| Restenosis after percutaneous transluminal coronary angioplasty (PTCA) is wound healing to excess. During restenosis, the expression level of osteopontin (OPN) is correlated to the degree of neointimal hyperplasia after de-endothelium, and OPN plays an important role in the proliferation and migration of vascular smooth muscle cells (VSMCs). To block OPN induced neointimal hyperplasia, the anti-OPN antibody was used to prevent neointimal formation in this experiment and its mechanism was investigated.Methods1 Establishment of animal model: The intimal hyperplasia model was established by balloon catheter. SD rats were randomly divided into two groups, model groups (n=4) and treatment groups (n=5). Rats were anesthetized. The left carotid artery was deendothelialized by passage of a Fogey`s tube, which was inserted into the external carotid, passed down to the terminal of abdominal aortic, inflated, and drawn with a twisting motion up to aortic arch. This procedure was performed three times. The external carotid was tied off, leaving blood flow through the internal carotid artery. Immediately before the balloon injury, the anti-OPN antibody (the treatment group) and the normal rabbit serum (the model group) was injected via tail vein after operation, respectively, at two days intervals for seven times. 2 Preparation of experimental specimen: All the rats were killed at 21 days after de-endothelium. The blood specimen was collected for FCM assay of CD11a and CD11c in leycocytes. The caroid arteries and the aorta were separated for preparation of sections and extraction of protein and FCM assay. MMP-2 activity and the expression of MMP-2 and TIMP-2 in vessel wall were detected by gelatin zymogram analysis, Western blot and immunohistochemistry. The expression of ICAM-1 was detected by FCM assay.Results1 The general conditions of animals: The general conditions of two groups were fine. Rats weight had not changes during the experiment. After antibody or normal serum were administrated by tail vein injection, the body temperature of rats rised up the peak within 8 hours, and then recovered to the normal range slowly 24 hours later. There were no significant changes in body temperature of rats after the treatment was given the last four times. There was no significant difference in body temperature between the two groups.2 Morphological changes: When experiments ended, the carotid arteries were isolated and embedded in paraffin, and histological area measurements were made. The rats treated with anti-OPN antibody had significantly less neointimal thickening than normal serum treated rats (0.08±0.02 mm2 versus 0.18±0.03 mm2; p<0.05). The intimal area/medial area(I/M)declined in the treatment group(1.14±0.21 versus 0.50±0.21; p<0.05),the area of lumen increased in the treatment group (0.18±0.02 mm2 versus 0.26±0.01 mm2. p<0.05). The results suggested that the anti-osteopontin antibody could inhibit the proliferation of the intima and the stenosis of the lumen after the injury of artery. 3 Content and activity of MMP-2: MMP-2 activity in aorta extract was detected by gelatin zymogram analysis. The results showed that MMP-2 activity increased in vascular wall after injury, the levels of MMP-2 activity decreased significantly in the rats treated with anti-OPN antibody, compared with the model. Western blot analysis indicated that there was no significant difference in MMP-2 content between the two groups. The findings suggested that anti-OPN antibody inhibited MMP-2 activation but not the expression or secretion. The results of immumohistochemistry staining supported the conclusion and also indicated that there was no significant difference in TIMP-2 expression between the two groups.4 Expression of adhesive molecules: The expression of CD11a and CD11c on leucocyte surface and ICAM-1 in vascular cells was detected by FCM analysis. For the treatment group, the expression levels of CD11a decreased obviously, compared with the model group (6.19±0.41 versus 5.48±0.33,p<0.05), but there was...
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