreatment with 10 ␮g/mL ALK1Fc. Quantitative RT-PCR results are
normalized to glyceraldehyde 3-phosphate dehydrogenase and then to the untreated
(UT) sample. Gray squares represent individual data points (n ⫽ 3). (C) Expression
level of Smad6 in lymphatic endothelial cells stimulated with rhBMP9 150 pg/mL and
transfected with siRNA targeting the Alk1-7, Smad1, 4, 5, or endoglin (Eng). Gray
squares represent individual data points (n ⫽ 3).
to ALK1, lymphatic endothelial cells also express ACVR2B,
BMPR2, and endoglin, which are involved in ALK1 signaling
(Figure 1A).26,27 To test whether lymphatic endothelial cells are
responsive to the ALK1 ligands BMP9 or BMP10, HUVECs and
lymphatic endothelial cells were stimulated with rhBMP9 or
rhBMP10, and the expression of Smad6, an ALK1 target gene, was
assessed. Both HUVECs and the lymphatic cells robustly responded to BMP9 and BMP10 stimulation (Figure 1B). Phosphorylation of Smad1 and induction of the ALK1 target gene Id1 also
correlated with Smad6 induction; however, Smad6 was the most
sensitive readout of BMP9 stimulation (data not shown). rhBMP9
or rhBMP10 activity was effectively neutralized by pretreatment
with a soluble decoy receptor of ALK1 extracellular domain fused
to Fc portion of human IgG1 (ALK1Fc; Figure 1B). Furthermore,
we found that siRNA targeting ALK1, compared with other type I
receptors, had the most significant effect on BMP9 stimulation of
lymphatic endothelial cells (Figure 1C). siRNA targeting downstream signaling components of ALK1 signaling, including Endog-
To circumvent the embryonic lethality of ALK1 deficiency that
prevents the assessment of its role during lymphatic development,
we decided to block the ALK1 signaling using ALK1Fc in neonatal
mice. Analysis of P8 mouse retinas revealed that systemic treatment of neonatal mice with ALK1Fc (10 mg/kg, at P1, P3, and P5)
resulted in a dramatic increase in retinal vascular density, failed
remodeling of the primary retinal capillary plexus into a mature
vascular pattern, and defective arteriogenesis as indicated by the
loss of smooth muscle actin (SMA) staining (Figure 2A). These
findings are reminiscent of the vascular defects associated with
ALK1 deficiency during embryogenesis19 and suggest that ALK1Fc
is able to block ALK1 signaling in vivo. Interestingly, similar
treatment also resulted in prominent accumulation of chyle in the
abdominal cavity by P7 (supplemental Figure 2), which is indicative of a possible defect in the intestinal lymphatics. To directly
investigate the impact of ALK1Fc on lymphatic function, P5
neonatal mice treated with ALK1Fc (10 mg/kg, P1 and P3) or PBS
were injected subcutaneously with dextran–fluorescein isothiocyanate (FITC) into the tail tip. Drainage of the dextran–FITC through
the lymphatic network was analyzed 5 minutes after injection. In
the PBS-injected pups, a dextran–FITC-positive lymphatic collecting vessel was readily visible running the length of the tail (Figure
2B). In addition, the stereotypic honeycomb pattern of capillary
lymphatic vessels in the tail dermis was highlighted by the injected
dextran–FITC (Figure 2B). By comparison, ALK1Fc-treated pups
(10 mg/kg, P1 and P3) had similar dextran–FITC-positive staining
in the collecting vessel (Figure 2B top); however, the honeycomb
lymphatic vessels were completely absent (Figure 2B bottom).
These data suggest that ALK1Fc treatment results in the defective
development of lymphatic microvessels, whereas it has little effect
on the lymphatic collecting vessels that have already formed before
birth. LYVE1 staining of P6 tail dermis further confirmed the
impaired development of the honeycomb pattern of lymphatic
vessels. Individual LYVE1-positive cells were still present in
ALK1Fc-treated tail dermis; however, the cells failed to make the
necessary connections to form the honeycomb structure (Figure
2C). In addition to lymphatic vessels the tail dermis has an
extensive network of blood vessel capillaries. Unlike the lymphatic
microvessels that form postnatally, the preexisting blood capillaries
were unaffected (Figure 2C).
Because the first observation of lymphatic dysfunction was
chyle accumulation in the abdomen of ALK1Fc-treated pups, the
lymphatic network in the intestine was further investigated.
ALK1Fc treatment resulted in shorter villous length of the P6
neonates, where the extension of villus lacteals into the villi was
greatly reduced and in many cases was completely stalled (Figure
2D). Because further lymphatic capillary extension into the villi
accompanies the continuing growth of intestinal villi in early
postnatal mice (supplemental Figure 3), the observed defect is
likely attributable to a blockade of lymphatic development rather
than a regression of preexisting villus lacteals. However, the
preexisting lymphatic vascular network on the outer surface of the
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BLOOD, 25 FEBRUARY 2010 䡠 VOLUME 115, NUMBER 8
Figure 2. ALK1Fc causes vascular and lymphatic defects. (A) Isolectin-B4
(green) and SMA (red) staining of P8 retina from PBS- or ALK1Fc-treated pups
(10 mg/kg; P1 P3, P5). (B) Analysis of lymphatic function by dextran–FITC injection
into the tail tip (™) of a P5 pup treated with PBS or ALK1Fc (10 mg/kg, P1 and P3).
Images were captured 5 minutes after injection. (Top) Drainage of dextran–FITC into
the collecting lymphatic vessel that extends the length of the tail. Scale bar represents
1 mm. (Bottom) “Honeycomb” lymphatic vessels in the dermis adjacent to the
injection site (box). Scale bar represents 250 ␮m. (C) CD31 (blue, vasculature) and
LYVE1 (red, lymphatic) staining of the tail dermis from PBS- or ALK1Fc-treated pups
(10 mg/kg, P1 and P3) at P6. Scale bar represents 250 ␮m. (D) CD31 (blue,
vasculature) and LYVE1 (red, lymphatic) staining of the intestine from PBS- or
ALK1Fc-treated pups (10 mg/kg, P1 and P3) at P6. Scale bar represents 250 ␮m.
intestine28 appeared largely unaffected, except for apparent vessel
dilation (data not shown). Similar to the tail dermis the blood
vessels within the intestine villi appeared normal in the ALK1Fctreated pups (Figure 2D). These data demonstrate that ALK1Fc
affects neovascular development of both lymphatic and blood
vessels, whereas it has little effect on existing vessels.
The signaling axis of ALK1–ACVR2B/BMPR2–BMP9/10
regulates early postnatal lymphatic development
Similar to ALK1Fc we found ACVR2BFc and BMPR2Fc were
able to block both BMP9- and BMP10-induced Smad6 expression
(supplemental Figure 4), suggesting that ACVR2B and BMPR2 are
potential type II receptors for BMP9/10 signaling. Genetic deletion
of ACVR2B has been demonstrated to result in postnatal death
associated with cardiac and vertebral patterning defects.29 Genetic
deletion of BMPR2 results in embryonic lethality, and mice with
hypomorphic allele of BMPR2 die before birth as a result of
skeletal and cardiac defects.30,31 Mutations in BMPR2 are also
ALK1 REGULATES LYMPHATIC DEVELOPMENT
1657
associated with pulmonary hypertension.32 However, the effect of
postnatal inhibition of BMPR2- or ACVR2B-dependent signaling
on vascular or lymphatic development has not been established.
As demonstrated in Figure 3A, treatment of neonatal mice with
either ACVR2BFc or BMPR2Fc (10 mg/kg, P1 and P3) resulted in
increased retinal vascular density and decreased SMA staining at
P6, which resembled the effect of ALK1Fc. Importantly, injection
of ACVR2BFc or BMPR2Fc also resulted in lymphatic vessel
defects in P6 neonates, which were similar, yet to a less extent, to
those observed with ALK1Fc (Figure 3A). These findings demonstrated that the effect on lymphatic vessel is a pathway specific
effect rather than being unique to one reagent.
The results of in vivo experiments with ALK1Fc, ACVR2BFc,
and BMPR2Fc all suggest a role for ALK1 signaling during
lymphatic development. Because the ligands of TGF-␤ family
oftentimes engage more than one receptor system, in principle the
effect of a decoy soluble receptor, which functions by sequestering
ligands, might not be limited to its own signaling system.33 To
demonstrate that the observed lymphatic defects are directly
attributed to blocked ALK1 signaling, we took advantage of an
ALK1-specific neutralizing antibody. The ALK1 antibody can
inhibit the binding of BMP9 to immobilized mALK1 in an
enzyme-linked immunosorbent assay (Figure 3C) and to block the
up-regulation of SMAD6 expression after BMP9 stimulation in
endothelial cells (Figure 3B). In addition, this antibody is highly
selective and does not bind other TGF-␤ type I receptors in an
enzyme-linked immunosorbent assay (Figure 3D). Importantly,
treatment of neonates with the ALK1-neutralizing antibody (10 mg/
kg, P1 and P3) resulted in lymphatic defects similar to those as
observed with ALK1Fc treatment (Figure 3E-F). ALK1-neutralizing
antibody treatment resulted in a failure of the lymphatic endothelial
cells to make the necessary connections to form the honeycomb
structure in the tail (Figure 3E). In the ear the treatment with
ALK1-neutralizing antibody resulted in decreased branching of the
lymphatic vessels and lymphatic vascular density (Figure 3F).
Collectively, these data suggest that ALK1 pathway plays an
essential role in regulating the postnatal development of lymphatic
vasculature.
Despite the dramatic effect of ALK1 inhibition on lymphatic
development in vivo, inhibition or activation of ALK1 signaling
had no significant effect on the proliferation and migration of
lymphatic endothelial cells in 2-dimensional culture (data not shown).
However, lymphatic endothelial cells cultured in 3-dimensional fibrin
matrix exhibited increased spouting and proliferation upon treatment
with ALK1Fc, BMPR2Fc, and ACVR2BFc, whereas treatment with
other TGF-␤ type I-Fc and type II-Fc receptors had no affect (supplemental Figure 5 and data not shown). These results are consistent with the
notion that blockade of ALK1 signaling disrupts the balance of
proliferation and differentiation of endothelial cells.
ALK1 is involved in multiple stages of lymphatic development
When ALK1Fc treatment was started at P1, the lymphatic and
vascular defects were very dramatic and severe (Figures 2B,3A),
and in many cases very few lymphatic structures were present. We
next sought to examine the effects of delaying treatment on
lymphatic development to determine at what stage of lymphatic
development ALK1 signaling is required. When ALK1Fc treatment
was started at P3 (10 mg/kg, P3 and P5), a time point when
lymphatic vessels are present but the honeycomb structure is not
well established, the lymphatic defect was still dramatic, with
many missing connections, when analyzed at P8 (Figure 4A). The
blood vascular defect associated with ALK1Fc was significantly
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NIESSEN et al
BLOOD, 25 FEBRUARY 2010 䡠 VOLUME 115, NUMBER 8
Figure 3. The ALK1 pathway is directly involved in
lymphatic vessel development. (A) Analysis of lymphatic and vascular development at P6, after treatment
with ALK1Fc, ACVR2BFc, BMPR2Fc, or VEGFR3Fc
(10 mg/kg, P1 and P3). Lymphatic development is visualized by LYVE1 (green) staining in the tail dermis. Vascular development is visualized by isolectin-B4 (green) and
SMA (red) staining of the retina. Scale bar represents
250 ␮m. (B) Inhibition of rhBMP9 induced up-regulation
of Smad6 expression by the ALK1 immunized serum
(␣-ALK1-s) or affinity purified antibody (␣-ALK1-a),
whereas preimmune serum (Pre) has no effect. Red
boxes represent each data point (n ⫽ 3). (C) Inhibition of
rhBMP9 binding to ALK1 protein by treatment with the
ALK1 immunized serum (␣-ALK1-s) or affinity purified
antibody (␣-ALK1-a), whereas preimmune serum (Pre)
has no effect. Red boxes represent each data point
(n ⫽ 2). (D) ALK1-neutralizing antibody binds mouse
ALK1 but not ALK2, 3, 4, 5, 6, or 7. (E) Analysis of
lymphatic development at P6 after treatment with the
ALK1 neutralizing antibody (10 mg/kg, P1 and P3) in the
tail dermis. (F) Analysis of lymphatic development at P6
after treatment with the ALK1-neutralizing antibody
(10 mg/kg, P1 and P3) in the ear. Lymphatic development is visualized by LYVE1 (green) staining in the tail
dermis and ear. Scale bar represents 250 ␮m.
decreased if the initiation of the treatment was delayed (Figure 4A).
When ALK1Fc treatment was started at P8 (10 mg/kg, P8 and
P10), a time point when the honeycomb pattern is well established,
ALK1Fc failed to cause regression of the preexisting lymphatic
vessels, when analyzed at P12 (Figure 4B). By comparison,
treatment with VEGFR3Fc (10 mg/kg, P8 and P10) resulted in
significant regression of the honeycomb structure (Figure 4B). A
closer examination of the lymphatic vessels in the PBS- and
ALK1Fc-treated pups at P8 and P12 suggests a possible impairment in the maturation or remodeling of the lymphatic vessels. In
comparison with the control P8, the control P12 tail contained
fewer “ringed structures” in the honeycomb network, which
apparently have been remodeled into discreet vessels. In contrast,
the ringed structures were much more prevalent (arrows) in the P12
ALK1Fc-treated pups, reminiscent of the P8 control tails, suggesting that ALK1Fc blocked the remodeling process (Figure 4C).
Therefore, our data suggest ALK1 signaling is required for the
formation of initial lymphatic plexus as well as further remodeling
into mature network.
Distinct roles of ALK1 and VEGFR3 during lymphatic
development
Previous reports and Figure 4B have demonstrated that the
VEGFR3–VEGFC/D pathway is critically required for lymphatic
vessel development and survival.4,5 We sought to explore the effect
of ALK1 blockade on the dependence of lymphatic vessels on
VEGFR3-VEGFC/D signaling. At P4, a time point, when the
honeycomb pattern is being established, single-agent treatment of
ALK1Fc (10 mg/kg, P2) or VEGFR3Fc (1 mg/kg, P1) significantly
inhibited lymphatic vessel development (Figure 5A). The combination of both agents had a much more dramatic effect, with almost a
complete loss of lymphatic vessels (Figure 5A). We next analyzed
the effect of combined treatment on preexisting lymphatic vessels
at P8, when the honeycomb pattern is well established. Defects in
lymphatic development became evident 24 hours after administration of VEGFR3Fc, the defect was much more dramatic with a
treatment of 48 hours (Figure 5B). ALK1Fc (10 mg/kg, P3 and P5)
treatment results in a moderate lymphatic defect by P8 (Figure 5B).
However, the combination of ALK1Fc and VEGFR3Fc resulted in
a more profound effect on the lymphatic vascular network, with
accelerated regression of the preexisting lymphatic vessels (Figure 5B).
To investigate the fate of the lymphatic vessels after treatment
with ALK1Fc and VEGFR3Fc, endothelial cell apoptosis was
assessed by activated Caspase3 staining. The combined treatment
with both ALK1Fc and VEGFR3Fc resulted in a marked increase
in apoptotic lymphatic endothelial cells, suggesting that blockade
of ALK1 signaling sensitizes lymphatic vessels to VEGFC/D
depletion (Figure 5A). Interestingly, staining for podoplanin expression revealed that ALK1Fc treatment markedly blocked its expression (Figure 5A). By comparison, podoplanin expression was
normal in VEGFR3Fc-treated pups (Figure 5A). A similar block in
podoplanin expression by ALK1 inhibition was observed in the ear
and intestine lymphatic vessels (data not shown). Podoplanin has
been suggested to be a marker of terminally differentiated lymphatic vessels, suggesting ALK1 may regulate differentiation or
maturation of lymphatic vessels. It is possible that the failure to
induce Podoplanin expression contributes to the remodeling defects caused by ALK1 inhibition. Further experiments, however,
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BLOOD, 25 FEBRUARY 2010 䡠 VOLUME 115, NUMBER 8
Figure 4. ALK1 is involved in multiple stages of lymphatic development.
(A) Analysis of vascular and lymphatic development in P8 pups when ALK1Fc
treatment is started when the honeycomb pattern is being established (10 mg/kg,
P3 and P5). Lymphatic development is visualized by LYVE1 (green) staining in the tail
dermis, whereas vascular development is visualized by isolectin-B4 (green) staining
of the retina. Scale bar represents 500 ␮m (retina) and 250 ␮m (tail). (B) Analysis of
lymphatic development in P12 pups when ALK1 treatment is started when the
honeycomb structure is fully established (10 mg/kg, P8 and P10). Lymphatic
development is visualized by LYVE1 (green) staining in the tail dermis. Scale bar
represents 250 ␮m. (C) High-magnification images demonstrate the remodeling
process that occurs from P8 to P12 and the “ringed” structures that fail to be
remodeled in ALK1Fc-treated pups.
are required to test this hypothesis. In addition, ALK1Fc treatment
results in dilated lymphatic vessels in the intestinal outer wall (data
not shown), which is similar to what is reported to result from
podoplanin deficiency.9 These data suggest that ALK1 and VEGFR3
regulate different aspects of lymphatic development.
Interestingly, we found that ALK1Fc only modestly reduced the
expression of podoplanin in cultured endothelial cells as opposed
to its dramatic in vivo effect. Furthermore, BMP9 stimulation had
little effect on podoplanin expression (supplemental Figure 6). By
contrast the known ALK1 target genes SMAD6 and ID1 were
significantly changed in both conditions. These data suggest that
podoplanin is likely not a direct target of the ALK1 pathway and
that additional signaling cues are required for the decreased
podoplanin expression observed in vivo.
Discussion
ALK1, a member of the TGF-␤ receptor superfamily, is an
important regulator of blood vessel development.19 ALK1-deficient
mice die at the onset of lymphatic development, limiting the study
of its importance during this process.19 In the current study we
provide several lines of evidence suggesting that ALK1 pathway is
ALK1 REGULATES LYMPHATIC DEVELOPMENT
1659
Figure 5. Distinct roles of ALK1 and VEGFR3 during lymphatic development.
(A top) Analysis of lymphatic development in P4 pups after treatment with ALK1Fc
(10 mg/kg, P2), VEGFR3Fc (1 mg/kg, P1), or combined ALK1Fc and VEGFR3Fc.
Lymphatic development is visualized by LYVE1 staining (red), inset shows a greater
magnification image of the honeycomb structure that is disrupted by the treatments.
Scale bar represents 500 ␮m. (Middle) Analysis of podoplanin (green) and LYVE1
(red) expression after ALK1Fc, VEGFR3Fc, or combination treatments. Scale bar
represents 250 ␮m. (Bottom) Analysis of LYVE1 (green) and active Caspase3 (red)
expression after ALK1Fc, VEGFR3Fc, and combination treatment. Scale bar represents 250 ␮m. (B) Analysis of lymphatic development in P8 pups after ALK1Fc
(10 mg/kg, P3 and P5), VEGFR3Fc (10 mg/kg, P6 or P7), or combined ALK1Fc and
VEGFR3Fc treatment. Lymphatic development is visualized by LYVE1 staining (red).
Scale bar represents 250 ␮m.
also intimately involved in the regulation of lymphatic development. We show that the ALK1 pathway is functional in cultured
lymphatic endothelial cells. Lymphatic endothelial cells express
the key components of the ALK1 pathway. ALK1 target genes,
including Smad6, were up-regulated in lymphatic endothelial cells
upon stimulation with ALK1 ligands, BMP9 and BMP10. In vivo
blockade of ALK1 signaling with the use of soluble decoy
receptors or an ALK1-specific antibody resulted in dramatic
defects in lymphatic development in multiple organs. In the tail
skin of neonatal mice, the lymphatic defects caused by ALK1
inhibition were characterized by the inability of lymphatic endothelial cells to form the stereotypical honeycomb-like pattern of
capillary lymphatic vessels. We also show that ALK1 signaling is
required for the maturation and remodeling of lymphatic vessels in
the tail skin. In ALK1Fc-treated neonatal mice, the extension of
lymphatic capillary into the intestinal villi was invariably reduced
and in many cases was completely stalled. ALK1 inhibition was
associated with the marked loss of podoplanin expression. Podoplanin has been suggested to be a marker of terminally differentiated
lymphatic vessels. Podoplanin-deficient mice have defects in
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1660
BLOOD, 25 FEBRUARY 2010 䡠 VOLUME 115, NUMBER 8
NIESSEN et al
lymphatic development. It is possible that loss of podoplanin
expression contributes to the abnormal lymphatic development
upon ALK1 inhibition.
In our current study, inhibition of ALK1 signaling in neonatal
mice by the use of soluble decoy receptors resulted in failed
remodeling of the primary retinal plexus into mature vascular
pattern, defective arteriogenesis, and vessel dilation (Figure 2A and
data not shown), which is consistent with the vascular defects
resulting from ALK1 deficiency during embryogenesis. These
effects, however, are apparently limited to the neovascular development, the existing blood vessels are largely unaltered. For instance,
ALK1Fc has little effect on the skin microvessels or the capillary
within the villi of small intestines. Therefore, it is unlikely the
observed lymphatic defect is secondary to impaired blood vascular
endothelium.
Previous studies and our current work suggest that ALK1 has a
dual role, in both vascular and lymphatic development. Such
finding of dual action of a signaling pathway is not unprecedented.
VEGFR3, in addition to its well-established role in lymphangiogenesis, is also essential for the early blood vessel development in
embryos.34 Studies of gene-targeted mice have suggested that the
Tie2–angiopoiteins signaling axis not only is important for the
remodeling and stability of blood vascular endothelium but also
plays a role in lymphangiogenesis.13 Interestingly, the embryonic
lethality resulting from targeted deletion of EphrinB2, which is
essential for the angiogenic remodeling of blood vessels in early
embryo, initially precluded the recognition of its role in the
development of lymphatic vessels.14,15 It should be pointed out that
the phenotypic outcomes resulting from the disruption of these
pathways are oftentimes distinct between the 2 vascular systems.
BMP9 is a circulating factor predominantly expressed in liver,
and BMP10 is specifically expressed in the developing and adult
heart. Previous in vitro studies have suggested that BMP9 and
BMP10 are potential ligands for ALK1. BMP10-knockout mice die
during development as the result of cardiac defects before lymphatic development,35 and mice deficient in BMP9 have not been
reported. Future studies that use genetic models of mice deficient in
BMP9 and BMP10 or ligand specific antibodies will help elucidate
the relative contribution of the 2 ligands to ALK1-mediated
signaling in regulating angiogenesis and lymphatic angiogenesis.
The findings from experimental models of lymphatic metastasis
and clinicopathologic analyses of human cancer have suggested
that targeting the VEGFR3–VEGFC/D pathway might be a promising approach of anticancer therapy by reducing the metastatic
spread of tumor cells through tumor lymphatic vasculature. Unlike
VEGFR3 signaling, which is essential for the proliferation and
survival of lymphatic endothelial cells,5 our data demonstrate that
ALK1 signaling is apparently involved in controlling the differentiation of lymphatic endothelial cells to influence the lymphatic
vascular development and remodeling. Because inhibition of
ALK1 signaling sensitizes lymphatic endothelial cells to VEGFC
depletion, combined inhibition of both VEGFR3 and ALK1
pathways might be more effective at blocking lymphangiogenesis
and tumor metastasis.
In conclusion, our findings reveal a novel aspect of ALK1
signaling in regulating lymphatic development, in addition to its
known role in vascular development, and provide a rationale for
targeting ALK1 pathway to modulate lymphangiogenesis in human
diseases.
Acknowledgments
We kindly thank XiaoHuan Liang for reagents and Dr Greg
Plowman and Dr Weilan Ye for helpful discussions.
Authorship
Contribution: K.N. designed and performed the experiments and
wrote the paper; G.Z. designed and performed the experiments;
J.B.R. and H.C. performed the experiments; M.Y. designed the
experiments and wrote the paper; and all authors discussed the
results and commented on the manuscript.
Conflict-of-interest disclosure: The authors declare no competing financial interests.
Correspondence: Minhong Yan, Genentech Inc, Tumor Biology
and Angiogenesis, 1 DNA Way, MS 93B, South San Francisco, CA
94080; e-mail: [email protected].
References
1. François M, Caprini A, Hosking B, et al. Sox18
induces development of the lymphatic vasculature in mice. Nature. 2008;456(7222):643-647.
2. Wigle JT, Oliver G. Prox1 function is required for
the development of the murine lymphatic system.
Cell. 1999;98(6):769-778.
3. Oliver G, Alitalo K. The lymphatic vasculature:
recent progress and paradigms. Annu Rev Cell
Dev Biol. 2005;21:457-483.
4. Karkkainen MJ, Haiko P, Sainio K, et al. Vascular
endothelial growth factor C is required for sprouting of the first lymphatic vessels from embryonic
veins. Nat Immunol. 2004;5(1):74-80.
5. Mäkinen T, Jussila L, Veikkola T, et al. Inhibition
of lymphangiogenesis with resulting lymphedema
in transgenic mice expressing soluble VEGF
receptor-3. Nat Med. 2001;7(2):199-205.
6. Wigle JT, Harvey N, Detmar M, et al. An essential
role for Prox1 in the induction of the lymphatic
endothelial cell phenotype. EMBO J. 2002;21(7):
1505-1513.
7. Hong YK, Harvey N, Noh YH, et al. Prox1 is a
master control gene in the program specifying
lymphatic endothelial cell fate. Dev Dyn. 2002;
225(3):351-357.
8. Mahtab EA, Wijffels MC, Van Den Akker NM, et
al. Cardiac malformations and myocardial abnormalities in podoplanin knockout mouse embryos:
Correlation with abnormal epicardial development. Dev Dyn. 2008;237(3):847-857.
9. Schacht V, Ramirez MI, Hong YK, et al. T1alpha/
podoplanin deficiency disrupts normal lymphatic
vasculature formation and causes lymphedema.
EMBO J. 2003;22(14):3546-3556.
10. Huang SS, Liu IH, Smith T, Shah MR, Johnson
FE, Huang JS. CRSBP-1/LYVE-l-null mice exhibit
identifiable morphological and functional alterations of lymphatic capillary vessels. FEBS Lett.
2006;580(26):6259-6268.
11. Laakkonen P, Waltari M, Holopainen T, et al. Vascular endothelial growth factor receptor 3 is involved in tumor angiogenesis and growth. Cancer
Res. 2007;67(2):593-599.
role is rescued by angiopoietin-1. Dev Cell. 2002;
3(3):411-423.
14. Adams RH, Wilkinson GA, Weiss C, et al. Roles
of ephrinB ligands and EphB receptors in cardiovascular development: demarcation of arterial/
venous domains, vascular morphogenesis, and
sprouting angiogenesis. Genes Dev. 1999;13(3):
295-306.
15. Mäkinen T, Adams RH, Bailey J, et al. PDZ interaction site in ephrinB2 is required for the remodeling of lymphatic vasculature. Genes Dev. 2005;
19(3):397-410.
16. Massagué J. TGFbeta in cancer. Cell. 2008;134(2):
215-230.
17. Watabe T, Miyazono K. Roles of TGF-beta family
signaling in stem cell renewal and differentiation.
Cell Res. 2009;19(1):103-115.
12. Lohela M, Bry M, Tammela T, Alitalo K. VEGFs
and receptors involved in angiogenesis versus
lymphangiogenesis. Curr Opin Cell Biol. 2009;
21(2):154-165.
18. David L, Mallet C, Vailhe B, Lamouille S, Feige
JJ, Bailly S. Activin receptor-like kinase 1 inhibits
human microvascular endothelial cell migration:
potential roles for JNK and ERK. J Cell Physiol.
2007;213(2):484-489.
13. Gale NW, Thurston G, Hackett SF, et al.
Angiopoietin-2 is required for postnatal angiogenesis and lymphatic patterning, and only the latter
19. Urness LD, Sorensen LK, Li DY. Arteriovenous
malformations in mice lacking activin receptor-like
kinase-1. Nat Genet. 2000;26(3):328-331.
From www.bloodjournal.org by guest on June 17, 2017. For personal use only.
BLOOD, 25 FEBRUARY 2010 䡠 VOLUME 115, NUMBER 8
20. Seki T, Yun J, Oh SP. Arterial endotheliumspecific activin receptor-like kinase 1 expression
suggests its role in arterialization and vascular
remodeling. Circ Res. 2003;93(7):682-689.
21. Berg JN, Gallione CJ, Stenzel TT, et al. The activin receptor-like kinase 1 gene: genomic structure and mutations in hereditary hemorrhagic telangiectasia type 2. Am J Hum Genet. 1997;61(1):
60-67.
22. McAllister KA, Grogg KM, Johnson DW, et al. Endoglin, a TGF-beta binding protein of endothelial
cells, is the gene for hereditary haemorrhagic telangiectasia type 1. Nat Genet. 1994;8(4):345351.
23. David L, Mallet C, Mazerbourg S, Feige JJ, Bailly
S. Identification of BMP9 and BMP10 as functional activators of the orphan activin receptor-like
kinase 1 (ALK1) in endothelial cells. Blood. 2007;
109(5):1953-1961.
24. Chen YG, Massague J. Smad1 recognition and
activation by the ALK1 group of transforming
growth factor-beta family receptors. J Biol Chem.
1999;274(6):3672-3677.
25. Ridgway J, Zhang G, Wu Y, et al. Inhibition of Dll4
26.
27.
28.
29.
30.
ALK1 REGULATES LYMPHATIC DEVELOPMENT
signalling inhibits tumour growth by deregulating
angiogenesis. Nature. 2006;444(7122):10831087.
Blanco FJ, Santibanez JF, Guerrero-Esteo M,
Langa C, Vary CP, Bernabeu C. Interaction and
functional interplay between endoglin and ALK-1,
two components of the endothelial transforming
growth factor-beta receptor complex. J Cell
Physiol. 2005;204(2):574-584.
Upton PD, Davies RJ, Trembath RC, Morrell NW.
Bone morphogenetic protein (BMP) and activin
type II receptors balance BMP9 signals mediated
by activin receptor-like kinase-1 in human pulmonary artery endothelial cells. J Biol Chem. 2009;
284(23):15794-15804.
Kim KE, Sung HK, Koh GY. Lymphatic development in mouse small intestine. Dev Dyn. 2007;
236(7):2020-2025.
Oh SP, Li E. The signaling pathway mediated by
the type IIB activin receptor controls axial patterning and lateral asymmetry in the mouse. Genes
Dev. 1997;11(14):1812-1826.
Délot EC, Bahamonde ME, Zhao M, Lyons KM.
BMP signaling is required for septation of the out-
1661
flow tract of the mammalian heart. Development.
2003;130(1):209-220.
31. Beppu H, Kawabata M, Hamamoto T, et al. BMP
type II receptor is required for gastrulation and
early development of mouse embryos. Dev Biol.
2000;221(1):249-258.
32. Deng Z, Morse JH, Slager SL, et al. Familial primary pulmonary hypertension (gene PPH1) is
caused by mutations in the bone morphogenetic
protein receptor-II gene. Am J Hum Genet. 2000;
67(3):737-744.
33. Shi Y, Massague J. Mechanisms of TGF-beta signaling from cell membrane to the nucleus. Cell.
2003;113(6):685-700.
34. Tammela T, Zarkada G, Wallgard E, et al. Blocking VEGFR-3 suppresses angiogenic sprouting
and vascular network formation. Nature. 2008;
454(7204):656-660.
35. Chen H, Shi S, Acosta L, et al. BMP10 is essential for maintaining cardiac growth during murine
cardiogenesis. Development. 2004;131(9):22192231.
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2010 115: 1654-1661
doi:10.1182/blood-2009-07-235655 originally published
online November 10, 2009
ALK1 signaling regulates early postnatal lymphatic vessel development
Kyle Niessen, Gu Zhang, John Brady Ridgway, Hao Chen and Minhong Yan
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