Key Points

  • Platelets prevent plasma leakage during neutrophil diapedesis by binding to endothelial VWF and releasing Angpt1.

  • Platelet-released Angpt1 stimulates endothelial Tie-2, thereby activating the GEF FGD5, which strengthens cortical actin bundles.

Abstract

Neutrophil extravasation requires opening of the endothelial barrier but does not necessarily cause plasma leakage. Leaks are prevented by contractile actin filaments surrounding the diapedesis pore, keeping this opening tightly closed around the transmigrating neutrophils. We have identified the receptor system that is responsible for this. We show that silencing, or gene inactivation, of endothelial Tie-2 results in leak formation in postcapillary venules of the inflamed cremaster muscle at sites of neutrophil extravasation, as visualized by fluorescent microspheres. Leakage was dependent on neutrophil extravasation, because it was absent upon neutrophil depletion. We identified the Cdc42 GTPase exchange factor FGD5 as a downstream target of Tie-2 that is essential for leakage prevention during neutrophil extravasation. Looking for the Tie-2 agonist and its source, we found that platelet-derived angiopoietin-1 (Angpt1) was required to prevent neutrophil-induced leaks. Intriguingly, blocking von Willebrand factor (VWF) resulted in vascular leaks during transmigration, indicating that platelets interacting with endothelial VWF activate Tie-2 by secreting Angpt1, thereby preventing diapedesis-induced leakiness.

REFERENCES

REFERENCES
1.
Carden
DL
,
Smith
JK
,
Korthuis
RJ
.
Neutrophil-mediated microvascular dysfunction in postischemic canine skeletal muscle. Role of granulocyte adherence
.
Circ Res
.
1990
;
66
(
5
):
1436
-
1444
.
2.
Kubes
P
,
Suzuki
M
,
Granger
DN
.
Modulation of PAF-induced leukocyte adherence and increased microvascular permeability
.
Am J Physiol
.
1990
;
259
(
5 Pt 1
):
G859
-
G864
.
3.
Sumagin
R
,
Lomakina
E
,
Sarelius
IH
.
Leukocyte-endothelial cell interactions are linked to vascular permeability via ICAM-1-mediated signaling
.
Am J Physiol Heart Circ Physiol
.
2008
;
295
(
3
):
H969
-
H977
.
4.
DiStasi
MR
,
Ley
K
.
Opening the flood-gates: how neutrophil-endothelial interactions regulate permeability
.
Trends Immunol
.
2009
;
30
(
11
):
547
-
556
.
5.
Finsterbusch
M
,
Voisin
MB
,
Beyrau
M
,
Williams
TJ
,
Nourshargh
S
.
Neutrophils recruited by chemoattractants in vivo induce microvascular plasma protein leakage through secretion of TNF
.
J Exp Med
.
2014
;
211
(
7
):
1307
-
1314
.
6.
Baluk
P
,
Bolton
P
,
Hirata
A
,
Thurston
G
,
McDonald
DM
.
Endothelial gaps and adherent leukocytes in allergen-induced early- and late-phase plasma leakage in rat airways
.
Am J Pathol
.
1998
;
152
(
6
):
1463
-
1476
.
7.
Kim
MH
,
Curry
FR
,
Simon
SI
.
Dynamics of neutrophil extravasation and vascular permeability are uncoupled during aseptic cutaneous wounding
.
Am J Physiol Cell Physiol
.
2009
;
296
(
4
):
C848
-
C856
.
8.
Wessel
F
,
Winderlich
M
,
Holm
M
, et al
.
Leukocyte extravasation and vascular permeability are each controlled in vivo by different tyrosine residues of VE-cadherin
.
Nat Immunol
.
2014
;
15
(
3
):
223
-
230
.
9.
Heemskerk
N
,
Schimmel
L
,
Oort
C
, et al
.
F-actin-rich contractile endothelial pores prevent vascular leakage during leukocyte diapedesis through local RhoA signalling
.
Nat Commun
.
2016
;
7
(
1
):
10493
.
10.
Dumont
DJ
,
Gradwohl
G
,
Fong
GH
, et al
.
Dominant-negative and targeted null mutations in the endothelial receptor tyrosine kinase, tek, reveal a critical role in vasculogenesis of the embryo
.
Genes Dev
.
1994
;
8
(
16
):
1897
-
1909
.
11.
Sato
TN
,
Tozawa
Y
,
Deutsch
U
, et al
.
Distinct roles of the receptor tyrosine kinases Tie-1 and Tie-2 in blood vessel formation
.
Nature
.
1995
;
376
(
6535
):
70
-
74
.
12.
Fukuhara
S
,
Sako
K
,
Minami
T
, et al
.
Differential function of Tie2 at cell-cell contacts and cell-substratum contacts regulated by angiopoietin-1
.
Nat Cell Biol
.
2008
;
10
(
5
):
513
-
526
.
13.
Saharinen
P
,
Eklund
L
,
Miettinen
J
, et al
.
Angiopoietins assemble distinct Tie2 signalling complexes in endothelial cell-cell and cell-matrix contacts
.
Nat Cell Biol
.
2008
;
10
(
5
):
527
-
537
.
14.
Gamble
JR
,
Drew
J
,
Trezise
L
, et al
.
Angiopoietin-1 is an antipermeability and anti-inflammatory agent in vitro and targets cell junctions
.
Circ Res
.
2000
;
87
(
7
):
603
-
607
.
15.
Pizurki
L
,
Zhou
Z
,
Glynos
K
,
Roussos
C
,
Papapetropoulos
A
.
Angiopoietin-1 inhibits endothelial permeability, neutrophil adherence and IL-8 production
.
Br J Pharmacol
.
2003
;
139
(
2
):
329
-
336
.
16.
Baffert
F
,
Le
T
,
Thurston
G
,
McDonald
DM
.
Angiopoietin-1 decreases plasma leakage by reducing number and size of endothelial gaps in venules
.
Am J Physiol Heart Circ Physiol
.
2006
;
290
(
1
):
H107
-
H118
.
17.
Thurston
G
,
Rudge
JS
,
Ioffe
E
, et al
.
Angiopoietin-1 protects the adult vasculature against plasma leakage
.
Nat Med
.
2000
;
6
(
4
):
460
-
463
.
18.
Witzenbichler
B
,
Westermann
D
,
Knueppel
S
,
Schultheiss
HP
,
Tschope
C
.
Protective role of angiopoietin-1 in endotoxic shock
.
Circulation
.
2005
;
111
(
1
):
97
-
105
.
19.
Shen
J
,
Frye
M
,
Lee
BL
, et al
.
Targeting VE-PTP activates TIE2 and stabilizes the ocular vasculature
.
J Clin Invest
.
2014
;
124
(
10
):
4564
-
4576
.
20.
Frye
M
,
Dierkes
M
,
Küppers
V
, et al
.
Interfering with VE-PTP stabilizes endothelial junctions in vivo via Tie-2 in the absence of VE-cadherin
.
J Exp Med
.
2015
;
212
(
13
):
2267
-
2287
.
21.
Braun
LJ
,
Zinnhardt
M
,
Vockel
M
,
Drexler
HC
,
Peters
K
,
Vestweber
D
.
VE-PTP inhibition stabilizes endothelial junctions by activating FGD5
.
EMBO Rep
.
2019
;
20
(
7
):
e47046
.
22.
Thomas
MR
,
Storey
RF
.
The role of platelets in inflammation
.
Thromb Haemost
.
2015
;
114
(
3
):
449
-
458
.
23.
Morrell
CN
,
Aggrey
AA
,
Chapman
LM
,
Modjeski
KL
.
Emerging roles for platelets as immune and inflammatory cells
.
Blood
.
2014
;
123
(
18
):
2759
-
2767
.
24.
Gros
A
,
Ollivier
V
,
Ho-Tin-Noé
B
.
Platelets in inflammation: regulation of leukocyte activities and vascular repair
.
Front Immunol
.
2015
;
5
:
678
.
25.
Zarbock
A
,
Singbartl
K
,
Ley
K
.
Complete reversal of acid-induced acute lung injury by blocking of platelet-neutrophil aggregation
.
J Clin Invest
.
2006
;
116
(
12
):
3211
-
3219
.
26.
Hara
T
,
Shimizu
K
,
Ogawa
F
, et al
.
Platelets control leukocyte recruitment in a murine model of cutaneous arthus reaction
.
Am J Pathol
.
2010
;
176
(
1
):
259
-
269
.
27.
Petri
B
,
Broermann
A
,
Li
H
, et al
.
von Willebrand factor promotes leukocyte extravasation
.
Blood
.
2010
;
116
(
22
):
4712
-
4719
.
28.
Tamagawa-Mineoka
R
,
Katoh
N
,
Ueda
E
,
Takenaka
H
,
Kita
M
,
Kishimoto
S
.
The role of platelets in leukocyte recruitment in chronic contact hypersensitivity induced by repeated elicitation
.
Am J Pathol
.
2007
;
170
(
6
):
2019
-
2029
.
29.
Bernardo
A
,
Ball
C
,
Nolasco
L
,
Choi
H
,
Moake
JL
,
Dong
JF
.
Platelets adhered to endothelial cell-bound ultra-large von Willebrand factor strings support leukocyte tethering and rolling under high shear stress
.
J Thromb Haemost
.
2005
;
3
(
3
):
562
-
570
.
30.
Goerge
T
,
Kleinerüschkamp
F
,
Barg
A
, et al
.
Microfluidic reveals generation of platelet-strings on tumor-activated endothelium
.
Thromb Haemost
.
2007
;
98
(
2
):
283
-
286
.
31.
Zuchtriegel
G
,
Uhl
B
,
Puhr-Westerheide
D
, et al
.
Platelets guide leukocytes to their sites of extravasation
.
PLoS Biol
.
2016
;
14
(
5
):
e1002459
.
32.
Cloutier
N
,
Paré
A
,
Farndale
RW
, et al
.
Platelets can enhance vascular permeability
.
Blood
.
2012
;
120
(
6
):
1334
-
1343
.
33.
Goerge
T
,
Ho-Tin-Noe
B
,
Carbo
C
, et al
.
Inflammation induces hemorrhage in thrombocytopenia
.
Blood
.
2008
;
111
(
10
):
4958
-
4964
.
34.
Hillgruber
C
,
Pöppelmann
B
,
Weishaupt
C
, et al
.
Blocking neutrophil diapedesis prevents hemorrhage during thrombocytopenia
.
J Exp Med
.
2015
;
212
(
8
):
1255
-
1266
.
35.
Ho-Tin-Noé
B
,
Boulaftali
Y
,
Camerer
E
.
Platelets and vascular integrity: how platelets prevent bleeding in inflammation
.
Blood
.
2018
;
131
(
3
):
277
-
288
.
36.
Deppermann
C
,
Kraft
P
,
Volz
J
, et al
.
Platelet secretion is crucial to prevent bleeding in the ischemic brain but not in the inflamed skin or lung in mice
.
Blood
.
2017
;
129
(
12
):
1702
-
1706
.
37.
Gros
A
,
Syvannarath
V
,
Lamrani
L
, et al
.
Single platelets seal neutrophil-induced vascular breaches via GPVI during immune-complex-mediated inflammation in mice
.
Blood
.
2015
;
126
(
8
):
1017
-
1026
.
38.
Ho-Tin-Noé
B
,
Goerge
T
,
Cifuni
SM
,
Duerschmied
D
,
Wagner
DD
.
Platelet granule secretion continuously prevents intratumor hemorrhage
.
Cancer Res
.
2008
;
68
(
16
):
6851
-
6858
.
39.
Higgins
SJ
,
Purcell
LA
,
Silver
KL
, et al
.
Dysregulation of angiopoietin-1 plays a mechanistic role in the pathogenesis of cerebral malaria
.
Sci Transl Med
.
2016
;
8
(
358
):
358ra128
.
40.
Claxton
S
,
Kostourou
V
,
Jadeja
S
,
Chambon
P
,
Hodivala-Dilke
K
,
Fruttiger
M
.
Efficient, inducible Cre-recombinase activation in vascular endothelium
.
Genesis
.
2008
;
46
(
2
):
74
-
80
.
41.
Zhou
BO
,
Ding
L
,
Morrison
SJ
.
Hematopoietic stem and progenitor cells regulate the regeneration of their niche by secreting angiopoietin-1
.
eLife
.
2015
;
4
:
e05521
.
42.
Tiedt
R
,
Schomber
T
,
Hao-Shen
H
,
Skoda
RC
.
Pf4-Cre transgenic mice allow the generation of lineage-restricted gene knockouts for studying megakaryocyte and platelet function in vivo
.
Blood
.
2007
;
109
(
4
):
1503
-
1506
.
43.
Faust
N
,
Varas
F
,
Kelly
LM
,
Heck
S
,
Graf
T
.
Insertion of enhanced green fluorescent protein into the lysozyme gene creates mice with green fluorescent granulocytes and macrophages
.
Blood
.
2000
;
96
(
2
):
719
-
726
.
44.
Nawroth
R
,
Poell
G
,
Ranft
A
, et al
.
VE-PTP and VE-cadherin ectodomains interact to facilitate regulation of phosphorylation and cell contacts
.
EMBO J
.
2002
;
21
(
18
):
4885
-
4895
.
45.
Broermann
A
,
Winderlich
M
,
Block
H
, et al
.
Dissociation of VE-PTP from VE-cadherin is required for leukocyte extravasation and for VEGF-induced vascular permeability in vivo
.
J Exp Med
.
2011
;
208
(
12
):
2393
-
2401
.
46.
Wegmann
F
,
Petri
B
,
Khandoga
AG
, et al
.
ESAM supports neutrophil extravasation, activation of Rho, and VEGF-induced vascular permeability
.
J Exp Med
.
2006
;
203
(
7
):
1671
-
1677
.
47.
Gotsch
U
,
Borges
E
,
Bosse
R
, et al
.
VE-cadherin antibody accelerates neutrophil recruitment in vivo
.
J Cell Sci
.
1997
;
110
(
Pt 5
):
583
-
588
.
48.
Koblizek
TI
,
Runting
AS
,
Stacker
SA
,
Wilks
AF
,
Risau
W
,
Deutsch
U
.
Tie2 receptor expression and phosphorylation in cultured cells and mouse tissues
.
Eur J Biochem
.
1997
;
244
(
3
):
774
-
779
.
49.
Ebnet
K
,
Schulz
CU
,
Meyer Zu Brickwedde
MK
,
Pendl
GG
,
Vestweber
D
.
Junctional adhesion molecule interacts with the PDZ domain-containing proteins AF-6 and ZO-1
.
J Biol Chem
.
2000
;
275
(
36
):
27979
-
27988
.
50.
Davis
S
,
Aldrich
TH
,
Jones
PF
, et al
.
Isolation of angiopoietin-1, a ligand for the TIE2 receptor, by secretion-trap expression cloning
.
Cell
.
1996
;
87
(
7
):
1161
-
1169
.
51.
Sundberg
C
,
Kowanetz
M
,
Brown
LF
,
Detmar
M
,
Dvorak
HF
.
Stable expression of angiopoietin-1 and other markers by cultured pericytes: phenotypic similarities to a subpopulation of cells in maturing vessels during later stages of angiogenesis in vivo
.
Lab Invest
.
2002
;
82
(
4
):
387
-
401
.
52.
Li
JJ
,
Huang
YQ
,
Basch
R
,
Karpatkin
S
.
Thrombin induces the release of angiopoietin-1 from platelets
.
Thromb Haemost
.
2001
;
85
(
2
):
204
-
206
.
53.
Boulaftali
Y
,
Hess
PR
,
Getz
TM
, et al
.
Platelet ITAM signaling is critical for vascular integrity in inflammation
.
J Clin Invest
.
2013
;
123
(
2
):
908
-
916
.
54.
Mammoto
T
,
Parikh
SM
,
Mammoto
A
, et al
.
Angiopoietin-1 requires p190 RhoGAP to protect against vascular leakage in vivo
.
J Biol Chem
.
2007
;
282
(
33
):
23910
-
23918
.
55.
David
S
,
Ghosh
CC
,
Mukherjee
A
,
Parikh
SM
.
Angiopoietin-1 requires IQ domain GTPase-activating protein 1 to activate Rac1 and promote endothelial barrier defense
.
Arterioscler Thromb Vasc Biol
.
2011
;
31
(
11
):
2643
-
2652
.
You do not currently have access to this content.