Biological Prevention And Physical Angiogenesis Of Restenosis After Vascular Grafting

Atherosclerosis obliterans(ASO) is the mostly common encounted ischemic disease of the lower extremity associated with major morbidity and mortality at present.The treatment for ASO includes autologous and artificial vessel bypass.But whatever kind of transplantation peorfomed,the rate of anastomotic restenosis(Rs) 6 months later makes about 30%-50%.After restenosis formation,the eventual result without any exception is the development of the lower limb gangene,due to the progression of atherosclerosis.Because of A number of patients have to receive the amputation owing to progressed gangrene of the lower extremity,there has been a imperative need to develop new effective therapies incuding the successful prevention intimal hyperplasia(IH) and salvaging ischemic limb after restenosis.Based on new understanding of the pathophsiological processes involved in restenosis ,our study attention is concentrated on reducation of extracellular matrix(ECM) deposition and therapeutic angiogenesis of ischemia which thus potentially open a new idea in the ongoing search for novel approaches for treatment of restenosis and ischemia.PartⅠ Inhibitor of Matrix Metallproteinase Prevents Restenosis after Vascular Grafting Objective To examine the effects of TIMP-2 transfered on the proliferation,matrix metallproteinase(MMP),ECM synthesis ,protein kinase-C(PKC)signaling pathway in cultured injured rabbit VSMC and to investigate its mechanism. To study the inhibition effect of Doxycycline on MMP and IH in rat model of autogenous vein-to-artery grafting and to testify its prevention effect of Rs.To provide the theory fundamental of TIMP-2 and Doxycycline experienced in clinical prevention of Rs.Methods In vitro,the rabbit VSMCs isolated freshly were activated by injure.The gene of TIMP-2 was transfected into injured VSMC via Lipofectin method ,then the transfection was confirmed by Western blot.Cell counting,immunocytochemical detecting of incorporated BrdU were used to evaluate the effect on proliferation of transfected-VSMC. MMPs were determined by zymography assay.Procollagen type Ⅳ was observed by immunocytochemical stain. The expressions of PKCα,ERK mRNA and protein were determined by RT-PCR and western blot. NF-κB was detected by electrophoretic mobility shift assay (EMSA).In vivo,the rat model of IH wasestablished by following procedure:separated jugular vein was transplantated into carotid artery of the same side by end-to-end anastomoses.Heparin acted as control.Zymography assay was applied to evaluate the levels of MMPs in grafted vessel treated with Doxycycline.Intimal area and I/M were indentified indirectly using HE stain,Van Gieson stain.Results In vitro, The injured VSMC showed that proliferation inceased by 2.8-fold , enzyme activity of MMP-2/9 enhanced by 2-fold/3-fold , procollagen type Ⅳ sythesis developed by 1.6-fold.Transfection of TIMP-2 decreased injured VSMC proliferation by 1.3/1.6-fold, inhibited enzyme activity of MMP-2/9 by 3-fold/2-fold ,weakened the immunocytochemical stainings of procollagen type Ⅳ by 3-fold. Normal VSMC showed the lower level expressions of ERK ,PKCα and NF-κB.Injury induced the high level expressions of them.However , in injured VSMC tranfected with TIMP-2 vector, the expressions of ERK, PKCα and NF-κB were suppressed. In vivo, the activity of MMP expressed at 7 days after grafting, peaked at 28 days , then progressively lasted by 56 days .Doxycycline reduced the activity of MMP-9 in the grafted vessel by 1.76-fold at 28 days and by 2-fold at 56 days .Image analysis showed that by contrast, the intimal:medial ratio(I/M) and intimal areas were significantly reduced at 28, 56 days after grafting in the doxycycline-treated rats and heparin-treated rats . Imparing effect appeared at 28 days after grafting ,peaked at 56 days. The inhibition effect of Doxycycline was by far significant than that of heparin. At 28 ,56 days after grafting ,I/M of heparin-treated rats was 1.87-fold,1.64-fold of that of Doxycycline-treated rats; intimal area of heparin-tre...