Key Points

  • Platelet activation supports lymphatic vessel growth during wound healing through release of the lymphangiogenic factor VEGFC.

  • Thrombin and plasmin support lymphatic vessel growth through proteolytic activation of the lymphangiogenic factors VEGFC and VEGFD.

Abstract

Hemostasis associated with tissue injury is followed by wound healing, a complex process by which damaged cellular material is removed and tissue repaired. Angiogenic responses are a central aspect of wound healing, including the growth of new lymphatic vessels by which immune cells, protein, and fluid are transported out of the wound area. The concept that hemostatic responses might be linked to wound healing responses is an old one, but demonstrating such a link in vivo and defining specific molecular mechanisms by which the 2 processes are connected has been difficult. In the present study, we demonstrate that the lymphangiogenic factors vascular endothelial growth factor C (VEGFC) and VEGFD are cleaved by thrombin and plasmin, serine proteases generated during hemostasis and wound healing. Using a new tail-wounding assay to test the relationship between clot formation and lymphangiogenesis in mice, we find that platelets accelerate lymphatic growth after injury in vivo. Genetic studies reveal that platelet enhancement of lymphatic growth after wounding is dependent on the release of VEGFC, but not VEGFD, a finding consistent with high expression of VEGFC in both platelets and avian thrombocytes. Analysis of lymphangiogenesis after full-thickness skin excision, a wound model that is not associated with significant clot formation, also revealed an essential role for VEGFC, but not VEGFD. These studies define a concrete molecular and cellular link between hemostasis and lymphangiogenesis during wound healing and reveal that VEGFC, the dominant lymphangiogenic factor during embryonic development, continues to play a dominant role in lymphatic growth in mature animals.

REFERENCES

REFERENCES
1.
Iannacone
M
.
Platelet-mediated modulation of adaptive immunity
.
Semin Immunol
.
2016
;
28
(
6
):
555
-
560
.
2.
Nurden
AT
.
The biology of the platelet with special reference to inflammation, wound healing and immunity
.
Front Biosci (Landmark Ed)
.
2018
;
23
:
726
-
751
.
3.
Opneja
A
,
Kapoor
S
,
Stavrou
EX
.
Contribution of platelets, the coagulation and fibrinolytic systems to cutaneous wound healing
.
Thromb Res
.
2019
;
179
:
56
-
63
.
4.
Drew
AF
,
Liu
H
,
Davidson
JM
,
Daugherty
CC
,
Degen
JL
.
Wound-healing defects in mice lacking fibrinogen
.
Blood
.
2001
;
97
(
12
):
3691
-
3698
.
5.
Kim
SJ
,
Davis
RP
,
Jenne
CN
.
Platelets as modulators of inflammation
.
Semin Thromb Hemost
.
2018
;
44
(
2
):
91
-
101
.
6.
Blair
P
,
Flaumenhaft
R
.
Platelet alpha-granules: basic biology and clinical correlates
.
Blood Rev
.
2009
;
23
(
4
):
177
-
189
.
7.
Wartiovaara
U
,
Salven
P
,
Mikkola
H
, et al
.
Peripheral blood platelets express VEGF-C and VEGF which are released during platelet activation
.
Thromb Haemost
.
1998
;
80
(
1
):
171
-
175
.
8.
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
.
9.
Bray
PF
,
McKenzie
SE
,
Edelstein
LC
, et al
.
The complex transcriptional landscape of the anucleate human platelet
.
BMC Genomics
.
2013
;
14
(
1
):
1
.
10.
Randolph
GJ
,
Ivanov
S
,
Zinselmeyer
BH
,
Scallan
JP
.
The lymphatic system: integral roles in immunity
.
Annu Rev Immunol
.
2017
;
35
(
1
):
31
-
52
.
11.
Cifarelli
V
,
Eichmann
A
.
The intestinal lymphatic system: functions and metabolic implications
.
Cell Mol Gastroenterol Hepatol
.
2019
;
7
(
3
):
503
-
513
.
12.
Kim
H
,
Kataru
RP
,
Koh
GY
.
Regulation and implications of inflammatory lymphangiogenesis
.
Trends Immunol
.
2012
;
33
(
7
):
350
-
356
.
13.
Alitalo
K
.
The lymphatic vasculature in disease
.
Nat Med
.
2011
;
17
(
11
):
1371
-
1380
.
14.
Yang
Y
,
Oliver
G
.
Development of the mammalian lymphatic vasculature
.
J Clin Invest
.
2014
;
124
(
3
):
888
-
897
.
15.
Koltowska
K
,
Betterman
KL
,
Harvey
NL
,
Hogan
BM
.
Getting out and about: the emergence and morphogenesis of the vertebrate lymphatic vasculature
.
Development
.
2013
;
140
(
9
):
1857
-
1870
.
16.
Joukov
V
,
Pajusola
K
,
Kaipainen
A
, et al
.
A novel vascular endothelial growth factor, VEGF-C, is a ligand for the Flt4 (VEGFR-3) and KDR (VEGFR-2) receptor tyrosine kinases
.
EMBO J
.
1996
;
15
(
2
):
290
-
298
.
17.
Jeltsch
M
,
Kaipainen
A
,
Joukov
V
, et al
.
Hyperplasia of lymphatic vessels in VEGF-C transgenic mice
.
Science
.
1997
;
276
(
5317
):
1423
-
1425
.
18.
Ober
EA
,
Olofsson
B
,
Mäkinen
T
, et al
.
Vegfc is required for vascular development and endoderm morphogenesis in zebrafish
.
EMBO Rep
.
2004
;
5
(
1
):
78
-
84
.
19.
Joukov
V
,
Sorsa
T
,
Kumar
V
, et al
.
Proteolytic processing regulates receptor specificity and activity of VEGF-C
.
EMBO J
.
1997
;
16
(
13
):
3898
-
3911
.
20.
Bui
HM
,
Enis
D
,
Robciuc
MR
, et al
.
Proteolytic activation defines distinct lymphangiogenic mechanisms for VEGFC and VEGFD
.
J Clin Invest
.
2016
;
126
(
6
):
2167
-
2180
.
21.
Jeltsch
M
,
Jha
SK
,
Tvorogov
D
, et al
.
CCBE1 enhances lymphangiogenesis via A disintegrin and metalloprotease with thrombospondin motifs-3-mediated vascular endothelial growth factor-C activation
.
Circulation
.
2014
;
129
(
19
):
1962
-
1971
.
22.
Alders
M
,
Hogan
BM
,
Gjini
E
, et al
.
Mutations in CCBE1 cause generalized lymph vessel dysplasia in humans
.
Nat Genet
.
2009
;
41
(
12
):
1272
-
1274
.
23.
Hogan
BM
,
Bos
FL
,
Bussmann
J
, et al
.
Ccbe1 is required for embryonic lymphangiogenesis and venous sprouting
.
Nat Genet
.
2009
;
41
(
4
):
396
-
398
.
24.
Janssen
L
,
Dupont
L
,
Bekhouche
M
, et al
.
ADAMTS3 activity is mandatory for embryonic lymphangiogenesis and regulates placental angiogenesis
.
Angiogenesis
.
2016
;
19
(
1
):
53
-
65
.
25.
Bos
FL
,
Caunt
M
,
Peterson-Maduro
J
, et al
.
CCBE1 is essential for mammalian lymphatic vascular development and enhances the lymphangiogenic effect of vascular endothelial growth factor-C in vivo
.
Circ Res
.
2011
;
109
(
5
):
486
-
491
.
26.
McColl
BK
,
Baldwin
ME
,
Roufail
S
, et al
.
Plasmin activates the lymphangiogenic growth factors VEGF-C and VEGF-D [published correction appears in J Exp Med. 2003;198(7):following 1126]
.
J Exp Med
.
2003
;
198
(
6
):
863
-
868
.
27.
Ackermann
M
,
Wettstein
R
,
Senaldi
C
, et al
.
Impact of platelet rich plasma and adipose stem cells on lymphangiogenesis in a murine tail lymphedema model
.
Microvasc Res
.
2015
;
102
:
78
-
85
.
28.
Zou
Z
,
Enis
DR
,
Bui
H
, et al
.
The secreted lymphangiogenic factor CCBE1 is essential for fetal liver erythropoiesis
.
Blood
.
2013
;
121
(
16
):
3228
-
3236
.
29.
Ventura
A
,
Kirsch
DG
,
McLaughlin
ME
, et al
.
Restoration of p53 function leads to tumour regression in vivo
.
Nature
.
2007
;
445
(
7128
):
661
-
665
.
30.
Baldwin
ME
,
Halford
MM
,
Roufail
S
, et al
.
Vascular endothelial growth factor D is dispensable for development of the lymphatic system
.
Mol Cell Biol
.
2005
;
25
(
6
):
2441
-
2449
.
31.
Jakus
Z
,
Gleghorn
JP
,
Enis
DR
, et al
.
Lymphatic function is required prenatally for lung inflation at birth
.
J Exp Med
.
2014
;
211
(
5
):
815
-
826
.
32.
Jha
SK
,
Rauniyar
K
,
Karpanen
T
, et al
.
Efficient activation of the lymphangiogenic growth factor VEGF-C requires the C-terminal domain of VEGF-C and the N-terminal domain of CCBE1
.
Sci Rep
.
2017
;
7
(
1
):
4916
.
33.
Schmaier
AA
,
Stalker
TJ
,
Runge
JJ
, et al
.
Occlusive thrombi arise in mammals but not birds in response to arterial injury: evolutionary insight into human cardiovascular disease
.
Blood
.
2011
;
118
(
13
):
3661
-
3669
.
34.
Rutkowski
JM
,
Moya
M
,
Johannes
J
,
Goldman
J
,
Swartz
MA
.
Secondary lymphedema in the mouse tail: Lymphatic hyperplasia, VEGF-C upregulation, and the protective role of MMP-9
.
Microvasc Res
.
2006
;
72
(
3
):
161
-
171
.
35.
Tabibiazar
R
,
Cheung
L
,
Han
J
, et al
.
Inflammatory manifestations of experimental lymphatic insufficiency
.
PLoS Med
.
2006
;
3
(
7
):
e254
.
36.
Wong
VW
,
Sorkin
M
,
Glotzbach
JP
,
Longaker
MT
,
Gurtner
GC
.
Surgical approaches to create murine models of human wound healing
.
J Biomed Biotechnol
.
2011
;
2011
:
969618
.
37.
Romer
J
,
Bugge
TH
,
Pyke
C
, et al
.
Impaired wound healing in mice with a disrupted plasminogen gene
.
Nat Med
.
1996
;
2
(
3
):
287
-
292
.
38.
Hong
YK
,
Lange-Asschenfeldt
B
,
Velasco
P
, et al
.
VEGF-A promotes tissue repair-associated lymphatic vessel formation via VEGFR-2 and the alpha1beta1 and alpha2beta1 integrins
.
FASEB J
.
2004
;
18
(
10
):
1111
-
1113
.
39.
Ito
M
,
Yang
Z
,
Andl
T
, et al
.
Wnt-dependent de novo hair follicle regeneration in adult mouse skin after wounding
.
Nature
.
2007
;
447
(
7142
):
316
-
320
.
40.
Gurtner
GC
,
Werner
S
,
Barrandon
Y
,
Longaker
MT
.
Wound repair and regeneration
.
Nature
.
2008
;
453
(
7193
):
314
-
321
.
41.
Italiano
JE
, Jr
.,
Battinelli
EM
.
Selective sorting of alpha-granule proteins
.
J Thromb Haemost
.
2009
;
7 Suppl 1173-176
.
42.
Klement
GL
,
Yip
TT
,
Cassiola
F
, et al
.
Platelets actively sequester angiogenesis regulators
.
Blood
.
2009
;
113
(
12
):
2835
-
2842
.
43.
Battinelli
EM
,
Markens
BA
,
Italiano
JE
Jr
.
Release of angiogenesis regulatory proteins from platelet alpha granules: modulation of physiologic and pathologic angiogenesis
.
Blood
.
2011
;
118
(
5
):
1359
-
1369
.
44.
Hess
PR
,
Rawnsley
DR
,
Jakus
Z
, et al
.
Platelets mediate lymphovenous hemostasis to maintain blood-lymphatic separation throughout life
.
J Clin Invest
.
2013
;
124
(
1
):
273
-
284
.
45.
Watson
SP
,
Lowe
K
,
Finney
BA
.
Platelets in lymph vessel development and integrity
.
Adv Anat Embryol Cell Biol
.
2014
;
214
:
93
-
105
.
46.
Janardhan
HP
,
Trivedi
CM
.
Establishment and maintenance of blood-lymph separation
.
Cell Mol Life Sci
.
2019
;
76
(
10
):
1865
-
1876
.
47.
Carramolino
L
,
Fuentes
J
,
Garcia-Andres
C
,
Azcoitia
V
,
Riethmacher
D
,
Torres
M
.
Platelets play an essential role in separating the blood and lymphatic vasculatures during embryonic angiogenesis
.
Circ Res
.
2010
;
106
(
7
):
1197
-
1201
.
48.
Saaristo
A
,
Tammela
T
,
Farkkilā
A
, et al
.
Vascular endothelial growth factor-C accelerates diabetic wound healing
.
Am J Pathol
.
2006
;
169
(
3
):
1080
-
1087
.
49.
Saaristo
A
,
Tammela
T
,
Timonen
J
, et al
.
Vascular endothelial growth factor-C gene therapy restores lymphatic flow across incision wounds
.
FASEB J
.
2004
;
18
(
14
):
1707
-
1709
.
50.
Veikkola
T
,
Jussila
L
,
Makinen
T
, et al
.
Signalling via vascular endothelial growth factor receptor-3 is sufficient for lymphangiogenesis in transgenic mice
.
EMBO J
.
2001
;
20
(
6
):
1223
-
1231
.
51.
Rissanen
TT
,
Markkanen
JE
,
Gruchala
M
, et al
.
VEGF-D is the strongest angiogenic and lymphangiogenic effector among VEGFs delivered into skeletal muscle via adenoviruses
.
Circ Res
.
2003
;
92
(
10
):
1098
-
1106
.
52.
Küchler
AM
,
Gjini
E
,
Peterson-Maduro
J
,
Cancilla
B
,
Wolburg
H
,
Schulte-Merker
S
.
Development of the zebrafish lymphatic system requires VEGFC signaling
.
Curr Biol
.
2006
;
16
(
12
):
1244
-
1248
.
53.
Stacker
SA
,
Stenvers
K
,
Caesar
C
, et al
.
Biosynthesis of vascular endothelial growth factor-D involves proteolytic processing which generates non-covalent homodimers
.
J Biol Chem
.
1999
;
274
(
45
):
32127
-
32136
.
54.
Nurmi
H
,
Saharinen
P
,
Zarkada
G
,
Zheng
W
,
Robciuc
MR
,
Alitalo
K
.
VEGF-C is required for intestinal lymphatic vessel maintenance and lipid absorption
.
EMBO Mol Med
.
2015
;
7
(
11
):
1418
-
1425
.
You do not currently have access to this content.