Effect of transplantation of hRAMP1 gene-modified bone marrow MSCs on postangioplasty neointima formation in rabbits

AIM: To explore the effect of transplantation of human receptor activity-modifying protein 1 ( hRAMP1 ) gene-modified bone marrow mesenchymal stem cells (MSCs) on neointima formation after carotid balloon angioplasty in carotid atherosclerosis rabbits. METHODS: MSCs were collected through density gradient centrifugation and adherent culture. MSCs were transfected with adenovirus vector carrying hRAMP1 gene to generate hRAMP1 gene-modified MSCs (hRAMP1-MSCs). All animals with carotid atherosclerosis and balloon angioplasty were randomly divided into hRAMP-MSCs group, MSCs group and control group. After the model was established, MSCs transfected with pAd2-EGFP-hRAMP1 or pAd2-EGFP and PBS were injected to the ear vein,respectively. The injured carotid arteries were harvested to detect the homing of MSCs,reendothelialization and neointima thickness 7 d, 14 d and 28 d after cell transplantation. The plasma samples were collected for detecting vascular endothelial growth factor (VEGF) by ELISA. The expression of endothelial nitric oxide synthase (eNOS) in injured carotid arteries was measured by Western blotting. RESULTS: The expression of CD31 and EGFP was observed in the neointima at different time points in hRAMP1-MSCs group and MSCs group. Compared to control group, the reendothelialization of carotid significantly increased in both hRAMP1-MSCs group and MSCs group at different time points (P<0.05), and that in hRAMP1-MSCs group showed better than that in MSCs group (P<0.05). The area of neointima and the rate of restenosis were lower in hRAMP1-MSCs group and MSCs group than those in control group, and those in hRAMP1-MSCs group were significantly lower than those in MSCs group. The plasma level of VEGF and the expression of eNOS in the injured carotid arteries were significantly higher in both hRAMP1-MSCs group and MSCs group than those in control group at different time points (P<0.05), and those in hRAMP1-MSCs group were better than those in MSCs group (P<0.05). In the injured carotid arteries, the expression level of proliferating cell nuclear antigen (PCNA) in hRAMP1-MSCs group was the lowest,with the middle level in MSCs group and the highest level in control group. CONCLUSION: The hRAMP1 gene-modified MSCs are better in promoting reendothelialization and attenuating neointima than natural MSCs. The recombinant hRAMP1 adenovirus vectors dont affect the differentiation potential of MSCs into endothelial cells.These findings indicate that the modified stem cells have the potency of more effective reendothelialization to decrease restenosis after angioplasty.     

The feasibility of reendothelialization of the injured arterial wall by autologus endothelial cell transplantation and their effects on neointima proliferation

AIM: To investigate the feasibility of reendothelialization of the injured arterial wall by autologous endothelial cell transplantation and their influences on neointima proliferation. METHODS: New Zealand white rabbits (n=30) were subjected to bilateral iliofemoral artery balloon injury. Cultured, autologous venous endothelial cells were immediately transplanted into one vessel(transplantation group), whereas the contralateral artery received medium only(control group). Reendothelialization of the injured arterial wall was analysed 4 hours or 4 days after cell transplantation by fluorescent tracing、scanning electron microscope(SEM) and Evans blue staining. Pathology analysis was employed 28 days after cell transplantation to evaluate neointima proliferation. RESULTS: The transplanted endothelial cells had adhered into the aterial wall 4 hours after transplantation and began to attach and spread 4 days later. A number of fluorescent labeling endothelial cells were observed in the endothelial injured arterial wall. The vessels in control group were stained nearly completely by Evans blue, whereas about 60% area was not stained in transplantation group. Pathological examination demostrated that neointimal area and maximal intima thickness in transplantation group significant decreased than those in control. CONCLUSION: Autologus endothelial cells were effectively transplanted into the injured arterial wall by balloon catheter, and it can relieve neointima proliferation in the long time.     

苯妥英钠对大鼠颈总动脉球囊损伤后内膜增生的影响

目的研究苯妥英钠促进创伤修复的特殊药理学作用在大鼠颈总动脉球囊损伤后内膜修复过程中的影响。方法采用大鼠颈总动脉内膜球囊损伤模型,术后大鼠分为苯妥英钠组和损伤组。术后28天处死大鼠,取双侧颈总动脉,石蜡包埋、切片后进行血管病理学染色分析。结果术后28天,苯妥英钠组内膜面积(0.154±0.018mm2比0.204±0.054mm2,P<0.01)、内膜/中膜面积比(1.70±0.08比2.26±0.46,P<0.01)、再狭窄率(59.5%±3.2%比75.9%±13.3%,P<0.01)均小于损伤组,管腔面积(0.106±0.024mm2比0.063±0.034mm2,P<0.01)大于损伤组;内膜细胞密度(72.18±20.08/cm2比84.85±10.77/cm2,P<0.05)、增殖细胞核抗原阳性细胞数(9.89±7.63个/200倍视野比23.03±13.95个/200倍视野,P<0.01)和α-平滑肌肌动蛋白阳性细胞数(30.91±20.05/200倍视野比61.81±16.57个/200倍视野,P<0.01)均小于损伤组;新生内膜中有少量胶原组织增生,但组间血管胶原面积和密度均无统计学差异(P>0.05)。结论大鼠颈总动脉球囊损伤后28天,苯妥英钠可以抑制损伤血管内膜增生过程中的细胞增生,促进细胞外基质合成,对血管损伤后内膜增生和管腔狭窄具有抑制作用。     

Inhibitory effects of Am80 on proliferation in vascular endothelial cells and neointima hyperplasia of carotid arteries

AIM: To explore the inhibitory effects of Am80 on the proliferation in the vascular endothelial cells (VECs) and neointima hyperplasia of the carotid arteries after balloon injury in the rats. METHODS: The proliferation of EA-hy926 cells were detected by cell counting and MTS assay after the cells were treated with various doses of Am80 for 24 h. The cell cycle was analyzed by flow cytometry after the cells were stained with PI. The mRNA expression of cyclinB1, P21 and matrix metalloproteinase (MMP)-2 in the EA-Hy926 cells was detected by real-time PCR. The changes of neointima hyperplasia in the carotid arteries were observed under microscope with hemotoxylin and eosin (HE) staining, and the expression of cyclinB1 was examined by the method of immunohistochemistry. RESULTS: The proliferation of EA-Hy926 cells was obviously inhibited in a dose-dependent manner when the cells were treated with various doses of Am80 for 24 h. The cell cycle was arrested at G₂/S stage in response to Am80 treatment. The mRNA expression of P21 was increased, however, the mRNA expression of cyclinB1 and MMP-2 was decreased when the cells were treated with Am80 at 4 μmol/L for various times. In addition, the vivo experiment demonstrated that Am80 not only significantly reduced neointimal hyperplasia and the thickness ratio of intima to tunicae media compared with injured group, but also inhibited cyclinB1 expression in the carotid arteries. CONCLUSION: Am80 inhibits the proliferation of VECs and neointima hyperplasia in the carotid arteries after balloon injury by promoting P21 expression and decreasing cyclinB1 expression.     

Am80 inhibits neointima hyperplasia by promoting interaction of KLF4 with RARα

AIM: To investigate the inhibitory effect of Am80 on neointima hyperplasia in carotid arteries after balloon injury and to observe the interaction between Krüppel-like factor 4 (KLF4) and retinoic acid receptor α (RARα).METHODS: Neointima hyperplasia in carotid arteries was observed by hemotoxylin and eosin staining. The expression of KLF4 and cyclin D1 was examined by immunostaining and Western blotting analysis. To detect the interaction between KLF4 and RARα in the vascular tissue, the injured arteries were harvested, and the protein extracts were prepared and subjected to co-immunoprecipitation assay.RESULTS: Compared with injured group, Am80 significantly reduced neointimal hyperplasia and the thickness ratio of intima to media. Am80 not only up-regulated KLF4 or RARα expression in caro-tid arteries, but also increased the interaction between KLF4 and RARα at tissue levels.CONCLUSION: Am80 inhibits neointima hyperplasia in carotid arteries after balloon injury by promoting the interaction between KLF4 and RARα.