Neuropilin-1 and Neovascularization Are Associated With the Vulnerability of Carotid Plaques: New Insights Into Atherosclerosis Plaques Development

Abstract

Background and Objective: Intraplaque neovascularization was considered as an important indicator of plaque vulnerability, neuropilin-1 (NRP1) acted as a coreceptor for vascular endothelial growth factor with an essential role in angiogenesis, the mechanism of NRP1 in plaque vulnerability remained unknown. This article aimed to investigate the relationship between NRP1 and neovascularization with carotid atherosclerotic plaque vulnerability. Besides, new insights into the NRP1 was attained by regulating the development of atherosclerosis plaques. Methods: In this study, 3 plaques of carotid endarterectomy (CEA) and 3 normal external carotid arteries which were obtained from trimmed external carotid artery of high flow intracranial - external artery bypass grafting was first compared by RNA sequencing, functional analysis was used to screen out the genes significantly different related to neovascularization in plaques. A total of 65 plaques from CEA were registered and divided into stable group and vulnerable group by histology. NRP1 and its associated protein expression were analyzed by western blot and co-immunoprecipitation assay. CCK-8 and tube formation assays were used to analyze cell viability and angiogenesis. Transwell migration and Wound healing assays were utilized to assess the motility of HMEC-1 cells. Immunohistochemistry of CD31 and CD68 were used to assess neovascularization and inflammation respectively. Findings: 7110 differentially expressed genes were identified in RNA sequencing. NRP1 was up-regulated compared to normal external carotid artery and related to angiogenesis. The protein levels of NRP1 was elevated in vulnerable plaques. Knockdown of NRP1 inhibited HMEC-1 cells proliferation, angiogenesis and migration in vitro. The protein levels of phospho-FAK and the molecules of integrin α3, α6, αV and β1 were also up-regulated in vulnerable plaques (all P < 0.05). In immunohistochemical results, the expression of CD31 and CD68 were elevated in vulnerable plaques and associated with the plaque vulnerability (P = 0.038, P = 0.015). Interpretation: The current study demonstrated that NRP1 played an important role in vulnerable plaques with angiogenesis. Meanwhile, NRP1 may interact with integrin to activate the FAK signaling pathway to increase neovascularization of plaques. Besides, neovascularization was also associated with plaques instability and inflammatory infiltrate. Funding Statement: This work was supported by cadre health care research project of Jiangsu Province (No. BJ17010), people’s livelihood science and technology demonstration project of Suzhou (No. SS202061, No. SS201859), thirteen major projects of the ministry of science and technology of China (No. 2017YFC0114302). Declaration of Interests: The authors declare no competing interests. Ethics Approval Statement: This study was approved by the local ethics committee (NO: 2019124) and all enrolled subjects had signed written informed consent.