Impact and related mechanism of exogenous receptor activity modifying protein 1 on calcitonin gene-related peptide modified bone marrow mesenchymal stem cells on the migration of vascular smooth muscle cells in vitro

Abstract

To investigate the impact of calcitonin gene-related peptide (CGRP) modified bone marrow mesenchymal stem cell (MSC) on the migration of vascular smooth muscle cell (VSMC) and related mechanisms. The MSC and VSMC were isolated from rats and cultured, CGRP was transfected to MSC with the high expression lentivirus vector, VSMC was transfected with high expression lentivirus vector of receptor activity modifying protein 1 (RAMP1) and the silence expression lentivirus vector of RAMP1. Then MSC was co-cultured with VSMC. Experimental groups were as follows: (1) Ang II group (MSC + VSMC + Ang II); (2) MSC(CGRP+) group (MSC(CGRP+) + VSMC + Ang II); (3) MSC(CGRP+) RAMP1(-) group (MSC(CGRP+) + VSMC(RAMP1-) + Ang II); (4) MSC(CGRP+) RAMP1(+) group (MSC(CGRP+) + VSMC(RAMP1+) + Ang II); (5) RAMP1(+) group (MSC + VSMC(RAMP1+) + Ang II). Transwell assay was applied to detect the migration of smooth muscle cells, Western blot was applied to detect the protein expression of cells in various groups. VSMC migration number was significantly lower in MSC(CGRP+) group compared with Ang II group (50.8 ± 2.6 vs. 71.4 ± 2.3, P < 0.05), but higher than in MSC(CGRP+) RAMP1(+) group (50.8 ± 2.6 vs. 30.4 ± 3.0, P < 0.05). When RAMP1 expression reduced in VSMC, compared with MSC(CGRP+) RAMP1(+) group, VSMC migration increased in the MSC(CGRP+) RAMP1(-) group compared to MSC(CGRP+)RAMP1(+) (69.0 ± 5.6 vs. 30.4 ± 3.0, P < 0.05) and was similar to Ang II group (69.0 ± 5.6 vs. 71.4 ± 2.3, P > 0.05) and RAMP1(+) group (71.6 ± 3.4). According to the result of Western blot, P-P65 protein expression in MSC(CGRP+) group was lower than that in Ang II group (0.475 ± 0.022 vs.0.642 ± 0.035, P < 0.05). P-P65 protein expression in MSC(CGRP+)RAMP1(-) group was higher than that in MSC(CGRP+) RAMP1(+) group (0.670 ± 0.030 vs. 0.373 ± 0.041, P < 0.05), and there was no difference between MSC(CGRP+)RAMP1(-) group and Ang II group (P > 0.05). P-P65 protein expression was similar between RAMP1(+) group (0.643 ± 0.039) and Ang II group (P > 0.05). CGRP inhibits VSMC migration through RAMP1. NF-κB and RAMP1 play crucial role in the inhibiting effects of CGRP on VSMC migration. Thus, RAMP1-CGRP signaling inhibits VSMC migration through NF-κB signal pathways.