[Effects of CGRP and CGRP receptor modified mesenchymal stem cells on proliferation and phenotypic transformation of vascular smooth muscle cells].

OBJECTIVE: To explore the effects and mechanism of secretory calcitonin gene-related peptide (CGRP) and CGRP receptor modified mesenchymal stem cells on proliferation and phenotypic transformation of vascular smooth muscle cell. METHODS: Firstly (Lenti-GFP-CGRP, referred to CGRP (+/+)) MSCs were transfected with high expression lentivirus vector of CGRP (MSCs(CGRP+/+)). Protein secretion in the above-mentioned MSCs(CGRP+/+) supernatant was detected with enzyme-linked immunosorbent assay (ELISA). And then MSCs(CGRP+/+) was co-cultured with VSMCs(RAMP1)(+/+) and VSMCs(RAMP1-/-) respectively. Experimental groups were as follows: MSCs +VSMCs MSCs(CGRP+/+) +VSMCs, MSCs(CGRP+/+) +VSMCs(RAMP1)(+/+) and MSCs(CGRP+/+) + VSMCs (RAMP1-/-). Flow cytometry was applied to detect the cycle variation of smooth muscle cells, thiazolyl blue tetrazolium bromide method for detecting the proliferation of smooth muscle cells and Western blot for examining the expression changes of spectrin α-SM-actin and synthetic protein OPN in each group respectively. RESULTS: After the transfection of CGRP(+/+), MSCs(CGRP+/+) secreted and expressed CGRP protein, the secretory volume of CGRP protein in MSCs(CGRP+/+) increased significantly compared with the control group (19.530 ± 0.498 vs 3.133 ± 0.160 and 3.120 ± 0.001, P < 0.05) . After a 72 h co-culturing with VSMCs, the proliferation of VSMCs in MSCs(CGRP+/+) +VSMCs(RAMP1)(+/+)group declined significantly (0.270 ± 0.263 vs 0.413 ± 0.070, P < 0.05) and the number of cells staying in G0 phase significantly increased (93.51% ± 0.38% vs 84.48% ± 0.31%, P < 0.05) , the expression of contractile phenotype protein α-SM-actin increased and intermediate phenotype OPN declined significantly as compared with MSCsCGRP(+/+) +VSMCs group { (α-SM-actin 102 946 ± 3847 vs 51 759 ± 635, P < 0.05), OPN (26 026 ± 2595 vs 44 201 ± 2811, P < 0.05) }; but compared with MSCs(CGRP+/+)+VSMCs(RAMP1)(+/+)group, the proliferation of VSMCs in MSCs(CGRP+/+) +VSMCs(RAMP1)-/-group significantly increased (0.601 ± 0.04 vs 0.270 ± 0.263, P < 0.05) while the number of cells staying in G0 phase significantly declined (78.57% ± 0.68% vs 93.51% ± 0.38%, P < 0.05) . The expression of contractile phenotype protein α-SM-actin declined while intermediate phenotype OPN increased significantly in MSCs(CGRP+/+)+VSMCs(RAMP1)-/-group {α-SM-actin (34 400 ± 2179 vs 102 946 ± 3847, P < 0.05), OPN (53 933 ± 1192 vs 26 026 ± 2595, P < 0.05)}. CONCLUSIONS: CGRP gene-modified MSCs may secrete target protein stably. And CGRP-modified MSCs inhibits VSMCs phenotypic transformation and cell proliferation. It is probably associated with enhanced CGRP effect due to an up-regulation of CGRP receptor.

[Effect of gene modified mesenchymal stem cells overexpression human receptor activity modified protein 1 on inflammation and cardiac repair in a rabbit model of myocardial infarction].

OBJECTIVE: To investigate the effect of mesenchymal stem cells (MSCs) overexpressing human receptor activity modified protein 1 (hRAMP1) by adenovirus vector on infarction related inflammation and cardiac repair in a rabbit model of myocardial infarction (MI). METHODS: Thirty rabbits underwent coronary artery ligation for 60 minutes followed by 24 hours reperfusion and divided into MSC(hRAMP1) group (intravenously injection of MSCs transfected with adenovirus vector encoding hRAMP1 gene enhanced green fluorescent protein, EGFP, n = 10), MSC(null) group (MSCs transfected with adenovirus vector encoding only EGFP without hRAMP1 gene, n = 10) and control group (equally volume of phosphate buffered saline, PBS, n = 10). The plasma level of tumor necrosis factor-α (TNF-α) and interleukin-10 (IL-10) were quantified by ELISA assay at before and 1, 3, 7, 28 days after induction of MI. The expression of nuclear factor-κB (NF-κB) and hRAMP1 in infracted myocardium were measured by Western blot at 1, 3, 7, 28 day following MI. The area of MI and collagen deposition and fibrosis were evaluated by TTC staining and Masson staining, respectively. RESULTS: Area of MI and collagen content were significantly reduced in MSC(hRAMP1) group compared those in MSC(null) and control group [(10.1 ± 2.9)% vs. (30.6 ± 2.7)% and (22.5 ± 3.2)%, P < 0.05]. Myocardial expression of NF-κB and plasma TNF-α[7 days after transplantation: (97.2 ± 6.7) pg/ml vs. (207.6 ± 12.7) pg/ml and (153.2 ± 9.9) pg/ml, P < 0.05] were also lower while plasma level of IL-10 [7 days after transplantation: (238.5 ± 17.5) pg/ml vs. (177.3 ± 19.8) pg/ml and (244.6 ± 27.3) pg/ml, P < 0.05] was significantly higher in MSC(hRAMP1) group than in MSC(null) and control group. CONCLUSION: MSCs overexpression hRAMP1 could further reduce area of MI possibly through inhibiting the myocardial expression of NF-κB and reducing the plasma TNF-α level and raising plasma IL-10 level.

[Effects of hRAMP1 modified mesenchymal stem cells on restenosis and heart function in rabbit model of carotid angioplasty and myocardial infarction].

OBJECTIVE: To observe the effects of mesenchymal stem cells (MSC) modified by human receptor activity-modifying protein 1 (hRAMP1) on restenosis and cardiac function post-myocardial infarction (MI) and explore the therapeutic safety for gene modification of MSC. METHODS: The double-injury rabbit model with MI reperfusion and sacculus damaged atherosclerotic carotid were established according to the previous study. MSC were transfected with adenovirus vector with enhanced green fluorescence protein (EGFP) and then introduced into rabbit model. The animals were randomly divided into Ad-EGFP-hRAMP1-MSC transfection group (hRAMP1-MSC group, n = 24) and Ad-EGFP-MSC transfection group (MSC group, n = 24), and PBS transfection group (C group, n = 24). At Day 28 post-transplantation, the injured carotid arteries and infarction myocardium were harvested to detect the expression of EGFP-positive MSC and assess organization morphology by hematoxylin and eosin, triphenyltetrazolium chloride or immunohistochemical stains and heart function by echocardiography. RESULTS: On flow cytometry, most cells expressed CD29 and CD90 while few ones expressed CD45. MSC with EGFP and a continuous expression of CD31 were found in intima of damaged carotid of both hRAMP1-MSC and MSC, but the expression of EGFP was not found in the control group. At Day 28 post-transplantation, the improvement of heart function and the decrease of infarct size were found in the hRAMP1-MSC and MSC groups compared with that in the control group(EF: 60.6% ± 1.5%,50.8% ± 3.2% vs 38.2% ± 2.0%, infarct size: 20.7% ± 1.4%, 33.2% ± 3.7% vs 35.6% ± 2.7%, all P < 0.05), especially much higher in hRAMP1-MSC group. At Day 28 post-transplantation, the area of intima hyperplasia and the rate of neointima and media in the hRAMP1-MSC group were lower than those in the MSC and C groups (0.15 ± 0.05 and 0.33 ± 0.08 vs 0.77 ± 0.11, 0.24 ± 0.07 and 0.51 ± 0.11 vs 1.09 ± 0.23, all P < 0.05). Also the expression of α-SMA was found in the hyperplasia intima. The expression of proliferating cell nuclear antigen significantly decreased in the hRAMP1-MSC group than those of the MSC and control groups (0.120 ± 0.028 vs 0.366 ± 0.013 and 0.627 ± 0.049, both P < 0.05). And it was much lower in the MSC group than that in the control group. CONCLUSIONS: Compared with MSC alone, hRAMP1-modified MSC have the potency not only of more improvement on cardiac function and the recovery of damaged endothelium, but also of significant inhibition of the proliferation of vascular smooth muscle cells. The recombinant hRAMP1 adenovirus vectors do not affect the differentiation potential MSC into endothelial cells.

[Effect of mesenchymal stem cells on cardiac function and restenosis of injured artery after myocardial infarction].

OBJECTIVE: Although earlier studies have shown that the transplantation of mesenchymal stem cells (MSCs) might improve cardiac functions after myocardial infraction, its role on vascular restenosis after percutaneous coronary intervention (PCI) remains controversial. The aim of this study was to investigate the effects of MSCs on the restenosis of injured artery following balloon angioplasty in a rabbit model with both myocardial infarction reperfusion and atherosclerotic stenosis carotid artery by balloon injury. METHODS: After the animal model was established for myocardial infraction reperfusion and atherosclerotic stenosis carotid artery by balloon injury, the rabbits received an intravenous transplantation of MSCs. And an equal volume of phosphate buffered solution was administered for the control group. The animal vascular tissue and myocardium tissue were excised at different time points post-transplantation and used to detect the homing of MSCs and the expressions of platelet-endothelial cell adhesion molecule-1 (CD31) and proliferating cell nuclear antigen (PCNA) by immunohistochemical staining. Four weeks later, vascular restenosis was analyzed by angiography of bilateral carotid arteries and the vascular tissues were used for histological studies. RESULTS: At one week post-transplantation, the 4',6-diamidino-2-phenylindole (DAPI)-labeled MSCs could be detected in myocardial infarction and injured intima. And the intimal expression of CD31 was observed at 2 weeks in the MSCs transplantation group. Yet the expression of PCNA was significantly lower in the MSCs transplantation group than that in the control group (50.5% ± 3.6% vs 23.4% ± 2.8%, P < 0.05). At 4 week post-transplantation, the neointimal area of injured vessels and the vascular restenosis were significantly lower in the MSCs transplantation group than those in the control group (0.092 ± 0.009 vs 0.189 ± 0.007, P < 0.05; 41.7 ± 3.7 vs 61.3 ± 1.6, P < 0.05). Furthermore the MSCs transplantation group demonstrated improved cardiac functions, reduced myocardial infarct size (21.7% ± 2.2% vs 34.3% ± 1.8%, P < 0.05) and significantly increased capillary density around infarction foci (33.6% ± 2.1% vs 20.8% ± 2.6%, P < 0.05) versus the control group. CONCLUSION: The transplantation of MSCs plays significant roles in cardiac repairing in terms of improved cardiac functions, accelerated repair of injured vessels, suppression of neointimal hyperplasia and reduced restenosis of injured vessels.