Effect Of Hyperlipidemia On Vascular Inflammation After De-endothelialization

Objective Hyperlipidemia is an important risk factor for atherosclerosis (AS). AS is an inflammatory disease of cardiovascular system, and inflammation is present in the whole pathological process of AS. Percutaneous transluminal coronary angioplasty (PTCA) has been widely used for the treatment of atherosclerotic coronary artery lesions, but restenosis remains a significant limitation to the long-term efficacy of the procedure. Elastic recoil of the arterial wall, neointimal hyperplasia and vascular remodeling are the main mechanisms for post-PTCA restenosis, and inflammation plays a very important role in neointimal hyperplasia and vascular remodeling. NF-κB activation and NF-κB-mediated iNOS expression exist in the inflammation of AS caused by hyperlipidemia, but the effect of hyperlipidemia on the vascular inflammation after de-endothelialization remains unclear. To confirm the role of hyperlipidemia in the couse of neointimal hyperplasia, the present study investigated the effects of hyperlipidemia on the expression of NF-κB and iNOS as well as neointimal hyperplasia by balloon de-endothelialization. Methods 1 Establishment of de-endothelialization model and grou-ping Male SD rats were randomly divided into three groups: normal group, control group and hyperlipid group. Normal group was fed with a general diet. Control group received arterial de-endothelialization procedure and was fed with a general diet. Hyperlipid group was fed with a high-fat diet for 7 days before de-endothelialization procedure, and for 21 days after the procedure. The rats of normal group were killed at 21 days after being fed with a general diet, and those of control group and hyperlipid group were killed at 21 days after the procedure. Blood samples were taken from all rats, and serum was separated for detection of levels of lipids. The aortas were collected for section preparation, immunohistochemistry for NF-κB and iNOS, and Western blot analysis of NF-κB at the end of experiment. 2 Serum lipid levels assay We performed as kit description with fully automatic biochemical analyzer. 3 Immunohistochemical staining of NF-κB P65 and iNOS The paraffin sections were treated with xylene and alchohol. After washing with PBS, 3% H2O2 diluted with methanol was applied for 10 min to block endogeneous peroxidase, and microwave antigen retrieve for 10 min, and then blocking solution was applied for 10 min after washing. Mouse anti-rat NF-κB P65 monoclonal antibody was used as a primary antibody for staining NF-κB P65. Rabbit anti-rat iNOS polyclonal antibody was used as a primary antibody for staining iNOS. The negative control sections were treated by PBSinstead of primary antibody. Biotinylated immunoglobulin was used as a secondary antibody, and reacted for 20 min. Streptavidine horseradish peroxidase and DAB were used to detect target proteins. 4 Western blot analysis of NF-κB P65 The extracts from areries of three group rats were separated on 8% SDS-PAGE, and then blotted onto PVDF membrane. The membrane was immunologically reacted with mouse monoclonal anti-rat NF-κB P65 antibody. Results 1 Determination of the levels of serum lipids Compared with normal group and control group, the levels of serum TC, TG and LDL-C increased significantly in hyperlipid group (P < 0.01). The levels of serum HDL-C had no difference among three groups (P > 0.05). The results suggest that the high-fat diet had led to significant hyperlipidemia. 2 The morphological changes The morphological obser-vation of the aorta section showed that widespread neointimal hyperplasia and lumen stenosis were formed on day 21 after de-endothelialization, compared with smooth intima and orderly arranged media VSMC in the normal rats. At the same time, VSMC occupied the superiority in the thickened area. These results indicated that the model of rat vascular stenosis after de-endothelialization is successfully established. Moreover, foam cells were observed in the thickening neointima in hyperlipid group, suggesting that hyperlipidemia results inpathological effect...