| Objective and BackgroundRestenosis, caused by balloon or stenting during Percutaneous coronary interventional therapy (PCI), was always having been urgently tackled one of the important clinical issues. Mesenchymal stem cells (MSCs) transplantation therapy has the function in not only improving heart function after myocardial infarction (MI) but also accelerating reendothelialization of injuried artery and inhibiting neointimal hyperplasia. However, the effectiveness of native MSCs therapy was not able to defend against reactive neointimal hyperplasia resulted from balloon or stenting destroying artery endothelium. The experimental results in our study team and others indicated that the disadvantage of MSCs transplantation is duing to its insufficiency function in inhibiting the proliferation of vascular smooth muscle cells while MSCs contributed to the recovery of damaged endothelium. So, what should we do to enhance the potency of MSCs to inhibit the proliferation of VSMCs through the methods of the gene modification, which must be paid more attention in the next study. Calcitonin gene related protein (CGRP) and its receptor subunit, receptor activity modifying protein-1 (RAMP1), have the potency of inhibiting the proliferation and promoting the apoptosis of VSMCs in vivo and vitro. RAMP1 is the key signal peptide to regulate the function of CGRP, and exogenous RAMP1 could reinforce its potency of protection against of proliferation. Thus, we found double-injury model of MI reperfusion and sacculus damaged atherosclerotic carotid through mimesis the PCI therapy pattern in the patients with MI, and explored whether hRAMP1 modified MSCs develop more favorable to inhibit the VSMCs proliferation while MSCs have the therapeutic potency on heart function and endothelial protection through cell transplantation of modified or natural MSCs, which could supply for the evidence of the therapy of Restenosis post-PCI.Methods Part One:VSMCs was isolated from rabbit's aortic artery and cultured with tissue adherence in vitro. VSMCs were simulated by incubated with angiotensinⅡafter the transfection with Ad-EGFP-hRAMP1, and divided into three groups:hRAMP1 group, empty-adenovirus vector group and control group, and each group including four subgroups of 24h,48h,72h and 96h. hRAMP1 protein was detected by immunocytochemistry, and the proliferation potency of VSMCs by cell count and MTT, and the apoptosis of VSMCs by flow cytometry and TUNEL assay.Part Two:MSCs were collected through density gradient centrifugation and adherent culture, P3 of MSCs was made use of the experiment. Double-injury Rabbit model with MI reperfusion and sacculus damaged atherosclerotic carotid were made according to the previous study. MSCs were transfected with adenovirus vector with enhanced green fluorescent protein (EGFP), or with both EGFP and hRAMP1, and then MSCs were transplanted into rabbit model. All animals were randomly divided into three groups: Ad-EGFP-hRAMP1-MSCs transfection group (hRAMP1-MSCs group, n=24), and Ad-EGFP-MSCs transfection group (MSCs group, n=24), and control group (n=20). At the different time-points of cell transplantation after 7d,14d and 28d. the injured carotid arteries and infarction myocardium was harvested to detect EGFP positive MSCs and hRAMP1 expression, and to assess organization morphology by HE stain, TCC stain or immunohistochemical stains, and heart function by UCG, and cell apoptosis by flow cytometry and TUNEL assay. Also, the plasma level of CRP and TNF-a was measured by ELISA.ResultsPart One:VSMCs were successfully infected by Ad-EGFP-hRAMP1 or Ad-EGFP, and the expression of EGFP was increasing, peaking at 72h and lasting for 96h. hRAMP1 protein expression of VSMCs significantly increased in hRAMP1 group compared with that in empty-adenovirus vector group and control group (P<0.05). Exogenous RAMP1 for 72h could inhibit the viability, continuous proliferation of VSMCs induced by AngⅡ(P<0.05). Also, the level of VSMCs apoptosis was much higher in hRAMP1 group compared with other both groups (P<0.05). Part Two:(1) MSCs with EGFP marker and continuous CD31 expression were found in intima of damaged carotid of both hRAMP1-MSCs group and MSCs, but CD31 expression not found in control group. The expression of hRAMPl mRNA by RT-PCR in myocardium and damaged carotid intima of hRAMP1-MSCs groups. Also, the expression of hRAMP1 protein was found by Western blot in myocardium and damaged carotid intima of three groups, and hRAMP1 higher in hRAMP1-MSCs group than that in other two groups. The improvement of heart function and the decrease of infarct size were found 28d after cell transplantation in hRAMP1-MSCs group and MSCs groups compared with that in control group, especially much higher in hRAMP1-MSCs group. (2) In all groups 7d,14d,28d after sacculus damaged carotid, the area of hyperplasia of intima and the rate of neointima and media in hRAMP1-MSCs group was lower than that in MSCs group; and which in both groups of MSCs transplantation was much lower than that in control groups. also, the expression of a-SMA was found in the hyperplasia intima (3) The expression of PCNA and TNF-a were significantly decreased in hRAMP1-MSCs group than MSCs group and control groups (P<0.05), and that much lower in MSCs group compared with control group. Meanwhile, cells apoptosis rate was significantly increased in hRAMPl-MSCs group (P<0.05), MSCs group less compared with hRAMP1-MSCs group (P<0.05), Control group at least. Compared to control group, ELISA results show TNF-a and CRP concentration significantly decrease in hRAMP1-MSCs group and MSCs group Id and 7d after MSCs transplantation (P<0.05). Especially most significant reduction in hRAMP1-MSCs group (P<0.05). The level of TNF-a was still high in control group, but obviously decrease in hRAMPl-MSCs group and MSCs group (P<0.05), especially in hRAMPl-MSCs group. The level of CRP gradually decrease into the normal level 14d after cell transplantation, and the difference among three groups was not statistically significant.Conclusion:(1) Exogenous RAMP1 gene modified VSMCs significantly showed the inhibition of VSMCs proliferation induced by AngⅡand the promotion of cell apoptosis, which could be the new therapeutic target of some vascular proliferation disease.(2) Compared to natural MSCs, hRAMP1 gene modified MSCs have the potency not only of more improvement on cardiac function and the recovery of damaged endothelium, but also of significant inhibition the proliferation of VSMCs and promotion of cell apoptosis, and of obviously reduction of inflammation. The recombinant hRAMP1 adenovirus vectors don't affect the differentiation potential VSMCs into endothelial cells, which shows that modified stem cells have the potency of more effective inhibition of hyperplasia intima, and thus was made use of decreased Restenosis. |
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