Page 220 - Haematologica - Vol. 105 n. 6 - June 2020

C. Kroone et al.
vascular injury.7-10 Upon injury to the vessel wall, TF is exposed to blood coagulation factors. The TF-factor VIIa complex catalyzes the proteolytic activation of coagula- tion factor X, leading to generation of the multi-purpose enzyme thrombin, which converts fibrinogen into fibrin, activates platelets, induces thrombus formation, and initi- ates protease-activated receptor (PAR) signaling.11,12 It has been demonstrated that TF expression is induced on vas- cular cells such as EC and SMC as well as on immune cells such as monocytes and may play a pivotal role in a variety of pathological conditions, including acute coro- nary syndromes, thrombosis, sickle cell disease, diabetes, anti-phospholipid antibody syndrome, septic shock, and cancer.2,4,13-20 Furthermore, TF is detectable in macrophages, pericytes and adventitial fibroblasts of nor- mal arteries.21
Inflammatory mediators such as tumor necrosis factor- α (TNF-α) and pro-thrombotic factors promoting throm- bus formation (for example thrombin) have been shown to increase TF expression in vascular cells including EC and SMC.22-24 The regulation of TF transcription in EC and SMC, and circulating cells has been described extensively and involves numerous transcription factors such as acti- vating protein-1 (AP-1) and nuclear factor-κB (NFκB).25,26 In order to identify individuals at risk of thrombosis and to design innovative therapeutic strategies inhibiting thrombus formation in the above-mentioned pathological conditions, it is crucial to identify key factors regulating TF expression and activity in EC and SMC.
LIM-only protein FHL2 is a member of the four and a half LIM (FHL) protein family and is composed of an N- terminal half LIM domain followed by four complete LIM domains.27-31 LIM domains contain double zinc finger structures that mediate protein-protein interactions and, unlike other zinc finger structures, show no affinity for DNA. Rather, FHL2 has been shown to interact with a plethora of proteins including nuclear receptors such as Nur77, liver X receptors, androgen receptor, estrogen receptor, and other transcription factors such as AP-1 and NFκB.27-31 FHL2 is a multifunctional protein and acts as a transcriptional coactivator or corepressor in a cell- and context-dependent manner. Cumulative evidence shows that FHL2 is implicated in a range of physiological and pathological processes, such as cell proliferation, differen- tiation, migration, and apoptosis, bone formation, wound healing and inflammation.27-31 FHL2 is highly expressed in vascular cells including EC and SMC,28-31 which is relevant for the current study.
In this study, we investigated the impact of FHL2 on venous thrombosis using ferric chloride (FeCl3)-induced vascular injury of murine mesenteric vessels. We also demonstrated that FHL2 inhibits TF expression and activ- ity in EC and SMC. Insight into the molecular mecha- nisms governing this regulation involves evidence that FHL2 regulates TF gene expression in an AP-1- and NFκB- dependent manner. Furthermore, we found that FHL2 physically interacts with TF, together modulating local thrombus formation in mice in response to vascular injury. Finally, we identified that the single nucleotide polymorphism (SNP) rs4851770 in the FHL2 gene is asso- ciated with venous thrombosis in humans.
Methods
The methods are described in detail in the Online Supplement.
In vivo thrombosis in mesenteric veins
Five-week old male FHL2-knockout (KO) mice and respective
wildtype (WT) littermate mice (C57BL/6; n=8 per group) were anesthetized by isoflurane (2% for induction, 1.6-1.8% to main- tain anesthesia during imaging) and thrombus formation in the mesenteric veins was provoked as described previously.32 Animals were handled in accordance with national and European animal experimental protocols.
Cell culture and transfection
HEK293T, human umbilical vein endothelial cells (HUVEC), murine and human SMC, and murine macrophages were cultured as described previously.28-30,33,34 Human microvascular endothelial cells (HMEC-1) were cultured in MCDB131 medium (Gibco, Blijswijk, the Netherlands) supplemented with 10% fetal calf serum, epidermal growth factor, penicillin/streptomycin, and L- glutamine. The human endothelial cell line ECRF was cultured in EGM2 medium (Lonza, Basel, Switzerland).
Real-time polymerase chain reaction, western blot, protein stability and co-immunoprecipitation assays
Real-time polymerase chain reaction analysis, western blot, protein stability and co-immunoprecipitation assays were per- formed as described previously.28,29
FHL2 knockdown
Recombinant lentiviral particles encoding FHL2 and short hair-
pin RNA (shRNA) targeting FHL2 were produced, concentrated, and titrated as described previously.24
Tissue factor activity and luciferase assays
TF activity in HUVEC and SMC was assayed as previously described.35
Luciferase assays were performed as described previously using TF-promoter luciferase reporter plasmids and full-length FHL2 or FHL2 variants.28,29,36
Single nucleotide polymorphism association in patients with venous thromboembolism
To study the association between FHL2 and VTE, a two-step validation study was designed, consisting of discovery and repli- cation phases. For the discovery phase, 18 FHL2 SNP (listed in Table 1) were tested. In collaboration with the International Network against VENous Thrombosis (INVENT) consortium, the association between these candidate SNP and venous thrombosis was assessed. Details about the design of the INVENT genome- wide association study have been previously published.37 To obtain replication evidence, SNP rs4851770 (P<0.000201) was selected for genotyping in The Thrombophilia, Hypercoagulability and Environmental Risks in Venous Thromboembolism (THE-VTE) case-control study including 676 patients with a first objectively diagnosed episode of deep venous thrombosis aged 18-70 years and 368 control subjects. This two- center, case–control study has been previously described.38 Human studies were performed with patients’ consent and approval from the institutional review boards of participating research centers and hospitals.
Statistical analysis
All statistical analyses were carried out with GraphPad Prism software (GraphPad Software, San Diego, CA, USA). Comparisons between two groups were done with the Student t test for unpaired variables. Comparisons between more than two
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