Page 226 - Haematologica - Vol. 105 n. 6 - June 2020

C. Kroone et al.
(Table 1). Next, we sought for replication in the THE-VTE study. Individuals homozygous for the rs4851770C C- allele had a mildly increased risk of venous thrombosis compared with individuals homozygous for the T-allele (OR: 1.2; 95% CI: 0.7-1.4). The risk of idiopathic venous thrombosis was more pronounced (OR:1.53 (95% CI: 0.96 - 2.45) (Table 2).
Discussion
Thrombosis-associated pathologies, such as VTE, myocardial infarction and stroke, are major causes of morbidity and mortality worldwide. TF is a key player in the extrinsic pathway of coagulation14 and although many experimental studies have helped us to understand the coagulation process, the regulation of TF expression and activity is incompletely understood. In the current study, we demonstrated that deficiency of FHL2 exacerbates thrombus formation in response to FeCl3-induced vascu- lar injury. We revealed that FHL2 inhibits the expression of TF in activated vascular cells, including EC and SMC. Furthermore, we showed that FHL2 interacts with the intracellular domain of TF and inhibits TF activity. Additionally, we found that the FHL2 rs4851770 poly- morphism is associated with venous thrombosis in humans.
We previously demonstrated that FHL2-KO mice devel- op larger SMC-rich lesions in the murine carotid artery lig- ation model and here we observed that these lesions com- prised, even 4 weeks after ligation, more thrombi than lesions in WT mice.30 The carotid artery ligation model is, however, not suitable for quantitative assessment of changes in venous thrombosis. For that reason, in this study we used the FeCl3-induced vascular injury model and established that FHL2-KO mice do indeed show exac- erbated venous thrombus formation compared to WT mice.
SMC are the major source of TF in the FeCl3-induced model of vascular injury. Although there are some con- trasting data on TF expression in EC, many studies demonstrated that TF is highly induced in endothelium under inflammatory conditions. Moreover, enhanced activity of TF is transient and directly correlates with increased mRNA levels.22-24 In the current study we found that overexpression of FHL2 inhibits TF expression, whereas FHL2 deficiency results in higher TF levels and activity. These observations were corroborated in HUVEC and microvascular HMEC-1 cells indicating that FHL2- mediated regulation of TF is not limited to larger vessels but may also occur in microvessels.
Thrombin is generated after TF exposure and is known to promote vascular neointima formation through multi- ple mechanisms, including activation of platelets and induction of SMC proliferation.41,42 Given that FHL2 inhibits SMC proliferation28,30 and our current observation that the level of active TF is increased in SMC deficient in FHL2, we hypothesize the following mechanism: in FHL2-KO mice enhanced TF expression promotes throm- bin generation that in turn accelerates SMC proliferation causing enhanced lesion formation in the carotid artery ligation model. In addition to increased TF expression lev- els, we also observed that FHL2-KO SMC produce more pro-inflammatory cytokines than WT SMC, which may
also contribute to the pro-thrombotic phenotype of FHL2- KO mice (data not shown). Further studies are warranted to investigate the hypothesis that thrombin actually medi- ates the increased SMC-rich lesion formation observed in FHL2-KO mice.
It is well established that the human TF gene contains binding sites for the transcription factors NFκB, AP-1, Sp- 1 and Egr-1.25,26 Interestingly, it has been reported that FHL2 is associated with these four transcription factors in multiple cell types in distinct contexts.29,31,43 Here, we demonstrated that FHL2 regulates TF-promoter activity through modulation of both NFκB and AP-1. The relative contribution of FHL2 in regulation of the activity of Egr-1 and Sp1 on the TF promoter is difficult to assess, because deletion of the respective response elements completely abrogates the activity of this promoter, as has been shown before. We found that FHL2 physically interacts with full- length TF, but not with the cytoplasmic tail-deleted mutant of TF (∆CT-TF) indicating that FHL2 interacts intracellularly at the cell membrane with TF. This finding may relate to the interaction of FHL2 with several integrin units such as α3β1, which form a complex with TF.44,45 It is not unlikely that FHL2 either stabilizes or abolishes the integrin α3β1-TF complex, thereby affecting downstream signaling. Further studies are required to investigate the exact role of FHL2 in such TF complexes.
We explored the association between FHL2 polymor- phisms (Table 1) and venous thrombosis risk demonstrat- ing that rs4851770 is associated. FHL2 was not previously known as a direct thrombo-modulator, although it has been shown to modulate the thrombosis-associated genes PAI-146 and eNOS.47 At present, we can only speculate on the functional implication of the FHL2 polymorphism rs4851770 in venous thrombosis. We postulate that this polymorphism affects the binding of specific transcription factors on the FHL2 promoter, resulting in modulation of FHL2 expression and, as a consequence, regulates TF activity.
In summary, we demonstrated that FHL2 is a novel regu- lator of TF in vascular EC and SMC. Furthermore, we report that FHL2 regulates TF promoter activity partly through NFκB and AP-1. Finally, we showed that FHL2 physically interacts with full-length TF. This work reinforces the bio- logical significance of FHL2 as a regulator of venous throm- bosis, which may be of significance to identify individuals at risk of VTE, and suggest that enhancement of FHL2 expression may even be a target for intervention.
Acknowledgments
We thank Kamran Bakhtiari and Joost C.M. Meijers (Dept. of Plasma Proteins, Sanquin Research, Amsterdam, the Netherlands) for assistance with the TF activity assays. We also thank Dr. Nigel Mackman (University of North Carolina) for TF-promoter luciferase reporter plasmids.
Funding
This work was supported by the research program of the BioMedical Materials Institute, co-funded by the Dutch Ministry of Economic Affairs as a part of Project P1.02 NEXTREAM. This work was also supported by the Rembrandt Institute for Cardiovascular Research (RICS grant 2013), the Netherlands CardioVascular Research Initiative: the Dutch Heart Foundation (CVON: 2012-08) and the Dutch Lung Foundation (5.2.17.198JO).
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