Tissue Kallikrein Inhibits Intimal Hyperplasia After Carotid Artery Balloon Injury In Rabbits And Its Underlying Mechanisms

Objective:Strategies against restenosis after percutaneous transluminal angioplasty and stenting (PTAS) are lacking. Previous studies have shown that human tissue kallikrein (hTK) gene transfer could attenuate neointima formation induced by balloon catheter. As a tissue kallikrein in vivo, human urinary kallidinogenase (HUK) is widely used for preventing ischemia/reperfusion injury. However, the efficacy of HUK on the neointima formation has not been investigated. We therefore explored whether HUK could prevent balloon catheter-induced intimal hyperplasia in rabbits on high-fat diets and its underlying mechanisms.Methods:Thirty-six male New Zealand white rabbits were allocated into 3 groups (n=12 in each group). Negative control group:animals without operation or treatment; vehicle-treated group:animals with balloon injury plus vehicle; HUK-treated group: animals with balloon injury plus HUK. Balloon injury operations were given to the last two groups animals after two weeks of high-fat diet, followed by the administration of normal saline and HUK.Blood samples were collected and separated at day 0 (before operation),7,14, and 28. Plasma total cholesterol (TC) and triglyceride (TG) was measured by enzymatic Plasma total TC and TG were measured by enzymatic using an automatic system. Rabbits injured carotid arteries were collected, processed into paraffin-embedded cross sections, and subjected to staining with hematoxylin and eosin staining and used for immunohistochemical analysis at day 7, 14, and 28 after surgery. Positive staining in areas of Mac in relation to neointima and the integral optical density (IOD) of VSMCs in neointima were measured. The neointimal area/media area (I/M) ratio was calculated. The positive staining for each antibody was determined and the sections were analyzed using Image-Pro Plus6.0. Local inflammatory response was evaluated by detecting gene expression of tumor necrosis factor-a (TNF-a), interleukin-1β (IL-1β), and transforming growth factor-β1 (TGF-β1), with real time quantitative PCR, plus the invasion of macrophages with immumohistochemical staining.The effect of HUK in total endothelial nitric oxide synthase (eNOS), p-eNOS, and TGF-β1-Smad2/3 signaling pathway activity were examined by western blotting.Results:Neointima hyperplasia, macrophage adhesion and invasion of macrophages, pro-inflammatory factors, and VSMCs proliferation and migration were observed in balloon injured arteries. HUK attenuated neointima formation and atherosclerosis development by depressing the secretion of pro-inflammatory factors, adhesion and invasion of macrophages, VSMCs proliferation and migration. The expression of TGF-β1 and phosphorylated Smad2/3 significantly increased after vascular injury, but was down regulated after HUK treatment. Notably, HUK enhanced the phosphorylation of eNOS in injured arteries.Conclusion:Our results indicated that HUK inhibited intimal hyperplasia via downloading the expression of TGF-β1, increasing the activity of eNOS and attenuating VSMCs proliferation and migration from media into intima synchronously. HUK may be a potential therapeutic agent for preventing restenosis after vascular injury.