Role of CGRP modified mesenchymal stem cells in restenosis in rat model with carotid angioplasty

Abstract

To explore the role of calcitonin gene-related peptide (CGRP) in the proliferation of vascular smooth muscle cells (VSMCs) and restenosis. The high-expression CGRP lentivirus [Lenti-green fluorescent protein (GFP)-CGRP] was constructed. And mesenchymal stem cells (MSCs) were transfected with Lenti-GFP-CGRP, Lenti-GFP and phosphate buffer saline (PBS). Reverse transcription-polymerase chain reaction (RT-PCR) and enzyme-linked immunosorbent assay (ELISA) were used to determine the CGRP gene and protein expression level of smooth muscle cells in each group respectively. Then MSCs were co-cultured with VSMCs. Experimental groups were MSCs(CGRP+/+)+VSMCs, MSCs(CGRP-/-)+VSMCs and MSCs(PBS)+VSMCs groups. The method of methyl thiazolyl tetrazolium (MTT) was employed to detect the proliferation of smooth muscle cells. Sacculus damaged atherosclerotic carotid was prepared according to the previous study. MSCs were transfected with Lv-CGRP-EGFP and Lv-CGRP and then transplanted into rat model. At Day 7 post-transplantation, injured carotid artery was harvested to detect the C expression of GRP by Western blot and the expression of CD31 by immunofluorescence. At Day 28 post-transplantation, injured carotid artery was harvested to assess organization morphology by hematoxylin and eosin stain and detect the expression of proliferating cell nuclear antigen (PCNA) by immunohistochemical stain. As compared with control and Lenti-GFP groups, the expressions of CGRP protein and CGRP mRNA increased at 72 h after transfecting with Lenti-GFP-CGRP (P < 0.05). After 72-hour co-culturing with VSMCs, the proliferation ability of VSMCs was the lowest in MSCs(CGRP+/+)+VSMCs group versus other three groups (P < 0.05). At Day 7 post-transplantation, as compared with control and Lenti-GFP groups, the expression of CGRP proteins increased significantly in MSCs-CGRP group (P < 0.05). A continuous expression of CD31 was found in damaged carotid intima in MSCs-CGRP group, but not in control group. At Day 28 post-transplantation, the area of intimal hyperplasia was smaller in MSCs-CGRP group than that in control and Lenti-GFP groups. Also the expression of PCNA decreased more in MSCs-CGRP group than control and Lenti-GFP groups (P < 0.05). CGRP protein and CGRP mRNA are expressed after CGRP transfection. And CGRP can suppress the proliferation of VSMCs and reduce intimal hyperplasia so as to facilitate a recovery of damaged endothelium.