Tissue factor pathway inhibitor-2 is induced by fluid shear stress in vascular smooth muscle cells and affects cell proliferation and survival

Abstract

Vascular smooth muscle cells (SMCs) are exposed to fluid shear stress (FSS) after interventional procedures such as balloon-angioplasty. Whereas the effects of hemodynamic forces on endothelial cells are explored in detail, the influence of FSS on smooth muscle cell function is poorly characterized. Here, we investigated the effect of FSS on SMC gene expression and function. Laminar FSS of arterial level (14 dynes/cm(2)) was applied to SMC cultures for 24 hours in a parallel-plate flow chamber. The effect of FSS on gene expression was first screened with microarray technology, and results further verified by real time polymerase chain reaction (RT-PCR) and immunoblotting. Tissue factor pathway inhibitor-2 (TFPI-2) and caspase-3 protein expression was studied in the rat carotid artery after balloon-injury, and the effect of TFPI-2 on SMC DNA synthesis and apoptosis was examined in vitro. Microarrays identified TFPI-2 as one of the most differentially expressed gene by FSS in cultured SMCs (P < .001). Gene set enrichment analysis revealed significant regulation of genes linked to proliferation, apoptosis, and cell cycle regulation. TFPI-2 induction was confirmed by RT-PCR and immunoblotting demonstrating a more than 400-fold (P < .001) increase in TFPI-2 mRNA in SMCs exposed to FSS compared with static controls, and a consistent protein upregulation. Functionally, SMC proliferation was decreased by FSS (P < .001), and recombinant TFPI-2 was found to inhibit SMC proliferation (P < .001) and induce SMC apoptosis as indicated by activation of caspase-3 (P < .01). In vivo, TFPI-2 expression was found to be upregulated 5, 10, and 20 hours (P < .01) after rat carotid balloon injury, and immunohistochemistry demonstrated TFPI-2 protein in FSS-exposed luminal SMCs, co-localized with caspase-3 in the rat carotid neointima. FSS influenced gene expression associated with cell growth and apoptosis in cultured SMCs and strongly induced expression of TFPI-2 mRNA and protein. TFPI-2 was expressed in luminal, FSS-exposed SMCs together with caspase-3 in the rat carotid neointima after balloon injury. Functionally, TFPI-2 may play a role in vessel wall repair by regulating SMC proliferation and survival. Further studies are needed to elucidate the mechanisms by which TFPI-2 controls SMC function.