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Vascular smooth muscle cells (VSMCs) proliferation and inflammation activation serve as two primary causal factors in restenosis which limits the efficacy of vascular percutaneous intervention. Calpastatin/calpain pathway has attacked more and more attention in vascular remodeling. However, the concrete roles of calpastatin and calpain in vascular restenosis have not been reported to date, no matter the relating mechanism. The present study was designed to elucidate the detailed actions of calpastatin and calpain in restenosis, with animal ligation model and platelet-derived growth factor (PDGF)-BB-pretreated cell model.
Carotid restenosis mice were rendered by ligating left carotid artery, while VSMCs were interfered with by PDGF-BB. Specific small interfering RNA (siRNA) against calpain-1 or -2 were administrated into VSMCs to inhibit their function. Cell proliferation and migration were determined by assays of cell counting kit-8 staining, transwell, and scratch wound healing, respectively. Moreover, the levels of protein and mRNA were assessed by immunohistochemical staining/western blots and quantitative real-time polymerase chain reaction (qRT-PCR), respectively.
In the ligated carotid artery, there was a marked increase in expressions of calpain-1 and calpain-2, whereas calpastatin content was obviously inhibited. With ligated calpastatin transgenic (TG) mice, we revealed that calpastatin induction attenuated the lumen narrowing gradually, which peaked on days 14-21 [day 14: p<0.05, (1.1±0.3 vs. 2.4±0.2 Intima/Media Ratio); day 21: p<0.05, (1.8±0.3 vs. 2.9±0.4 Intima/Media Ratio)]. Cell proliferation and collagen synthesis were also suppressed in TG mice [Proliferating cell nuclear antigen positive cells: p<0.01, (17.6±6.4 vs. 41.2±4.5%); CollagenI: p<0.05, (0.04±0.006 vs. 0.08±0.005 Integral Optical Density/Area)], as well as expressions of calpain-1, calpain-2, matrix metalloproteinase2 (MMP2) and transforming growth factor-β1 (TGF-β1). Consistently, in cultured neonatal murine VSMCs pretreated with PDGF-BB, calpastatin overexpression and specific siRNA against calpain-1 or -2 successfully inhibited the proliferation and migration of VSMCs and the synthesis of collagen, and reduced expressions of MMP2 and TGF-β1. Moreover, simvastatin improved restenosis indicators by suppressing calpain/MMP2/TGF-β1 signaling. However, MMP2 supplementation eliminated the vascular protection of calpastatin induction and simvastatin.
Calpastatin/calpain pathway plays an important role in vascular restenosis by preventing neointimal hyperplasia in the early stage, which may be partly mediated by MMP2/TGF-β1 signaling.