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VASCULAR BIOLOGY
Novel function for blood platelets and podoplanin in developmental separation of
blood and lymphatic circulation
*Pavel Uhrin,1 *Jan Zaujec,1 Johannes M. Breuss,1 Damla Olcaydu,1 Peter Chrenek,1 Hannes Stockinger,2 Elke Fuertbauer,2
Markus Moser,3 Paula Haiko,4 Reinhard Fässler,3 Kari Alitalo,4 Bernd R. Binder,1 and Dontscho Kerjaschki5
1Department of Vascular Biology and Thrombosis Research, Center for Biomolecular Medicine and Pharmacology, Medical University of Vienna, Vienna,
Austria; 2Department of Molecular Immunology, Center for Physiology, Pathophysiology and Immunology, Medical University of Vienna, Vienna, Austria;
3Department of Molecular Medicine, Max Planck Institute of Biochemistry, Martinsried, Germany; 4Molecular/Cancer Biology Laboratory and Department of
Pathology, Haartman Institute and Helsinki University Hospital, Biomedicum Helsinki, University of Helsinki, Helsinki, Finland; and 5Clinical Institute for
Pathology, Medical University of Vienna, Vienna, Austria
During embryonic development, lymph
sacs form from the cardinal vein, and
sprout centrifugally to form mature lymphatic networks. Separation of the lymphatic from the blood circulation by a
hitherto unknown mechanism is essential for the homeostatic function of the
lymphatic system. O-glycans on the lymphatic endothelium have recently been
suggested to be required for establishment and maintenance of distinct blood
and lymphatic systems, primarily by me-
diating proper function of podoplanin.
Here, we show that this separation process critically involves platelet activation
by podoplanin. We found that platelet
aggregates build up in wild-type embryos
at the separation zone of podoplaninⴙ
lymph sacs and cardinal veins, but not in
podoplaninⴚ/ⴚ embryos. Thus, podoplaninⴚ/ⴚ mice develop a “nonseparation”
phenotype, characterized by a bloodfilled lymphatic network after approximately embryonic day 13.5, which, how-
ever, partially resolves in postnatal mice.
The same embryonic phenotype is also
induced by treatment of pregnant mice
with acetyl salicylic acid, podoplaninblocking antibodies, or by inactivation of
the kindlin-3 gene required for platelet
aggregation. Therefore, interaction of endothelial podoplanin of the developing
lymph sac with circulating platelets from
the cardinal vein is critical for separating
the lymphatic from the blood vascular
system. (Blood. 2010;115(19):3997-4005)
Introduction
The lymphatic vascular system supplements the blood circulation
by draining extravasated cells, proteins, and fluids from the tissues
back to the blood circulation, which necessitates separation of the
lymphatic from the blood circulatory systems during development.1,2 The lymphatic vasculature starts to develop in mouse
embryos at around embryonic day (E) 10.5, when the cardiovascular system is already functioning. Focally, clusters of endothelial
cells in the cardinal vein commit to the lymphatic phenotype, and
sprout to form the primary lymphatic sacs,1-4 from where part of the
peripheral lymphatic vasculature is generated by further centrifugal
growth. In addition, lymphatic vessels also may be generated in the
periphery from precursor cells5 in embryos, as well as in adult
tissues in pathologic conditions.6 The lymphatic circulation subsequently becomes separated from the blood circulation by currently
unknown mechanisms, and only the connection between the veins
in the neck region and the thoracic ducts is left open in adults4,7 to
permit the entrance of lymph into the blood stream.
Endothelial cells of lymphatic vessels are characterized by the
expression of specific markers, such as the abundant lymphatic
endothelial membrane mucoprotein podoplanin and the CD44
hyaluronan receptor-related protein Lyve-1.1 The former is expressed under the control of the transcription factor Prox-1,8-10 a
“master” control gene for lymphatic development. Disruption of
the Prox-1 or VEGF-C gene or of certain other genes specific to the
lymphatic endothelium10 results in a so called “lymphatic” pheno-
type characterized by the absence or by severe defects of lymphatic
vessels.8,11-14 In contrast, deletion of Lyve-115 had no consequence
on lymphatic development. Unexpectedly, however, disruption of
Syk or SLP-76,16 genes involved in the development of T lymphocytes17,18 or B lymphocytes19,20 and, importantly, also for the
function of platelets21-23 resulted in a “nonseparation” phenotype
that was characterized by blood filling of the lymphatic vessels and
hemorrhages. A similar phenotype was found in endothelial cell
O-glycan deficiency that in turn decreased podoplanin expression.24
Here, we confirm a role for podoplanin25,26 in the separation of
the lymphatic from the venous system during development and
attribute its role in this process to its interaction with platelets.
Podoplanin-deficient mice were first generated in the 129/SvEv
background,27 but study of their postnatal vascular development
was impossible, because they died at birth from respiratory failure
resulting from lack of podoplanin in lung alveolar type I cells.28
These embryos show defects in lymphatic vessel patterning,
diminished lymphatic transport, congenital lymphedema, and lymphatic dilatation, but they appeared not to have misconnections
between blood and lymphatic vessels.29 Re-evaluation of these
mice by Fu et al,24 however, revealed that they had disorganized
and blood-filled lymphatic vessels at birth.24
We have generated podoplanin⫺/⫺ mice in a different background30 and backcrossed them into C57bl/6 mice. We found that a
Submitted April 20, 2009; accepted January 4, 2010. Prepublished online as Blood
First Edition paper, January 28, 2010; DOI 10.1182/blood-2009-04-216069.
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.
*P.U. and J.Z. contributed equally to this article.
The online version of this article contains a data supplement.
BLOOD, 13 MAY 2010 䡠 VOLUME 115, NUMBER 19
© 2010 by The American Society of Hematology
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BLOOD, 13 MAY 2010 䡠 VOLUME 115, NUMBER 19
UHRIN et al
fraction of these mice survive without developing of a pulmonary
phenotype, allowing more detailed studies. These podoplanin⫺/⫺
mice showed a full-blown “nonseparation” phenotype in line with
the report by Fu et al.24 The observation that podoplanin induces
platelet activation31,32 by interacting with the platelet membrane
lectin CLEC-232 allowed us to demonstrate that local podoplanininduced platelet aggregation and subsequent closure of the junction
between the cardinal vein and the developing lymph sacs are
responsible for separating of the lymphatic from the blood vessels.
This “platelet hypothesis” of vessel separation was corroborated by
several different means, indicating that the absence of podoplanin
interferes directly with platelet aggregation and results in the
“nonseparation” phenotype.
Methods
Generation of knockout mice
Podoplanin-deficient mice in the 129S/v ⫻ Swiss background were generated by disrupting the coding region of the podoplanin gene, and were
characterized as described.30 These mice were backcrossed onto the
C57BL/6 background for 6 generations and also bred into the VEGFR-3
gene–targeted strain that directs ␤-galactosidase expression into lymphatic
endothelial cells.33 Kindlin-3–deficient mice were generated as described.34
Animal care and all experimental procedures were approved by the
Animal Experimental Committee of the Medical University of Vienna,
and by the Austrian Ministry of Science (License No. 1321/115, and
66.009/0103-C/Gl/2007).
Immunohistochemistry
Tissue samples were snap-frozen in liquid N2-cooled OCT (Miles), and
10-␮m cryostat sections were prepared. For immunofluorescence, the
following primary antibodies were used: rabbit polyclonal anti–mouse
Lyve-1 (Abcam Ltd), rat anti–mouse monoclonal CD31 (Becton Dickinson), hamster monoclonal anti–mouse podoplanin antibody (Acris), rabbit
polyclonal anti–Prox-1 antibody (AngioBio Co), rat anti–mouse monoclonal Ter119 antibody (eBioscience), and monoclonal rat anti–mouse antibody directed against the activated form of mouse platelet integrin ␣IIb␤3
(clone JON/A-PE; Emfret-Analytics). The nuclei were counterstained with
DAPI (Sigma-Aldrich). Secondary reagents were goat anti–rabbit IgG
(Invitrogen) labeled with Alexa Fluor 488 or 568, goat anti–mouse IgG
labeled with Alexa Fluor 488 or 647 (Invitrogen), conjugated biotinylated
rabbit anti–hamster polyclonal IgG (Acris), and streptavidin Alexa Fluor
488 or 568 (Invitrogen). Fluorescent imaging was performed on a Zeiss
LSM 510 META confocal system in multitrack mode, using a 40⫻/1.3 plan
Neofluar oil objective, pinhole sizes between 0.7 and 1.5 ␮m and the
appropriate standard laser-filter combinations (images were handled with
the Zeiss AIM software package; Zeiss), or on a motorized Olympus AX70
microscope by 10⫻ and 20⫻ UPlanApo air objective lenses and using a
cooled F-View II Camera and the CellP imaging software (Olympus Soft
Imaging Solutions). Some tissue samples were also fixed in 4% formaldehyde, and processed for standard paraffin embedding, followed by immunohistochemical detection of Lyve-1 or podoplanin, as described.26 ␤-galactosidase activity in transgenic podoplanin⫹/⫹ and podoplanin⫺/⫺ mice was
detected as described.33
In vitro platelet adhesion assay
Podoplanin-expressing NIH-3T3 cells were generated by retroviral transfection, using human podoplanin cDNA cloned into pBMN-Z plasmid (kindly
provided by Gerry Nolan, Stanford University) and transfected into the
Phoenix-Eco packaging cell line (Gentaur). Retrovirus-transduced cells
were purified by fluorescence-activated cell sorter (FACS)–sorting and
single-cell cloning. Cells transfected with empty vector were used as
controls. Human umbilical vein endothelial cells (Technoclone) that were
devoid of podoplanin and human dermal lymphatic endothelial cells were
prepared as described.35
For the purification of mouse platelets, blood was drawn from the
periorbital sinus of mice into heparinized capillaries and centrifuged at
230g for 7 minutes, and the platelet-rich fraction was collected. Human
platelet concentrates were obtained from the Department of Blood Grouping and Transfusion Medicine, Medical University of Vienna. A total of
3 ⫻ 106 platelets were suspended in 70 ␮L of phosphate-buffered saline
(PBS), and injected via microinjection needles at a flow rate of 3 ␮L/minute
for 20 minutes onto confluent monolayers of NIH-3T3 cells or lymphatic or
blood endothelial cells cultured in 24-well plates that contained 2 mL of
medium. For inhibition experiments, rabbit IgG directed against human
podoplanin36 was added at a concentration of 50 ␮g/mL to the incubation
media. In other experiments, the isolated platelets were preincubated in
500 ␮g/mL acetyl salicylic acid for 3 hours at room temperature before
infusion into the culture dishes. Platelet movement and aggregate formation
were recorded by video microscopy with an F-View camera (Olympus) and
an Olympus IX70 microscope using the AnalySIS software (Soft Imaging
System).
In vivo inhibition of formation of platelet aggregates
A total of 2 mg of rabbit anti–human podoplanin IgG or control preimmune
IgG from the same rabbit were injected intravenously into ketamine/xylazineanesthetized pregnant podoplanin⫹/⫹ females at E10.5, and the embryos
were harvested at E13.5. In other experiments, the drinking water of
pregnant podoplanin⫹/⫹ mothers was supplemented with acetyl salicylic
acid (500 mg/L, 250 mg/L, or 75 mg/L) starting from approximately E8.5
until E16.5, and embryos were collected at E13.5, E14.5, and E16.5. The
amount of acetyl salicylic acid consumed by a mouse was estimated to be
45 to 70 mg/kg/d, 25 to 33 mg/kg/d, and 6 to 9 mg/kg/d, respectively.
Functional tracer experiments
Podoplanin⫹/⫹ and podoplanin⫺/⫺ mice aged 3 days and 8 weeks were
injected with 50 ␮L or 250 ␮L of a 1% Chicago sky blue 6B (SigmaAldrich) in PBS into the periorbital sinus under ketamine/xylazine anesthesia. At 1 minute after injection, the abdominal wall was dissected and fixed
in 4% formalin, and micrographs were taken with a stereo microscope SZ40
(Olympus).
Double-labeling experiments were performed in 8-week-old podoplanin⫹/⫹ and podoplanin⫺/⫺ mice. We injected 30 ␮L of 1% Chicago sky blue
6B in PBS intradermally into the hind footpad, followed 2 minutes later by
intravenous injection of Indian ink (Winsor & Newton) into the periorbital
sinus. After 1 minute, the skin of the hind limb and the hips were peeled off,
and micrographs were taken.
Podoplanin⫹/⫹ and podoplanin⫺/⫺ mice aged 3 days were also injected
with 50 ␮L of FITC-labeled Bandeiraea simplicifolia lectin (1 ␮g/␮L in
PBS; Sigma-Aldrich) into the periorbital sinus under ketamine/xylazine
anesthesia. At 1 minute after injection, the abdominal skin was snap-frozen
in liquid N2-cooled OCT, and FITC-lectin–perfused tissue sections were
additionally stained for Lyve-1 and Ter119.
Blood chemistry
Blood samples were collected by decapitation of postnatal day (P) 1 to P5
mice. Activated partial thromboplastin time was determined with a
commercial kit (Technoclone). Hematocrit was determined by standard
procedure. Triglycerides were analyzed enzymatically, using a commercial
kit (Roche Diagnostics).
Statistical analysis
Results are given as means plus or minus SD. Statistical significance was
calculated by paired and unpaired t test, or by analysis of variance
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BLOOD, 13 MAY 2010 䡠 VOLUME 115, NUMBER 19
PODOPLANIN ACTIVATE PLATELETS IN FORMING LYMPHATIC
3999
Results
129S/v ⫻ Swiss background. Podoplanin-deficient neonates were
smaller (Figure 1A-B), and approximately 55% died during the first
postnatal week. Importantly, however, approximately 20% survived (Figure 1B), had normal weights and life spans, and were
fertile.
Generation and gross phenotype of podoplaninⴚ/ⴚ mice
Development of vascular abnormalities in podoplaninⴚ/ⴚ mice
We generated podoplanin-deficient mice in a mixed 129S/
v ⫻ Swiss background by disrupting the entire coding region of the
podoplanin gene. Our strategy inactivated the podoplanin gene,
which has been previously shown by Northern and Western
blotting.30 We subsequently backcrossed the mice for 6 generations
onto the C57Bl/6 background. The phenotype of these backcrossed
podoplanin⫺/⫺ mice resembled the one observed in the mixed
In podoplanin⫺/⫺ embryos of the mixed as well as the C57BL/6
background, tortuous cutaneous blood-perfused vessels (Figure
1D; black arrows) were detected as early as at E13.5 in the jugular
and axillary areas, extending toward the hind limb and the
abdominal region (Figure 1E), and at E15 and E16.5 also toward
most of the lateral aspects (Figure 1F-G). These vessels were
Lyve-1⫹ and contained erythrocytes (Figure 1I), indicating their
(ANOVA) as indicated. Significance was assigned to P values less than .05.
Experiments were performed at least in triplicate if not stated otherwise.
A
C
B
P5.0
+/+
+/−
−/−
*
*
Time (s)
Weight (g)
4.5
4
3.5
3
2.5
2
1.5
1
0.5
0
days
1
2
3
4
5
60
50
40
30
20
10
0
+/+
80
20
0
-/-
+/+
+/−
−/−
60
40
12 3 4 5 6 7 142128 2 4 681012141618
days weeks
D
Figure 1. Macroscopic phenotype of podoplaninⴚ/ⴚ mice in
postnatal and embryonic development. (A-B) Podoplanin⫺/⫺
mice in postnatal development are significantly smaller (right
mouse) than podoplanin⫹/⫹ littermates (left mouse) or heterozygotes. Podoplanin⫺/⫺ mice also show ectatic vessels and some
bleeding in their skin. Approximately 20% of podoplanin⫺/⫺ mice
reach fertility age. (C) No significant differences between podoplanin⫺/⫺ and podoplanin⫹/⫹ mice are detected in their activated
partial thromboplastin time (aPTT), but a lower hematocrit is found
in P5 podoplanin⫺/⫺ mice. Values in panels B and C are
means ⫾ SD. (D-G) Blood-filled capillary network (black arrows)
that increases in size with age, and blood extravasations (white
arrows) are found in podoplanin⫺/⫺, but not in podoplanin⫹/⫹
embryos (H); Olympus SZ zoom stereo microscope, Sony DSC
W200 camera with adapter VAE-WD. Immunofluorescence staining (I) identifies the dermal blood-filled vessels of podoplanin⫺/⫺
embryos as Lyve-1⫹ lymphatics (green) that contain Ter119⫹
erythrocytes (red). Cell nuclei are stained with DAPI (blue);
Olympus AX70, 20 ⫻/0.7 UPlanApo air objective. Scale bar in
panel I equals 50 ␮m.
G
E13.5-/-
E16.5-/-
(%)
Survival (%)
100
E
E14.0-/-
H
E16.5+/+
months
F
60
50
40
30
20
10
0
aPTT
P > .05
n = 12
n = 12
+/+ –/–
Hematocrit
P < .05
n = 12
n = 11
+/+
–/–
E15.0-/-
I
E16.5-/-
Lyve-1 Ter119
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BLOOD, 13 MAY 2010 䡠 VOLUME 115, NUMBER 19
UHRIN et al
lymphatic origin and a connection to blood vessels. Cutaneous
bleedings (white arrows) were found mainly in the flank regions
(Figure 1E-G). Microvascular abnormalities disappeared postnatally after 10 to 14 days, and were absent from mice surviving
beyond this point. The cutaneous vasculature of podoplanin⫹/⫹ and
podoplanin⫹/⫺ littermates was devoid of aberrant vessels during the
entire developmental period (Figure 1H). Postnatally, some podoplanin⫺/⫺ animals developed chylothorax or chylous ascites and
exhibited portolymphatic shunts (supplemental Figure 1, available
on the Blood Web site; see the Supplemental Materials link at the
top of the online article). The thromboplastin time was similar
between wild-type and podoplanin⫺/⫺ littermates, whereas the
hematocrit was significantly reduced in podoplanin⫺/⫺ mice (Figure 1C), presumably due to bleeding into tissues.
Lymphatic vessels of podoplaninⴚ/ⴚ embryos exhibit a
“nonseparation” phenotype
The macroscopic dermal aspect of the abdominal skin of podoplanin⫹/⫹ P3 mice showed blood-filled veins and venules (Figure 2A).
The dermal lymphatics identified by double fluorescence for
A Podoplanin+/+ β-gal B
C
F
Podoplanin−/−
D Podoplanin−/−
Podoplanin+/+
Prox-1 Lyve-1
Podoplanin−/−
Lyve-1 Ter119
Prox-1 and Lyve-1 did not contain blood (Figure 2C). In contrast,
tortuous blood-filled blind-ending cutaneous vessels were encountered in podoplanin⫺/⫺ mice (Figure 2B). They were identified as
lymphatics, because their endothelial cells expressed Lyve-1,
Prox-1 (Figure 2D), and ␤-galactosidase when intercrossed with a
mouse strain that expresses this reporter under the VEGFR-3
promoter and thus, exclusively in lymphatic endothelial cells33
(Figure 2A-B insets). The erythrocyte-containing lymphatic vessels were indicative of a “nonseparation” phenotype of the
lymphatic and blood vasculatures. Tortuous blind-ending and
blood-filled lymphatic vessels were also found in the intestine and
in the thoracic walls (supplemental Figure 1).
Abnormal communications between the blood and the lymphatic circulatory systems were visualized upon injection of
Chicago sky blue intravenously into the periorbital sinus. In
podoplanin⫹/⫹ mice, the tracer appeared in the large subcutaneous
blood vessels, but not in lymphatic vessels, whereas in podoplanin⫺/⫺ mice, both vessel types were filled with the tracer (supplemental Figure 2). Interestingly, the tracer was absent from the
β-gal
E Podoplanin−/−
Chicago sky blue
G H Podoplanin+/+
FITC-lectin
Figure 2. Malformations and blood perfusion (the “nonseparation” phenotype) of cutaneous lymphatic vessels in podoplaninⴚ/ⴚ mice. (A) In a wild-type P3, mouse the en face view of
the dermal side of the skin of the flanks and abdomen shows
blood-filled veins of different calibers. The lymphatic system is
visualized by VEGFR-3–driven, lymphatic endothelium–specific
expression of ␤-galactosidase (inset). (B) By contrast, in P3
podoplanin⫺/⫺ mice, a fractal pattern of blood-filled, tortuous,
blind-ending vessels are found, besides occasional areas of
bleeding (white asterisk). These vessels are of lymphatic origin
because they express VEGFR-3–driven ␤-galatosidase (inset);
Olympus SZ zoom stereo microscope, Sony DSC W200 camera
with adapter VAE-WD. (C) Sections through the flank skin of P3
podoplanin⫹/⫹ mice show Lyve-1–expressing lymphatics (brown)
in the upper dermis. These vessels also express Prox-1 (red) on
top of Lyve-1 (green) by double immunofluorescence (inset).
(D) Skin of age-matched podoplanin⫺/⫺ mice contains extended,
randomly distributed lymphatic vessels. Their endothelial cells
express Prox-1 (red) and Lyve-1 (green), and their dilated lumen
contains erythrocytes (yellow) that also have leaked into the
perivascular space (white arrowhead; inset). (E) In a P3 podoplanin⫺/⫺ mouse, intravenously injected Chicago sky blue fills dermal
veins and arteries (“purple vessel” on the left side of the image;
Olympus SZ40); however, the blood-filled lymphatics (green
arrowheads) are not entered by the tracer, indicating that this
compartment is already sequestered from the circulation in this P3
mouse. Identification of perfused lymphatic vessels (F) by intravenous injection of FITC-lectin (green). Lyve-1 is blue and Ter119 is
red. FITC-lectin is not detectable in the small erythrocyte-filled
lymphatic capillaries (arrow in panel G). (H) Control injection of
FITC-lectin into wild-type mice. FITC-lectin is confined to blood
vessels and not detectable in large (arrow) or small lymphatic
vessels (the latter are not shown in this picture). Scale bars in
panels A, B, and E equal 300 ␮m; in panels C and D, 100 ␮m; in
panel D inset, 15 ␮m; and in panels F through H, 50 ␮m.
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BLOOD, 13 MAY 2010 䡠 VOLUME 115, NUMBER 19
PODOPLANIN ACTIVATE PLATELETS IN FORMING LYMPHATIC
subcutaneous tortuous blood-filled initial “lymphatic” microvasculature of P3 podoplanin⫺/⫺ mice, indicating that at this point the
microvessels had become disconnected from the circulation (Figure 2E). These findings were confirmed by intravenous injection of
FITC-lectin into P3 podoplanin⫺/⫺ mice, where the FITC signal
was detected primarily in large lymphatic vessels that were
Lyve-1⫹ and contained Ter119⫹ erythrocytes (Figure 2F). Such
FITC-lectin signal was absent from most of the capillaries positive
for Lyve-1 and Ter119 (Figure 2G). In contrast, in podoplanin⫹/⫹
mice, the FITC signal was restricted to blood vessels and absent
from Lyve-1⫹ vessels (Figure 2H). The communication of large
lymphatic vessels and veins persisted in adult podoplanin⫺/⫺ mice
(supplemental Figure 2).
Podoplanin induces platelet aggregation under flow conditions
in vitro
It has been reported previously that podoplanin can induce platelet
aggregation in vitro in a static assay system.37 To demonstrate that
podoplanin-expressing cells induce platelet aggregation under flow
A
conditions that resemble the in vivo situation, we expressed
podoplanin in NIH-3T3 fibroblasts at levels comparable with
dermal lymphatic endothelial cells (data not shown). Human or
mouse platelet suspensions were then continuously superfused onto
the surface of fibroblast or endothelial monolayers to produce
permanent laminar flow at speeds similar to that found in veins and
presumably also in the patent lymph sacs. In these conditions,
podoplanin-transfected NIH-3T3 fibroblasts triggered the formation of platelet aggregates already in 5 minutes, and aggregation
was complete in 20 minutes (Figure 3A), regardless of whether the
platelets were from wild-type or podoplanin-deficient mice. In
sharp contrast, neither wild-type nor podoplanin⫺/⫺ platelets from
mice (Figure 3B) or humans (data not shown) formed aggregates
on untransfected or mock-transfected NIH-3T3 fibroblasts. Furthermore, incubation of platelets with podoplanin⫹/⫹-transfected fibroblasts in the presence of a blocking rabbit anti-podoplanin IgG36
inhibited the formation of platelet aggregates. Finally, platelet
aggregation could also be induced on cultured lymphatic, but not
blood vascular endothelial cells (supplemental Table 1).
NIH-3T3/Podoplanin
0 min
20 min
C Podoplanin
B
NIH-3T3
0 min
Platelet integrin αIIbβ3
D
20 min
Lyve-1
Platelet integrin αIIbβ3
E12.5+/+
E12.5 +/+
Cardinal
vein
Cardinal
vein
Lymph sac
Lymph sac
E
Podoplanin Platelet integrin αIIbβ3
F
Lyve-1
Platelet integrin αIIbβ3
E13.5 +/+
Figure 3. Platelet aggregation is driven by podoplanin and
linked to separation of the lymph sacs from the cardinal
veins. (A-B) Still frames of movies showing coincubation under
flow conditions of isolated normal mouse platelets with monolayers of NIH-3T3 cells that transgenetically express podoplanin (A)
or NIH-3T3 cells that were transfected with an empty vector (B).
While large platelet aggregates form on the surfaces of podoplaninexpressing NIH-3T3 cells (red arrowheads), NIH-3T3 cells that
lack podoplanin fail to interact with platelets even after 20 minutes. Imaged on an Olympus IX50 using a 40⫻/0.65 air LCAch
(objective lens and a FViewII) camera (Olympus). (C-F) Direct
demonstration in E12.5 and E13.5 podoplanin⫹/⫹ embryos of
platelet thrombi (labeled for integrin ␣IIb␤3 in red) at the junction of
cardinal veins and lymphatic sacs that are marked by podoplanin
(green; C,E) or Lyve-1 (green; D,F). By contrast, platelets are
absent from the lymph sac’s orifice in E13.5 podoplanin⫺/⫺
embryos (G-H) as seen on serial sections (220 ␮m apart) stained
for Lyve-1 (green) and the platelet integrin ␣IIb␤3 (red). Note the
still persisting connection (G) between lymph sac and vein. In the
section 220 ␮m apart (H), the connection between lymph sac and
cardinal vein is no longer visible; in panels G and H, erythrocytes/
reticulocytes are present in the lymph sacs (Ter119 staining not
shown). Cell nuclei are stained with DAPI (blue); Olympus AX70,
10⫻/0.40 air UPlanApo. Scale bars in panels A and B equal
10 ␮m; in panels C through H, 50 ␮m.
E13.5 +/+
Lymph sac
Lymph sac
Cardinal
vein
G
Lyve-1
Platelet integrin αIIbβ3
Cardinal
vein
H
Lyve-1
Platelet integrin αIIbβ3
E13.5 −/−
E13.5 −/−
Cardinal vein
Lymph sac
bud
4001
Cardinal vein
Lymph sac
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4002
A
IgG
Podo IgG
B
BLOOD, 13 MAY 2010 䡠 VOLUME 115, NUMBER 19
UHRIN et al
ASA
C Kindlin-3-/-
Lyve-1
Ter119
Podo IgG
Lyve-1
Control IgG
Ter119
Podoplanin
Prox-1
Figure 4. Inhibition of platelet aggregation causes a “nonseparation” phenotype.
(A) E13.5 podoplanin⫹/⫹ embryo whose mother was injected with rabbit anti-podoplanin
IgG that blocks podoplanin-platelet interaction (left panel). Subtle vascular malformations
(arrowheads) similar to those in podoplanin⫺/⫺ mice are locally observed. Immunofluorescence staining (middle panel) demonstrates dermal Lyve-1⫹ lymphatic vessels (green)
containing erythrocytes (red; white arrow). Cell nuclei are stained with DAPI (blue). Right
panel shows macroscopically normal vasculature in podoplanin⫹/⫹ embryo whose mother
was injected with irrelevant rabbit IgG. (B) E14.5 podoplanin⫹/⫹ embryo whose mother was
treated with acetyl salicylic acid (ASA; 25 mg/kg/d) to inhibit platelet aggregation. Vascular
lesions reminiscent of those found in podoplanin⫺/⫺ mice are locally observed (left panel;
inset shows higher magnification). Immunofluorescence staining (right panel) shows that
the dermal vessels are Lyve-1⫹ lymphatics (green) and contain erythrocytes (Ter119 in
red). Cell nuclei are stained with DAPI (blue). (C) Kindlin-3⫺/⫺ E14.0 embryo that shows
blood-containing vascular malformations in the skin (left panel). Double immunofluorescence of dermal vessel (right panel); endothelium is positive for Prox1 (red nuclei) and
podoplanin (green), and the vessel is filled with erythrocytes (yellow). Arrowheads mark
extravasated erythrocytes (fluorescent images were taken on an Olympus AX70 with a
20⫻ objective lens; all other images with the Olympus SZ40). Scale bars in panels A and B
equal 50 ␮m; in panel C, 20 ␮m.
Platelet thrombi form at the orifices of embryonic lymph sacs
In E12.5 and E13.5 wild-type embryos, both podoplanin (Figure
3C,E) and Lyve-1 (Figure 3D,F) were expressed in large amounts
in the endothelial cells of forming lymph sacs. Aggregates of
platelets with activated ␣IIb␤3 integrins were encountered at the
separation zone of lymph sacs and the cardinal veins (Figure 3C-F),
but were absent from podoplanin⫺/⫺ embryos (Figure 3G-H). This
finding strongly suggests that podoplanin induces platelet aggregation also in vivo, which likely terminates the connection between
the lymph sacs and the blood vasculature. In podoplanin⫺/⫺
embryos, we detected no platelet aggregates at the “neck” region
(Figure 3G), but we found persisting connections between the
lymph sac and vein, and in erythrocytes in the lymph sac up to
220 ␮m away from the persisting connection (Figure 3H).
Interference with platelet aggregation/activation in vivo
produces a transient “nonseparation” phenotype
To further corroborate our hypothesis that podoplanin-induced platelet
aggregation is critical for the separation of lymph sacs from the blood
vasculature, we tested to see if different means of blocking platelet
aggregation in vivo would also interfere with the separation process. A
“nonseparation” phenotype was produced in E13.5 embryos by injecting blocking anti-podoplanin into pregnant mice, whereas control IgG
gave no phenotype (Figure 4A). The blood-filled vessels generated upon
anti-podoplanin antibody treatment were Lyve-1⫹, confirming their
lymphatic identity (Figure 4A middle panel).
Similarly, inhibition of platelet aggregation by treatment of pregnant
podoplanin⫹/⫹ mice with acetyl salicylic acid at approximately 50 mg/
kg per day or approximately 25 mg/kg per day from approximately E8.5
until E16.5 resulted in 50% (5 of 10) and approximately 45% (13 of 28),
respectively, of embryos exhibiting LYVE-1⫹ vessels in the skin
containing red blood cells (Figure 4B). The lower concentration of
acetyl salicylic acid (7.5 mg/kg/d) did not induce such phenotype in any
of the 14 embryos, and this phenotype was never observed in embryos
from mothers treated with the vehicle alone.
Finally, we studied kindlin-3–deficient mouse strains, which
display a prominent platelet aggregation defect due to an inability
to activate platelet integrins.34 Importantly, the lymphatic vessels of
kindlin-3–deficient embryos around E15 also contained blood like
the podoplanin-deficient animals (Figure 4C). Altogether, these
results demonstrate that platelet aggregation is required for efficient separation of blood and lymphatic vessels.
Discussion
Centrifugal lymphangiogenesis4 starts by focal expression of
lymphatic markers, such as Prox-1 and podoplanin, by endothelial
cells of cardinal veins, followed by sprouting and formation of
lymph sacs from which the peripheral lymphatics are generated.8
A critical step in this development is the separation of the newly
formed lymph sacs and the parental cardinal veins.
In this investigation, we have discovered the basic mechanism
necessary for the lymphatic and blood vascular separation during
embryonic development. Based on the vascular phenotype in
podoplanin⫺/⫺ mice generated in a mixed 129S/v ⫻ Swiss or
C57Bl/6 background, we ascribe a decisive role in this process to
the interaction of podoplanin that is expressed on the surface of
nascent lymphatic endothelial cells, with circulating platelets that
arrive from the blood stream of the cardinal vein (Figure 5).
Expression of genes related to the lymphatic phenotype of endothelial cells has provided crucial insights into the formation and outgrowth
of lymph sacs from the cardinal vein, but—with the exception of
podoplanin—no evidence for their involvement in the separation of the
2 circulatory systems was found. For example, disruption of the gene of
the homeobox transcription factor Prox-1 indicated its role in the
budding and migration of lymphatic endothelial cells from the cardinal
vein, leading to a complete absence of the lymphatic vascular system.8,38
VEGF-C in turn was required for migration and survival of Prox-1–
expressing endothelial cells and the formation of lymph sacs.14
Angiopoietin-2,13 ephrinB2,39 FoxC2,40 or ␣9 integrin11 play a role in
lymphatic vessel differentiation and maturation, but not in the lymphatic
sprouting and segregation of lymph sacs. Interestingly, in a report by
Johnson et al on conditional down-regulation of Prox1, scattered
blood-filled vessels were detected when Prox1 was excised in embryos
from venous lymphatic endothelial cell precursors at early developmental stages. A much larger number of scattered, superficial, blood-filled
vessels were observed in conditional mutant embryos in which Prox1
was excised from developing lymph sacs at later stages.41
A possible role for podoplanin in the separation of blood and
lymphatic vasculature was suggested by the findings that endothelial
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BLOOD, 13 MAY 2010 䡠 VOLUME 115, NUMBER 19
Podoplanin−/−
Podoplanin+/+
Figure 5. Schematic depiction of the mechanisms involved in separation of
lymph sacs from the cardinal veins, resulting in the “separation phenotype”
and “nonseparation phenotype.” The developmental default “separation” phenotype in podoplanin⫹/⫹ mice (right column) starts with formation of lymph sacs from the
cardinal vein around E12. Endothelial cells of the lymph sacs acquire the lymphatic
phenotype and express podoplanin (depicted by red “Y”-shaped objects) on their
surface that binds and activates circulating platelets (depicted in green). Platelet
thrombi develop around the orifice of the lymph sacs and lead to separation of the
lymphatic sac from the cardinal vein either directly by an occluding platelet aggregate
or indirectly by release of vasoconstrictive substances or growth factors released
from activated platelets recruiting mural cells. At the stage of E14 and later, the
separated lymph sacs are fully sequestered from the cardinal veins and sprout
centrifugally to form a separate lymphatic circulatory system. The “nonseparation”
phenotype in podoplanin⫺/⫺ mice (left column) is characterized by persisting patent
connections between the venous and the lymphatic systems. This is due to the
absence of podoplanin on the lymphatic sac’s endothelial cells, and thus the lack of
local platelet activation and aggregation at the lymph sac’s orifices. This results in
blood perfusion of the outgrowing lymphatic vessels.
cell O-glycan deficiency24 causes down-regulation of podoplanin expression and a “nonseparation” phenotype. Here, we confirm a central role
of the expression of podoplanin by lymphatic endothelial cells26 for the
separation of blood and lymphatic vasculature, specifically by its
capacity to bind, activate, and aggregate platelets.31 Podoplanin is
identical with aggrus that was previously discovered on the surface of
murine colon carcinoma cells and found to induce platelet aggregation.31
Consistent with this observation, we demonstrate that aggregation of
mouse and human platelets is induced in vitro under flow conditions by
cells expressing podoplanin (supplemental Table 1).
Importantly, the in vivo significance of platelet-podoplanin interaction for the development of the “separation” phenotype in vivo is
underscored by the direct immunohistologic demonstration of aggregated activated platelets at the orifices of sprouting lymphatic sacs on the
side of the cardinal veins. The occlusion of this vascular communication
is presumably due to the platelet aggregate itself serving as a plug,
and/or to mediators released from aggregated platelets, such as vasoconstricting serotonin or PDGF and TGF-␤, that attract mural cells.
Strong support for our “platelet hypothesis” of the separation of the
lymphatic from the blood vasculature is further provided by experiments
in which we interfere selectively with the function of platelets or with
their interaction with podoplanin.All manipulations generate a “nonseparation” phenotype. Specifically, we have used approaches to inhibit in
vivo podoplanin-platelet interaction by injecting blocking antipodoplanin antibody, or treating pregnant wild-type mice with large
dosages of acetyl salicylic acid. The effects of acetyl salicylic acid
treatment in mice cannot be directly related to humans, because of their
PODOPLANIN ACTIVATE PLATELETS IN FORMING LYMPHATIC
4003
clear species-specific differences in sensitivity for the effects of acetyl
salicylic acid.42,43 Most compelling were the results obtained with mice
lacking the platelet integrin-binding protein kindlin-3. These mice suffer
from a failure to aggregate platelets but display no alterations of
endothelial cells.34 The nonseparation phenotype in these animals is
transient, and is restricted to the time around E15. The further
phenotypic development of the kindlin-3 knockout mice is, however,
different from that of podoplanin knockouts, and is dominated by
hemorrhages, anemia, and their clinical consequences.44 Taken together
with the results obtained on podoplanin⫺/⫺ mice, these observations
strongly indicate that any interference with podoplanin-mediated platelet aggregation results in a “nonseparation” phenotype in embryos.
Failure to separate emerging lymphatic vessels from blood vessels
similar to that observed in our podoplanin⫺/⫺ mice was previously
described in mice lacking the hematopoietic signaling molecules Syk19
or SLP-76;16,18 however, the underlying mechanisms for this defect
were not elucidated, and the established roles of these proteins in
lymphocyte differentiation did not offer an explanation either. It was
speculated that vascular progenitor cells expressing these proteins
differentiate into endothelial cells after their recruitment to the sites of
separation, or that Syk or SLP-76 expressed by circulating hematopoietic cells convey signals critical for lymphatic or vascular endothelial
cell homing and differentiation.16,45 Our findings offer an alternative
simple explanation, as lack of Syk and/or SLP-76 strongly affect the
capacity of platelets to activate integrins, and to undergo aggregation
and activation.21-23,46 Therefore, formation of platelet plugs that
separate the cardinal vein and the lymph sacs is likely compromised in embryos with deficiencies of these proteins. Further
support for the validity of our “platelet hypothesis” of blood and
lymphatic vascular separation is provided by the finding that
ablation of other components of the intracellular Syk and SLP-76
pathways also results in a similar phenotype. This applies to
phospholipase-C␥2 that is phosphorylated by Syk and SLP76,16,21,22 and most probably also to Rap1b, an abundant small
GTPase in platelets acting downstream of phospholipase-C␥2 and
regulating the cross-talk between platelet integrin ␣2␤1 and integrin
␣IIb␤3.47,48 The link between podoplanin and this described pathway of platelet aggregation was revealed recently, because Syk,
SLP-76, and phospholipase-C␥2 in platelets act downstream of
CLEC-2,49 which was found to be the platelet receptor responsible
for platelet aggregation induced by podoplanin.32
In our study, we also show that mice lacking the platelet integrinbinding protein kindlin-3 and displaying a prominent platelet aggregation defect due to an inability to activate platelet integrins34 exhibit a
“nonseparation phenotype” similarly as the other measures interfering
with platelet aggregation/activation described here do. Collectively,
these findings provide evidence that expression of Syk and SLP-76 and
downstream components of their intracellular pathways, as well as other
components such as kindlin-3 that are essential for correct aggregation/
activation of platelets, are required for proper separation of the blood
and the lymphatic systems, and that defective podoplanin-driven platelet
activation is the dominating common causative factor for a “nonseparation” phenotype.
An open question might be the lack of a “nonseparation” phenotype
in NF-E2–deficient mice50 despite a 96% to 99% reduction in their
platelet count during adulthood.51 A possible explanation could be that
during the embryonic development, platelet counts in NF-E2–deficient
embryos are not decreased to the same extent as that reported for the
adult animals. In fact, the platelet counts in NF-E2–deficient embryos at
E15.5 were estimated to be still as high as 70 000 to 80 000/␮L,
compared with approximately 650 000 platelets/␮L in heterozygous
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4004
BLOOD, 13 MAY 2010 䡠 VOLUME 115, NUMBER 19
UHRIN et al
mutant pups (Figure 6A in Levin et al 51), which may be sufficient to
disconnect the lymphatic sac from the cardinal vein.
There are also indications that platelet-mediated separation also
plays a role in human pathologies. We have recently found that in
human inflammatory skin diseases, single endothelial cells of
venules express lymphatic markers, similar to the initial events that
precede lymph sac formation in cardinal veins.52 Moreover,
preliminary results on the healing of incisional wounds in podoplanin⫺/⫺ and wild-type mice provide evidence for transient, erroneous communications of newly formed lymphatic and blood microvessels, as aggregates of activated platelets are found in the
lymphatics of wild-type mice, and erythrocytes are found in the
lymphatics of podoplanin⫺/⫺ mice (supplemental Figure 3A-B). In
addition, the expression of podoplanin on the surface of angioma
endothelial cells26 in partly blood-perfused vascular tumors causes
thrombogenicity, and could provide a sink for platelets that causes
thrombocytopenic bleeding disorders53 in some cases (supplemental Figure 3C-D).
Taken together, our results identify the interaction of platelets
and podoplanin on the surface of lymphatic endothelial cells as a
novel central event in the separation of lymphatic and blood vessels
that integrates all currently known findings, including the vascular
phenotype observed in Syk- or SLP-76–deficient animals. The
relevance of this simple mechanism remains to be established for
lymphangiogenesis in human pathology such as in inflammation52
or vascular tumors.26
Acknowledgments
We thank Dr E. Molinari (deceased), Technoclone Inc, Vienna, for the
lipid analysis, Dr H. Kowalski (Pathology) for help in designing of the
podoplanin knockout construct, Mario Hilpert (Department of Vascular
Biology and Thrombosis Research) for excellent technical support, and
T. Nardelli (Department of Vascular Biology and Thrombosis Research)
and A. Jaeger (Pathology) for help with the artwork.
This work was supported by the Austrian Science Foundation
program project grant F005 Microvascular injury and repair 5-03 to
H.S., 5-07 to D.K., and 5-09 to B.R.B.; the European Union 6th
framework program Integrated Projects Lymphangiogenomics
(LSGH-CT-2004-503573 to D.K.) and Cancerdegradome (LSHCCT-2003-503297 to B.R.B.); and was performed partially within
the European Vascular Genomics Network of Excellence (EVGN),
contract no. LSHM-CT-2003-503254 (B.R.B. laboratory).
Authorship
Contribution: P.U, J.Z., and P.C. generated the podoplanin⫺/⫺ mice
and analyzed the gross phenotype; J.M.B. and D.O. performed
immunohistochemistry and performed platelet experiments; H.S.
and E.F. provided the podoplanin-expressing NIH-3T3 fibroblasts;
M.M. and R.F. provided the kindlin-3⫺/⫺ mice; P.H. and K.A.
produced data on the ␤-galactosidase–expressing mice under the
VEGFR-3 promoter; B.R.B. designed the experiments and wrote
the manuscript together with D.K., P.U., and J.M.B.; and D.K.
provided lymphatic endothelial cells, data from the tufted angioma,
and the podoplanin antibody. The podoplanin knockout mouse is a
joint project of the Kerjaschki and Binder laboratories.
Conflict-of-interest disclosure: The authors declare no competing financial interests.
The current address for P.C. is Animal Production Research
Centre and SUA FBFS Nitra, Nitra, Slovak Republic.
Correspondence: Bernd R. Binder, Schwarzspanier Str 17,
A-1090 Vienna, Austria; e-mail: [email protected].
References
1. Alitalo K, Carmeliet P. Molecular mechanisms of
lymphangiogenesis in health and disease. Cancer Cell. 2002;1(3):219-227.
2. Adams RH, Alitalo K. Molecular regulation of angiogenesis and lymphangiogenesis. Nat Rev Mol
Cell Biol. 2007;8(6):464-478.
3. van der Putte SC. The early development of the
lymphatic system in mouse embryos. Acta Morphol Neerl Scand. 1975;13(4):245-286.
4. Oliver G, Detmar M. The rediscovery of the lymphatic system: old and new insights into the development and biological function of the lymphatic vasculature. Genes Dev. 2002;16(7):773783.
5. Schneider M, Othman-Hassan K, Christ B, Wilting J.
Lymphangioblasts in the avian wing bud. Dev Dyn.
1999;216(4-5):311-319.
6. Kerjaschki D, Huttary N, Raab I et al. Lymphatic
endothelial progenitor cells contribute to de novo
lymphangiogenesis in human renal transplants.
Nat Med. 2006;12(2):230-234.
7. Sabin FR. On the origin of the lymphatic system
from the veins, and the development of the lymph
hearts and thoracic duct in the pig. Am J Anat.
1902;1:367-389.
8. Wigle JT, Oliver G. Prox1 function is required for
the development of the murine lymphatic system.
Cell. 1999;98(6):769-778.
9. 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.
10. Petrova TV, Makinen T, Makela TP, et al. Lymphatic endothelial reprogramming of vascular en-
dothelial cells by the Prox-1 homeobox transcription factor. EMBO J. 2002;21(17):4593-4599.
the adaptor protein SLP-76. Cell. 1998;94(2):229238.
11. Huang XZ, Wu JF, Ferrando R, et al. Fatal bilateral chylothorax in mice lacking the integrin
alpha9beta1. Mol Cell Biol. 2000;20(14):52085215.
19. Cheng AM, Rowley B, Pao W, Hayday A, Bolen
JB, Pawson T. Syk tyrosine kinase required for
mouse viability and B-cell development. Nature.
1995;378(6554):303-306.
12. Yuan L, Moyon D, Pardanaud L, et al. Abnormal
lymphatic vessel development in neuropilin 2 mutant mice. Development. 2002;129(20):47974806.
20. Turner M, Mee PJ, Costello PS, et al. Perinatal
lethality and blocked B-cell development in mice
lacking the tyrosine kinase Syk. Nature. 1995;
378(6554):298-302.
13. Gale NW, Thurston G, Hackett SF, et al. Angiopoietin-2 is required for postnatal angiogenesis
and lymphatic patterning, and only the latter role
is rescued by Angiopoietin-1. Dev Cell. 2002;3(3):
411-423.
21. Clements JL, Lee JR, Gross B, et al. Fetal hemorrhage and platelet dysfunction in SLP-76-deficient
mice. J Clin Invest. 1999;103(1):19-25.
14. 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.
15. Gale NW, Prevo R, Espinosa J, et al. Normal lymphatic development and function in mice deficient
for the lymphatic hyaluronan receptor LYVE-1.
Mol Cell Biol. 2007;27(2):595-604.
16. Abtahian F, Guerriero A, Sebzda E, et al. Regulation of blood and lymphatic vascular separation
by signaling proteins SLP-76 and Syk. Science.
2003;299(5604):247-251.
22. Gross BS, Lee JR, Clements JL, et al. Tyrosine
phosphorylation of SLP-76 is downstream of Syk
following stimulation of the collagen receptor in
platelets. J Biol Chem. 1999;274(9):5963-5971.
23. Poole A, Gibbins JM, Turner M, et al. The Fc receptor gamma-chain and the tyrosine kinase Syk
are essential for activation of mouse platelets by
collagen. EMBO J. 1997;16:2333-2341.
24. Fu J, Gerhardt H, McDaniel JM, et al. Endothelial
cell O-glycan deficiency causes blood/lymphatic
misconnections and consequent fatty liver disease in mice. J Clin Invest. 2008;118(11):37253737.
17. Clements JL, Yang B, Ross-Barta SE, et al. Requirement for the leukocyte-specific adapter protein SLP-76 for normal T cell development. Science. 1998;281(5375):416-419.
25. Breiteneder-Geleff S, Matsui K, Soleiman A, et al.
Podoplanin, novel 43-kd membrane protein of
glomerular epithelial cells, is down-regulated in
puromycin nephrosis. Am J Pathol. 1997;151(4):
1141-1152.
18. Pivniouk V, Tsitsikov E, Swinton P, Rathbun G, Alt
FW, Geha RS. Impaired viability and profound
block in thymocyte development in mice lacking
26. Breiteneder-Geleff S, Soleiman A, Kowalski H, et
al. Angiosarcomas express mixed endothelial
phenotypes of blood and lymphatic capillaries:
From www.bloodjournal.org by guest on June 16, 2017. For personal use only.
BLOOD, 13 MAY 2010 䡠 VOLUME 115, NUMBER 19
27.
28.
29.
30.
31.
32.
33.
34.
35.
podoplanin as a specific marker for lymphatic endothelium. Am J Pathol. 1999;154(2):385-394.
Ramirez MI, Millien G, Hinds A, Cao Y, Seldin DC,
Williams MC. T1alpha, a lung type I cell differentiation gene, is required for normal lung cell proliferation and alveolus formation at birth. Dev Biol.
2003;256(1):61-72.
Rishi AK, Joyce-Brady M, Fisher J, et al. Cloning,
characterization, and development expression of
a rat lung alveolar type I cell gene in embryonic
endodermal and neural derivatives. Dev Biol.
1995;167(1):294-306.
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.
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.
Kato Y, Fujita N, Kunita A, et al. Molecular identification of Aggrus/T1alpha as a platelet aggregationinducing factor expressed in colorectal tumors.
J Biol Chem. 2003;278(51):51599-51605.
Suzuki-Inoue K, Kato Y, Inoue O, et al. Involvement of the snake toxin receptor CLEC-2, in podoplanin-mediated platelet activation, by cancer
cells. J Biol Chem. 2007;282(36):25993-26001.
Iljin K, Karkkainen MJ, Lawrence EC, et al.
VEGFR3 gene structure, regulatory region, and
sequence polymorphisms. FASEB J. 2001;15(6):
1028-1036.
Moser M, Nieswandt B, Ussar S, Pozgajova M,
Fassler R. Kindlin-3 is essential for integrin activation and platelet aggregation. Nat Med. 2008;
14(3):325-330.
Kriehuber E, Breiteneder-Geleff S, Groeger M, et
al. Isolation and characterization of dermal lymphatic and blood endothelial cells reveal stable
and functionally specialized cell lineages. J Exp
Med. 2001;194(6):797-808.
PODOPLANIN ACTIVATE PLATELETS IN FORMING LYMPHATIC
36. Kerjaschki D, Regele HM, Moosberger I, et al.
Lymphatic neoangiogenesis in human kidney
transplants is associated with immunologically
active lymphocytic infiltrates. J Am Soc Nephrol.
2004;15(3):603-612.
37. Kaneko M, Kato Y, Kunita A, Fujita N, Tsuruo T,
Osawa M. Functional sialylated O-glycan to platelet aggregation on Aggrus (T1alpha/Podoplanin)
molecules expressed in Chinese hamster ovary
cells. J Biol Chem. 2004;279(37):38838-38843.
4005
Slp-76 mutant mice reveal a cell-autonomous hematopoietic cell contribution to vascular development. Dev Cell. 2006;11(3):349-361.
46. Judd BA, Myung PS, Leng L, et al. Hematopoietic
reconstitution of SLP-76 corrects hemostasis and
platelet signaling through alpha IIb beta 3 and
collagen receptors. Proc Natl Acad Sci U S A.
2000;97(22):12056-12061.
38. 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.
47. Chrzanowska-Wodnicka M, Smyth SS,
Schoenwaelder SM, Fischer TH, White GC.
Rap1b is required for normal platelet function and
hemostasis in mice. J Clin Invest. 2005;115(3):
680-687.
39. Makinen 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.
48. Bernardi B, Guidetti GF, Campus F, et al. The
small GTPase Rap1b regulates the cross talk between platelet integrin alpha2beta1 and integrin
alphaIIbbeta3. Blood. 2006;107(7):2728-2735.
40. Petrova TV, Karpanen T, Norrmen C, et al. Defective valves and abnormal mural cell recruitment
underlie lymphatic vascular failure in lymphedema distichiasis. Nat Med. 2004;10(9):974-981.
49. Suzuki-Inoue K, Fuller GL, Garcia A, et al. A novel
Syk-dependent mechanism of platelet activation
by the C-type lectin receptor CLEC-2. Blood.
2006;107(2):542-549.
41. Johnson NC, Dillard ME, Baluk P, et al. Lymphatic
endothelial cell identity is reversible and its maintenance requires Prox1 activity. Genes Dev.
2008;22(23):3282-3291.
50. Shivdasani RA, Rosenblatt MF, Zucker-Franklin
D, et al. Transcription factor NF-E2 is required for
platelet formation independent of the actions of
thrombopoietin/MGDF in megakaryocyte development. Cell. 1995;81(5):695-704.
42. Slone D, Siskind V, Heinonen OP, Monson RR,
Kaufman DW, Shapiro S. Aspirin and congenital
malformations. Lancet. 1976;1(7974):1373-1375.
43. Cappon GD, Cook JC, Hurtt ME. Relationship
between cyclooxygenase 1 and 2 selective inhibitors and fetal development when administered to
rats and rabbits during the sensitive periods for
heart development and midline closure. Birth Defects Res B Dev Reprod Toxicol. 2003;68(1):4756.
44. Krüger M, Moser M, Ussar S, et al. SILAC mouse
for quantitative proteomics uncovers kindlin-3 as
an essential factor for red blood cell function.
Cell. 2008;134(2):353-364.
45. Sebzda E, Hibbard C, Sweeney S, et al. Syk and
51. Levin J, Peng JP, Baker GR, et al. Pathophysiology of thrombocytopenia and anemia in mice
lacking transcription factor NF-E2. Blood. 1999;
94(9):3037-3047.
52. Gröger M, Niederleithner H, Kerjaschki D,
Petzelbauer P. A previously unknown dermal
blood vessel phenotype in skin inflammation.
J Invest Dermatol. 2007;127(12):2893-2900.
53. North PE, Kahn T, Cordisco MR, Dadras SS,
Detmar M, Frieden IJ. Multifocal lymphangioendotheliomatosis with thrombocytopenia: a
newly recognized clinicopathological entity.
Arch Dermatol. 2004;140(5):599-606.
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2010 115: 3997-4005
doi:10.1182/blood-2009-04-216069 originally published
online January 28, 2010
Novel function for blood platelets and podoplanin in developmental
separation of blood and lymphatic circulation
Pavel Uhrin, Jan Zaujec, Johannes M. Breuss, Damla Olcaydu, Peter Chrenek, Hannes Stockinger,
Elke Fuertbauer, Markus Moser, Paula Haiko, Reinhard Fässler, Kari Alitalo, Bernd R. Binder and
Dontscho Kerjaschki
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