• ECFCs of patients with low VWF exhibit decreased VWF secretion, transcriptional heterogeneity, and alterations in VWF mRNA content.

  • Single-cell RNA sequencing of ECFCs identified FLI1 and other genes as candidate regulators of VWF.

von Willebrand factor (VWF) plays a key role in normal hemostasis, and deficiencies of VWF lead to clinically significant bleeding. We sought to identify novel modifiers of VWF levels in endothelial colony-forming cells (ECFCs) using single-cell RNA sequencing (scRNA-seq). ECFCs were isolated from patients with low VWF levels (plasma VWF antigen levels between 30 and 50 IU/dL) and from healthy controls. Human umbilical vein endothelial cells were used as an additional control cell line. Cells were characterized for their Weibel Palade body (WPB) content and VWF release. scRNA-seq of all cell lines was performed to evaluate for gene expression heterogeneity and for candidate modifiers of VWF regulation. Candidate modifiers identified by scRNA-seq were further characterized with small-interfering RNA (siRNA) experiments to evaluate for effects on VWF. We observed that ECFCs derived from patients with low VWF demonstrated alterations in baseline WPB metrics and exhibit impaired VWF release. scRNA-seq analyses of these endothelial cells revealed overall decreased VWF transcription, mosaicism of VWF expression, and genes that are differentially expressed in low VWF ECFCs and control endothelial cells (control ECs). An siRNA screen of potential VWF modifiers provided further evidence of regulatory candidates, and 1 such candidate, FLI1, alters the transcriptional activity of VWF. In conclusion, ECFCs from individuals with low VWF demonstrate alterations in their baseline VWF packaging and release compared with control ECs. scRNA-seq revealed alterations in VWF transcription, and siRNA screening identified multiple candidate regulators of VWF.

1.
Ng
C
,
Motto
DG
,
Di Paola
J
.
Diagnostic approach to von Willebrand disease
.
Blood.
2015
;
125
(
13
):
2029
-
2037
.
2.
Lavin
M
,
Aguila
S
,
Schneppenheim
S
, et al
.
Novel insights into the clinical phenotype and pathophysiology underlying low VWF levels: the Low von Willebrand Factor in Ireland Cohort (LoVIC) Study
.
Blood.
2016
;
128
(
22
):
873
.
3.
Smith
NL
,
Chen
M-H
,
Dehghan
A
, et al;
Wellcome Trust Case Control Consortium
.
Novel associations of multiple genetic loci with plasma levels of factor VII, factor VIII, and von Willebrand factor: the CHARGE (Cohorts for Heart and Aging Research in Genome Epidemiology) Consortium
.
Circulation.
2010
;
121
(
12
):
1382
-
1392
.
4.
Sabater-Lleal
M
,
Huffman
JE
,
de Vries
PS
, et al;
INVENT Consortium; MEGASTROKE Consortium of the International Stroke Genetics Consortium (ISGC)
.
Genome-wide association transethnic meta-analyses identifies novel associations regulating coagulation factor VIII and von Willebrand factor plasma levels
.
Circulation.
2019
;
139
(
5
):
620
-
635
.
5.
van Loon
J
,
Dehghan
A
,
Weihong
T
, et al
.
Genome-wide association studies identify genetic loci for low von Willebrand factor levels [correction published in Eur J Hum Genet. 2016;24:1096]
.
Eur J Hum Genet.
2016
;
24
(
7
):
1035
-
1040
.
6.
Sanders
YV
,
van der Bom
JG
,
Isaacs
A
, et al;
WiN Study Group
.
CLEC4M and STXBP5 gene variations contribute to von Willebrand factor level variation in von Willebrand disease
.
J Thromb Haemost.
2015
;
13
(
6
):
956
-
966
.
7.
Zhu
Q
,
Yamakuchi
M
,
Ture
S
, et al
.
Syntaxin-binding protein STXBP5 inhibits endothelial exocytosis and promotes platelet secretion
.
J Clin Invest.
2014
;
124
(
10
):
4503
-
4516
.
8.
Swystun
LL
,
Lai
JD
,
Notley
C
, et al
.
The endothelial cell receptor stabilin-2 regulates VWF-FVIII complex half-life and immunogenicity
.
J Clin Invest.
2018
;
128
(
9
):
4057
-
4073
.
9.
Liu
J
,
Kanki
Y
,
Okada
Y
, et al
.
A +220 GATA motif mediates basal but not endotoxin-repressible expression of the von Willebrand factor promoter in Hprt-targeted transgenic mice
.
J Thromb Haemost.
2009
;
7
(
8
):
1384
-
1392
.
10.
Yuan
L
,
Nikolova-Krstevski
V
,
Zhan
Y
, et al
.
Antiinflammatory effects of the ETS factor ERG in endothelial cells are mediated through transcriptional repression of the interleukin-8 gene
.
Circ Res.
2009
;
104
(
9
):
1049
-
1057
.
11.
Liu
J
,
Yuan
L
,
Molema
G
, et al
.
Vascular bed-specific regulation of the von Willebrand factor promoter in the heart and skeletal muscle
.
Blood.
2011
;
117
(
1
):
342
-
351
.
12.
Peng
Y
,
Jahroudi
N
.
The NFY transcription factor functions as a repressor and activator of the von Willebrand factor promoter
.
Blood.
2002
;
99
(
7
):
2408
-
2417
.
13.
Yuan
L
,
Chan
GC
,
Beeler
D
, et al
.
A role of stochastic phenotype switching in generating mosaic endothelial cell heterogeneity
.
Nat Commun.
2016
;
7
(
1
):
10160
.
14.
Xiang
Y
,
Cheng
J
,
Wang
D
, et al
.
Hyperglycemia repression of miR-24 coordinately upregulates endothelial cell expression and secretion of von Willebrand factor
.
Blood.
2015
;
125
(
22
):
3377
-
3387
.
15.
Jaffe
EA
,
Hoyer
LW
,
Nachman
RL
.
Synthesis of antihemophilic factor antigen by cultured human endothelial cells
.
J Clin Invest.
1973
;
52
(
11
):
2757
-
2764
.
16.
Everett
LA
,
Cleuren
ACA
,
Khoriaty
RN
,
Ginsburg
D
.
Murine coagulation factor VIII is synthesized in endothelial cells
.
Blood.
2014
;
123
(
24
):
3697
-
3705
.
17.
Lin
Y
,
Weisdorf
DJ
,
Solovey
A
,
Hebbel
RP
.
Origins of circulating endothelial cells and endothelial outgrowth from blood
.
J Clin Invest.
2000
;
105
(
1
):
71
-
77
.
18.
Wang
J-W
,
Bouwens
EAM
,
Pintao
MC
, et al
.
Analysis of the storage and secretion of von Willebrand factor in blood outgrowth endothelial cells derived from patients with von Willebrand disease
.
Blood.
2013
;
121
(
14
):
2762
-
2772
.
19.
Starke
RD
,
Paschalaki
KE
,
Dyer
CEF
, et al
.
Cellular and molecular basis of von Willebrand disease: studies on blood outgrowth endothelial cells
.
Blood.
2013
;
121
(
14
):
2773
-
2784
.
20.
Ferraro
F
,
Mafalda Lopes da
S
,
Grimes
W
, et al
.
Weibel-Palade body size modulates the adhesive activity of its von Willebrand Factor cargo in cultured endothelial cells
[correction published in Sci Rep. 2016;6:33938].
Sci Rep.
2016
;
6
(
1
):
32473
.
21.
Baker
CD
,
Black
CP
,
Ryan
SL
,
Balasubramaniam
V
,
Abman
SH
.
Cord blood endothelial colony-forming cells from newborns with congenital diaphragmatic hernia
.
J Pediatr.
2013
;
163
(
3
):
905
-
907
.
22.
Lavin
M
,
Aguila
S
,
Schneppenheim
S
, et al
.
Novel insights into the clinical phenotype and pathophysiology underlying low VWF levels
.
Blood.
2017
;
130
(
21
):
2344
-
2353
.
23.
Ng
CJ
,
McCrae
KR
,
Ashworth
K
, et al
.
Effects of anti-β2GPI antibodies on VWF release from human umbilical vein endothelial cells and ADAMTS13 activity
.
Res Pract Thromb Haemost.
2018
;
2
(
2
):
380
-
389
.
24.
White-Adams
TC
,
Ng
CJ
,
Jacobi
PM
,
Haberichter
SL
,
Di Paola
JA
.
Mutations in the D’D3 region of VWF traditionally associated with type 1 VWD lead to quantitative and qualitative deficiencies of VWF
.
Thromb Res.
2016
;
145
:
112
-
118
.
25.
Consortium
TEP
;
ENCODE Project Consortium
.
An integrated encyclopedia of DNA elements in the human genome
.
Nature.
2012
;
489
(
7414
):
57
-
74
.
26.
10X Genomics
.
How does Cell Ranger correct for amplification bias?
https://kb.10xgenomics.com/hc/en-us/articles/115004037743-How-does-Cell-Ranger-correct-for-amplification-bias-. Accessed 7 April 2021.
27.
Qiu
X
,
Hill
A
,
Packer
J
,
Lin
D
,
Ma
YA
,
Trapnell
C
.
Single-cell mRNA quantification and differential analysis with Census
.
Nat Methods.
2017
;
14
(
3
):
309
-
315
.
28.
Trapnell
C
,
Cacchiarelli
D
,
Grimsby
J
, et al
.
The dynamics and regulators of cell fate decisions are revealed by pseudotemporal ordering of single cells
.
Nat Biotechnol.
2014
;
32
(
4
):
381
-
386
.
29.
Butler
A
,
Hoffman
P
,
Smibert
P
,
Papalexi
E
,
Satija
R
.
Integrating single-cell transcriptomic data across different conditions, technologies, and species
.
Nat Biotechnol.
2018
;
36
(
5
):
411
-
420
.
30.
Benaglia
T
,
Chauveau
D
,
Hunter
DR
,
Young
D
.
mixtools: an R package for analyzing finite mixture models
.
J Stat Softw.
2009
;
32
(
6
):1-29.  
31.
James
PD
,
Notley
C
,
Hegadorn
C
, et al
.
The mutational spectrum of type 1 von Willebrand disease: results from a Canadian cohort study
.
Blood.
2007
;
109
(
1
):
145
-
154
.
32.
Flood
VH
,
Friedman
KD
,
Gill
JC
, et al
.
No increase in bleeding identified in type 1 VWD subjects with D1472H sequence variation
.
Blood.
2013
;
121
(
18
):
3742
-
3744
.
33.
Butler
LM
,
Hallström
BM
,
Fagerberg
L
, et al
.
Analysis of body-wide unfractionated tissue data to identify a core human endothelial transcriptome
.
Cell Syst.
2016
;
3
(
3
):
287
-
301
.
34.
Krock
BL
,
Skuli
N
,
Simon
MC
.
Hypoxia-induced angiogenesis: good and evil
.
Genes Cancer.
2011
;
2
(
12
):
1117
-
1133
.
35.
Fryer
BH
,
Field
J
.
Rho, Rac, Pak and angiogenesis: old roles and newly identified responsibilities in endothelial cells
.
Cancer Lett.
2005
;
229
(
1
):
13
-
23
.
36.
Schwachtgen
JL
,
Janel
N
,
Barek
L
, et al
.
ETS transcription factors bind and transactivate the core promoter of the von Willebrand factor gene
.
Oncogene.
1997
;
15
(
25
):
3091
-
3102
.
37.
Nagai
N
,
Ohguchi
H
,
Nakaki
R
, et al
.
Downregulation of ERG and FLI1 expression in endothelial cells triggers endothelial-to-mesenchymal transition
.
PLoS Genet.
2018
;
14
(
11
):
e1007826
.
38.
Baratz
KH
,
Tosakulwong
N
,
Ryu
E
, et al
.
E2-2 protein and Fuchs’s corneal dystrophy
.
N Engl J Med.
2010
;
363
(
11
):
1016
-
1024
.
39.
Flood
VH
,
Schlauderaff
AC
,
Jacobi
PM
, et al
.
VWF interaction with type IV collagen is mediated through critical VWF A1 domain residues
.
Blood.
2013
;
122
(
21
):
29
.
40.
Groeneveld
DJ
,
van Bekkum
T
,
Dirven
RJ
, et al
.
Angiogenic characteristics of blood outgrowth endothelial cells from patients with von Willebrand disease
.
J Thromb Haemost.
2015
;
13
(
10
):
1854
-
1866
.
41.
Aird
WC
,
Edelberg
JM
,
Weiler-Guettler
H
,
Simmons
WW
,
Smith
TW
,
Rosenberg
RD
.
Vascular bed-specific expression of an endothelial cell gene is programmed by the tissue microenvironment
.
J Cell Biol.
1997
;
138
(
5
):
1117
-
1124
.
42.
Yamamoto
K
,
de Waard
V
,
Fearns
C
,
Loskutoff
DJ
.
Tissue distribution and regulation of murine von Willebrand factor gene expression in vivo
.
Blood.
1998
;
92
(
8
):
2791
-
2801
.
43.
Fujisawa
T
,
Tura-Ceide
O
,
Hunter
A
, et al
.
Endothelial progenitor cells do not originate from the bone marrow
.
Circulation.
2019
;
140
(
18
):
1524
-
1526
.
44.
Toshner
M
,
Dunmore
B
,
McKinney
E
, et al
.
Analyses of blood outgrowth endothelial cells reveal an endothelial HOX gene signature in human beings
.
Lancet.
2013
;
381
:
S108
.
45.
Nightingale
T
,
Cutler
D
.
The secretion of von Willebrand factor from endothelial cells; an increasingly complicated story
.
J Thromb Haemost.
2013
;
11
(
suppl 1
):
192
-
201
.
46.
McCormack
JJ
,
Lopes da Silva
M
,
Ferraro
F
,
Patella
F
,
Cutler
DF
.
Weibel-Palade bodies at a glance
.
J Cell Sci.
2017
;
130
(
21
):
3611
-
3617
.
47.
Schillemans
M
,
Karampini
E
,
Kat
M
,
Bierings
R
.
Exocytosis of Weibel-Palade bodies: how to unpack a vascular emergency kit
.
J Thromb Haemost.
2019
;
17
(
1
):
6
-
18
.
48.
Klarenbach
SW
,
Chipiuk
A
,
Nelson
RC
,
Hollenberg
MD
,
Murray
AG
.
Differential actions of PAR2 and PAR1 in stimulating human endothelial cell exocytosis and permeability: the role of Rho-GTPases
.
Circ Res.
2003
;
92
(
3
):
272
-
278
.
49.
Yuan
L
,
Janes
L
,
Beeler
D
, et al
.
Role of RNA splicing in mediating lineage-specific expression of the von Willebrand factor gene in the endothelium
.
Blood.
2013
;
121
(
21
):
4404
-
4412
.
50.
Hawke
L
,
Bowman
ML
,
Poon
M-C
,
Scully
MF
,
Rivard
GE
,
James
PD
.
Characterization of aberrant splicing of von Willebrand factor in von Willebrand disease: an underrecognized mechanism
.
Blood.
2016
;
128
(
4
):
584
-
593
.
51.
Mojiri
A
,
Alavi
P
,
Lorenzana Carrillo
MA
, et al
.
Endothelial cells of different organs exhibit heterogeneity in von Willebrand factor expression in response to hypoxia
.
Atherosclerosis.
2019
;
282
:
1
-
10
.
52.
Morini
MF
,
Giampietro
C
,
Corada
M
, et al
.
VE-cadherin-mediated epigenetic regulation of endothelial gene expression
.
Circ Res.
2018
;
122
(
2
):
231
-
245
.
53.
Toyama
T
,
Asano
Y
,
Miyagawa
T
, et al
.
The impact of transcription factor Fli1 deficiency on the regulation of angiogenesis
.
Exp Dermatol.
2017
;
26
(
10
):
912
-
918
.
54.
Asano
Y
,
Stawski
L
,
Hant
F
, et al
.
Endothelial Fli1 deficiency impairs vascular homeostasis: a role in scleroderma vasculopathy
.
Am J Pathol.
2010
;
176
(
4
):
1983
-
1998
.
55.
de Boer
S
,
Bowman
M
,
Notley
C
, et al
.
Endothelial characteristics in healthy endothelial colony forming cells; generating a robust and valid ex vivo model for vascular disease
.
J Thromb Haemost.
2020
;
18
(
10
):
2721
-
2731
.
56.
Pruss
CM
,
Golder
M
,
Bryant
A
, et al
.
Pathologic mechanisms of type 1 VWD mutations R1205H and Y1584C through in vitro and in vivo mouse models
.
Blood.
2011
;
117
(
16
):
4358
-
4366
.
You do not currently have access to this content.

Sign in via your Institution

Sign In