Regulation Of Calcitonin Gene-related Pep Tide And Receptor Activity-modifying Protein-1on Proliferation Of Vascular Smooth Muscle Cells

Objective and BackgroundVascular smooth muscle cells exist in the vascular intima, normally they can secrete and synthesize collagen and maintain the tension of blood vessel wall through mild contraction. Relevant studies show that the abnormal VSMC proliferation and migration of VSMCs plays an important role in the pathological process of hypertension, atherosclerosis, and vascular restenosis after percutaneous coronary intervention. Recently, studies on stem cell transplantation therapy indicate that transplantation of mesenchymal stem cells (MSCs) could improve the repair of damaged arterial endothelium and inhibit neointimal hyperplasia, while no direct effects on the inhibition of smooth muscle cells proliferation were indicated in these studies. Therefore, problems need to be solved in the future is how to modify or change MSCs to maximize its effect. Calcitonin gene related protein (CGRP) and its receptor subunit bind, which inhibit the proliferation in vivo and in vitro and reverse artery remodeling. It is confirmed that a lot of CGRP receptors expressed in the VSMCs, while CGRP could not bind to receptor separately, it worked through receptor activity modifying proteins (RAMPs). RAMP1is a member of the RAMPs family. RAMP1receptor expresses in VSMCs. Our group’s previous studies show that RAMP1can inhibit VSMCs proliferation in the vessels wall after percutaneous coronary interventional therapy, and promote the repair of damaged vessels, and reduce the expression of inflammation cytokines after vessels damaged, and inhibit endometrial hyperplasia as well as reduce restenosis after angioplasty. However, mechanisms are still unknown. We inferred that this is possibly related with the inhibition of VSMCs proliferation by RAMP1, which possiblely acts through the regulation of CGRP by RAMP1. We constructed the high expression level of RAMP1and RAMP1-silenced VSMCs and observed the effect of RAMP1on VSMCs proliferation in combined with CGRP and RAMP1intervention together, so as to investigate whether the mechanisms of effect of RAMP1on VSMCs could be achieved by the regulation of the sentivity of VSMCs to CGRP. Also, after we tested the transfection of MSCs by lentivirus carried CGRP, we want to know whether the amount of CGRP secretion and MSCs with high expression level could play a stable biology effect of CGRP, so as to further explore the effect of MSCs, which is modified by target gene, in the treatment of restenosis after angioplasty, and thus provided the basis for the experiments.MethodsPart ⅠThe high expression RAMP1lentivirus (Lenti-GFP-RAMP1, referred to RAMP1+/+), silent RAMP1lentivirus vector (Lenti-GFP-shRAMP1, referred to RAMP1+/+) and empty virus vector (Lenti-GFP, referred to GFP+/+) were constructed respectively. After isolation, the rat thoracic aorta VSMCs were obtained. After synchronization of cell cultures, the lentiviral vector, successfully constructed as above, were transfected to VSMCs. The experiment has four groups:RAMP1+/+transfected to VSMCs group (VSMCsRAMP1+group), RAMP1-/-transfected to VSMCs group (VSMCsRAMP1-/-group), GFP+/+empty virus transfected to VSMCs group (VSMCsGFP+/+group) and VSMCs control group. Each group was further divided into24h,48h,72h and96h subgroups. The flow cytometry was used to determine the transfection efficiency of lentivirus respectively; The ELIS A method was used to determine the protein secretion of each subgroup RAMP1at different time points to decide the best infection time point; RT-PCR and Western blot technique were used to determine the RAMP1genes and protein expression level of the smooth muscle cells in each group respectively. In addition, Ang II inducer of cell proliferation was added6h after lentivirus transfection to simulate intravascular VSMCs proliferation model. MTT method, flow cytometry and Western blot technique are applied to detect the proliferation, apoptosis, periodic variation of each subgroup cell and the expression changes of spectrin a-SM-actin (a-Smooth Muscle Antibody, a-SM-actin) and synthetic proteins osteopontin(OPN); the transfer ability of smooth muscle cells in each group was observed in the cell scratch test.Part IICGRP protein direct contact method:In order to observe the action pathway of RAMP1during CGRP inhibition of smooth muscle cell proliferation, above-mentioned VSMCsRAMP1+/+group, VSMCsRAMP1-/-group and normal VSMCs group were interfered by certain concentration of CGRP protein. Experimental groups were as follows:VSMCs control group, CGRP+VSMCs group, CGRP+VSMCs RAMP1+/+group, CGRP+VSMCsRAMP1-/-group. MTT, flow cytometry and Western blot technique were used to detect the proliferation, apoptosis, and cycle variation of vascular smooth muscle cells in each group, respectively.Cell co-culture method:To further validate the above experimental results, meanwhile, to observe whether CGRP can continue to maintain the original biological effects or not after CGRP+/+was transfected into MSCs. Firstly, the high expression lentivirus vector of CGRP (Lenti-GFP-CGRP, referred to CGRP+/+) were constructed and MSCs were transfected to become MSCs of high expression CGRP (MSCsCGRP+/+). Protein secretion in the above-mentioned MSCsCGRP+/+supernatant was detected using ELISA method, and then MSCsCGRP+" was co-cultured with VSMCsRAMP1+/+, VSMCsRAMP1-/-and normal VSMCs, respectively. Experimental groups were as follows:VSMCs control group, MSCs+VSMCs group, MSCsCGRP+/++VSMCs group, MSCs CGRP+/+VSMCsRAMP1+/+group, MSCsCGRP+/++VSMCs RAMP1-/-group. Flow cytometry and Western blot technique were applied to detect the apoptosis and cycle variation of smooth muscle cells, the expression changes of spectrin α-SM-actin and synthetic protein OPN in each group, respectively. ResultsPartⅠThe expression of green fluorescent proteins reached the highest level72hours after the transfection of cells with lentivirus. Compared with the control group, the expression of RAMP1genes and proteins increased significantly after the transfection of VSMCs with RAMP1+/+(P<0.05), while decreased significantly after the transfection of VSMCs with RAMP1-/-(P<0.05). Furthermore, the former transfection inhibited Angll-induced cell proliferation, boosted early apoptosis cells, made relatively more cells stay in the stationary phase, enhanced the expression of contractile protein a-SM-actin and lowered cell migration rate distinctively. The indications mentioned above were of significant difference when compared with the control group (P<0.05), suggesting that high expression of RAMP1clearly inhibited proliferation of VSMCs. On the contrary, the latter transfection promoted Ang Ⅱ-induced cell proliferation, reduced the number of cells in Go phase and early apoptosis cells, enhanced the expression of synthetic protein OPN and increased cell migration rate. The indications mentioned above were of significant difference when compared with the control group (P<0.05). On the other hand, tranfection with GFP+/+were of no significant effect on the proliferation, apoptosis and phenotype of cells, which was not significantly different from that of the control group (P>0.05).Part ⅡCGRP protein direct contact method:MTT results showed that the intervention of CGRP protein significantly inhibited the proliferation of VSMCs compared with the control group (P<0.05); flow cytometry detection results showed that CGRP protein intervention in the VSMCs could cause increased viable apoptotic cells, and the number of the cells that remain in the Go phase significantly increased when compared with the control group (P <0.05); when CGRP protein intervened VSMCsRAMP1+/+, the above-mentioned inhibition of CGRP significantly increased (P<0.05); when CGRP protein intervened VSMCsRAMP1the above-mentioned inhibition of CGRP decreased markedly (P<0.05).Cell co-culture method:After CGRP+/+transfected MSCs, the secretory volume of CGRP protein in the MSCsCGRP+/+supernatant detected by ELISA increased significantly when compared with the control group; after MSCsCGRP+/+co-cultured with VSMCs RAMP1+/+, VSMCsRAMP1-/-, VSMCs respectively, the effects made by CGRP protein are consistent with the results of CGRP protein direct contact method. Moreover, Western blot results also show that the co-culture with MSCsCGRP+/+can precipitate the increased expression of contractile phenotype protein a-SM-actin and the decreased expression of intermediate phenotype OPN in normal smooth muscle cells (P<0.05), when MSCsCGRP+co-cultured with VSMCs RAMP1+/+, the above phenotype changes significantly increased (P <0.05), when MSCsCGRP+/+co-cultured with VSMCsRAMP1-/-, the above phenotype changes significantly reduced (P<0.05).ConclusionPart ⅠHigh expression of RAMP1suppressed the proliferation of VSMCs induced by AngⅡ, promoted early apoptosis, and affected the phenotypic switch. However, these effects were decreased after silence of RAMP1in VSMCs, suggesting RAMP1has inhibitory effect on VSMCs.Part ⅡHigh expression of RAMP1enhanced the inhibitory effects of CGRP on the proliferation of VSMCs, promoted apoptosis, and inhibited the phenotype switch from contractile to synthetic phenotype; in contrast, silencing of RAMP1reduced these effects of CGRP on VSMCs. These results further demonstrate that RAMP1is a key peptide for CGRP to exert the biological effects; MSCsCGRP+/+can secrete high-level of CGRP, and enable CGRP to have its biological effects. Therefore, the above results provide in vitro support for in vivo transplantation of CGRP-modified MSCs.