SM22α Inhibits Neointimal Hyperplasia After Balloon Injury In Rat

Objective: Percutaneous transluminal balloon angioplasty has now been widely used in the treatment of obstructed atherosclerotic vessels. However, its overall benefits are interrupted by inducing local arterial restenosis.Smooth muscle 22 alpha (SM22α) is a differentiated VSMC marker, which is characterized by its smooth muscle tissue-specific and VSMC phenotype-specific expression pattern. The present study was designed to elucidate the effect of SM22αon neointimal hyperplasia after balloon injury and its mechanism of action.Methods:1 Establishment of the animal modelSD rats were anesthetized with urethane. The left carotid artery was de-endothelialized by a Fogarty's tube, which was inserted into the external carotid, passed down to the aortic arch, inflated, and drawn with a twisting motion up to the carotid bifurcation, where the balloon was deflated. This procedure was performed three times to ensure complete denudation. 20μl of pAd-SM22αadenovirus suspension was injected into the carotid artery after blockade of blood stream, which maintained for 20 min, and then recovered the blood stream.SD rats were randomly divided into three groups, including uninfected group, pAd group and pAd-SM22αgroup.2 Preparation of experimental specimenAll the rats were killed at different time intervals after de-endothelium. The injured carotid arteries were separated for preparation of sections and protein extracts. The sections were stained with hematoxylin and eosin to detect the suppression of neointimal hyperplasia by SM22α, and the thickness of neointima was measured. Immunohistochemistry were performed to detect the expression of the SM22α, PCNA, and p27, respectively. The protein extracts from left carotid artery were analyzed by Western blot to detect the expression of SM22α, p-Raf, p-MEK and p-ERK by Western blot.Results:1 The expression of pAd-SM22αin vivoWestern blot analysis showed that the level of SM22αin the carotid arteries of pAd-SM22αgroup was much higher than that in the uninfected group and the pAd group. Immunohistochemical staining also showed that the number of SM22αpositive cells in pAd-SM22αgroup were increased significantly, compared to uninfected group and pAd group. The results of immunohistochemistry staining were similar to that of Western blot, suggesting that the infection was successful. 2 Overexpression of SM22αinhibits neointimal hyperplasia To investigate the effect of gene transfer on neointimal formation, the intima to media area (I/M) ratio was evaluated by morphometric analysis in each cross-section of arteries at 14 days after balloon injuries. The I/M ratio was 2.43±0.21 in the uninfected group and 2.19±0.19 in the pAd group, whereas the ratio was reduced significantly in the pAd-SM22αgroup (0.68±0.12, P<0.05 versus uninfected and pAd groups). The results indicated that SM22αoverexpression significantly inhibits neointimal hyperplasia.3 Overexpression of SM22αinhibits PCNA expression, and up-regulates p27 levelTo demonstrate the mechanism by which SM22αinhibits neointimal hyperplasia, the expression of PCNA and p27 was detected by immunohistochemical staining. The results demonstrated that the level of PCNA in the pAd-SM22αgroup was lower than that in the uninfected group and the pAd group. Howerer, the expression of p27 was reduced, compared with the control. There was no significant difference in the two protein expression between the uninfected group and the pAd group.4 Overexpression of SM22αinhibits the phosphorylation of Raf-1, MEK1/2 and ERK1/2To clarify the signaling pathway of SM22αaction,Western blot was performed. The results showed that phosphorylation of Raf-1, MEK1/2 and ERK1/2 in the pAd-SM22αgroup was decreased, compared with the uninfected group and the pAd group (P<0.05). Conclusion:1 pAd-SM22αwas successfully infected into the carotid artery after balloon injury in rat.2 Overexpression of SM22αsignificantly inhibits neointimal hyperplasia induced by balloon injury.3 Overexpression of SM22αsignificantly inhibits PCNA expression, and up-regulates p27 expressive level.4 SM22αinhibits neointimal formation after balloon injury in rats via the blockade of Raf-1-MEK1/2-ERK1/2 signaling cascades phosphorylation.