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Defining Molecular Mechanism Promoting Neointimal Hyperplasia by CSN8 Hypomorphism
Author(s) -
Giri Samiksha,
Suo Chao,
Martin Douglas,
Wang Xuejun
Publication year - 2021
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.2021.35.s1.04143
Subject(s) - neointimal hyperplasia , cell growth , cop9 signalosome , vascular smooth muscle , downregulation and upregulation , microbiology and biotechnology , hyperplasia , neointima , knockout mouse , ligation , intimal hyperplasia , biology , medicine , endocrinology , cancer research , restenosis , biochemistry , smooth muscle , stent , receptor , protease , peptide hydrolases , gene , enzyme
Background Neointimal hyperplasia is a common pathological process narrowing the vessel lumen caused by the proliferation and migration of vascular smooth muscle cells (VSMCs). This proliferative process threatens literally every known vascular reconstructive process. The COP9 signalosome (CSN) is composed of 8 unique protein subunits (CSN1 through CSN8) that functions in regulating the assembly/disassembly of Cullin RING ligases (CRLs), through a process known as deneddylation. Interestingly, each or some of the 8 subunits may have deneddylation‐independent function. Downregulation of CSN8, the smallest CSN subunit accelerated the growth rate of cultured mouse embryonic fibroblasts whereas cardiac CSN8 deficiency caused massive cardiomyocyte necrosis in mice. A CSN inhibitor has shown very promising antitumor effects. Despite the strong evidence linking CSN to cell cycle regulation, direct study of CSN in VSMC proliferation is lacking. To fill this critical gap, we performed the present study to test the hypothesis that increased CSN5‐mediated nuclear exclusion of p27 contributes to promotion of VSMC proliferation in injured vessels by CSN8 hypomorphism (Figure 1). Methods and Results To produce neointimal hyperplasia in vivo , we subjected adult CSN8 hypomorphic and the littermate non‐hypomorphic control mice to left common carotid artery (LCCA) ligation and collected the LCCA segment proximal to the ligation for analyses. LCCA ligation induced significant increases in CSN8 proteins in wild type mice, which was attenuated in CSN8 hypomorphic mice. Compared to non‐hypomorphic controls, the CSN8 hypomorphic mice displayed more severe neointimal thickening at both 1‐ and 4‐week after ligation, a markedly greater increase in PCNA (proliferating cell nuclear antigen) as revealed by western blot analyses, and a significantly greater prevalence of Ki67‐positive VSMCs as detected by double immunostaining for Ki67 and Sm‐22 (a VSMC specific marker) at 1 week after LCCA ligation. In vitro , platelet derived growth factor (PDGF‐BB) stimulation induced a significant increase in PCNA in CSN8 hypomorphic VSMCs compared to the control VSMCs. Consistently, MTT cell proliferation assays also displayed an increased proliferation of VSMCs from hypomorphic mice in response to PDGF‐BB. Next, we found that LCCA ligation‐induced increases in p27 and p53 proteins were significantly less in hypomorphic mice than in the control. Furthermore, nuclear fractionation revealed that the nuclear to cytoplasmic ratio for p27 and CSN5 proteins were markedly smaller in hypomorphic mice; and native gel electrophoresis revealed a significant increase in the cytoplasmic CSN5 mini complex in hypomorphic mice. This suggests that increased cytoplasmic CSN5 as observed by nuclear fractionation is indeed the increase in the CSN5 associated minicomplex or free CSN5. Conclusion CSN8 hypomorphism increases VSMC proliferation ( in vivo and in vitro ) and promotes neointimal hyperplasia in injured arteries likely through the nuclear exclusion of p27 protein that is mediated by CSN5.