Expression Of RAGE In Autogenous Vein Graft Of Streptozotocin-induced Diabetic Rats And Modulation To Intimal Hyperplasia By Aminoguanidine

IntroductionCoronary atherosclerotic heart disease and lower limbs arteriosclerosis are critical diseases in the senior population and the use of vein grafts as bypass conduits is one of most effective treatments.Although vein grafts are successful in relieving symptoms in patients who suffer from severe ischemic arterial disease,long-term patency is still a critical problem hampered by intimal hyperplasia,a common response of a vein after grafted into the arterial circulation.It is well known that diabetes mellitus is associated with atherosclerosis.Diabetes mellitus is not only a risk factor for atherosclerosis,but is also associated with worse outcomes when the saphenous vein graft is used in coronary and infrageniculate reconstruction.Under diabetic conditions,advanced glycation end products(AGE) is known to accumulate at an extremely accelerated rate both in various tissues and in the circulation and has been implicated in the development of diabetic vascular complications.Receptor-dependent mechanisms are likely to be involved in the AGE-induced tissue dysfunction.The receptor for advanced glycation end products(RAGE) is a multi-ligand cell surface receptor belonging to the immunoglobulin super-family of cell surface molecules.RAGE consists in vascular endotheliocyte,vascular smooth muscle cell,and monocyte.The engagement of RAGE by AGE can induce cellular oxidant stress,attract inflammatory cells such as polymorphonuclear leukocytes,mononuclear phagocytes,and T-lymphocytes into the tissue provides a mechanism to sustain the inflammatory response and result in the injury of vessel.In this process,the activation of nuclear factor-kappa B(NF-κB) is the most important signal transmission pathway. Aminoguanidine is a kind of hydrazine compound which has nucleophilic function and can block the early glycation production from rearranging and dehydrating to form AGE,thus reducing the production of AGE.Article one illuminated the expression of RAGE in vein graft of diabetic rats and the relationship of RAGE and intimal hyperplasia.By examining the expression of RAGE,NF-κB p65 and vascular cell adhesion molecule-1(VCAM-1) in vein graft of diabetic rats,article two aimed at exploring the mechanism of vein graft intimal hyperplasia increasing of diabetic rats. Article three is to explore the modificatory effect to vein graft intimal hyperplasia of diabetic rats by blocking the engagement of AGE-RAGE.Materials and MethodsMaterials1.Animals:Sprague Dawley rats(body weight ranged from 250g to 280g) were obtained from laboratory animal centre of China Medical University.2.Main reagents:streptozotocin(STZ),aminoguanidine(AG),polyclonal antibody against rat RAGE and NK-κB p65,monoclonal antibody against rat VCAM-1 and PCNA,SP immunohistochemical kits,Trizol reagent,RT-PCR kit,RT-PCR primers of RAGE,NK-κB p65,VCAM-1 and GAPDH(reference).3.Main instruments:Accu-Chek blood glucose monitoring system,SXP-1B operating microscope,microsurgical instruments,-70℃deep low temperature refrigerator,JUNG RM2025 skiving machine,microscope with camera,TGL-16B high speed centrifugal,722 spectrophotometer,ultraviolet spectrophotometer,850 fluorospectrophotometer,DYY-6B and DYY-Ⅲ5 electrophoresis apparatus, Rotorgene2000 gene amplifier,computer image analysis system.Methods1.Diabetic animal:Fasting 24h,the rats were given a single intraperitoneal injection of 60 mg/kg of STZ dissolved in freshly prepared 0.1mol/L citrate buffer (pH4.0-4.5) immediately before administration.Forty-eight hours later,the blood glucose levels were sampled from tail veins and measured with Accu-Chek blood glucose monitoring system to ensure induction of diabetes(blood glucose≥16.7mmol/L).2.Rat venous intimal hyperplasia model:Rats were anesthetized by 10%chloral hydrate solution,i,p.injection,300 mg/kg.The right external jugular vein(≈0.8 cm) was grafted into the infrarenal abdominal aorta with 11/0 suture under SXP-1B operating microscope(10-fold).Twelve to sixteen interrupted sutures were required per end.The aorta was clamped for an average of 60 min.Ensuring the vein graft's patency before closing the muscle and skin.The rats were given intramuscular injection next three days.3.Tissue harvest:Vein grafts were harvested after the rats were anesthetized,the left external jugular veins were harvested as a control at the same time.Before rats were sacrificed,0.5 ml blood was collected from the abdominal aorta.Each sample was divided into two parts from center,proximal half was embedded in paraffin for morphological measure and distal half was freezed by liquid nitrogen for Western blot and reverse transcription-polymerase chain reaction(RT-PCR) analysis.4.Indexes examined:(1) Measured the thickness of hyperplastic intima though HE and Verhoeff staining and computer image analysis system to evaluate the degree of vein graft intimal hyperplasia.(2) Detected the expression of PCNA with immunohistochemistry to evaluate the degree of proliferation of VASC in vein graft.(3) Detected the expression of RAGE,NK-κB p65 and VCAM-1 protein with immunohistochemistry and Western blot.(4) Detected the expression of RAGE,NK-κB p65 and VCAM-1 mRNA with RT-PCR.(5) The serum AGE level was determined by fluorospectrophotometry.5.Data were expressed as mean±S.D.Means of different groups were compared with analysis of variance(ANOVA) and t-test.P＜0.05 was considered to indicate statistical significance.Results1.The results of histomorphology and immunohistochemistry of PCNA:The thickness of hyperplastic intima in vein graft of diabetic rats was increased with time after operation.The PCNA positive cells appeared at 24h after operation,rised to peak at 28d after operation and falled later.After 7 or 14 days surgery,the thickness of hyperplastic intima and the ratio of intima to media thickness in diabetic rats were significantly greater than non-diabetic rats(P＜0.05).Interesting,the thickness of hyperplastic intima and the ratio of intima to media thickness in diabetic rats drunk AG were significantly less than diabetic rats drunk distilled water(P＜0.05) and had no difference compared with non-diabetic rats(P＞0.05).Similar results were obtained for PCNA positive cell percentage.2.The results of immunohistochemistry and Western blot:The expression of RAGE was not found in normal vein of diabetic rats and appeared at 24h after operation in vein graft of diabetic rats.The expression of RAGE in vein graft of diabetic rats was increased with time,which increased slowly after the 28^th day.After 7 or 14 days surgery,the percentage of RAGE,NK-κB p65 and VCAM-1 positive cells in diabetic rats was significantly increased compare to non-diabetic rats(P＜0.05).There is no significant difference between diabetic rats drunk AG or distilled water in the percentage of RAGE positive cells(P＞0.05).The percentage of NF-κB p65-positive cells in diabetic rats drunk AG was significantly decreased compared to diabetic rats drunk distilled water after 7 and 14 days surgery(P＜0.05),and no difference compare to non-diabetic rats(P＞0.05).3.The results of RT-PCR:There was not the expression of RAGE mRNA in normal vein of diabetic rats.The expression of RAGE mRNA appeared at 24h after operation in vein graft of diabetic rats and till 28d.Later the rise was slow.After 7 or 14 days surgery,the expression of RAGE,NK-κB p65 and VCAM-1 mRNA in diabetic rats was significantly increased compare to non-diabetic rats(P＜0.05).There is no significant difference between diabetic rats drunk AG or distilled water in the expression of RAGE mRNA(P＞0.05).The expression of NF-κB p65 mRNA in diabetic rats drunk AG was significantly decreased compared to diabetic rats drunk distilled water after 7 and 14 days surgery(P＜0.05),and no difference compare to non-diabetic rats(P＞0.05).4.The results of serum AGE:The serum AGE level in diabetic rats was significantly more than which in non-diabetic rats(P＜0.05) 7 or 14 days after operation.After 7 and 14 days grafting,the serum AGE level in diabetic rats that dnmk distilled water significantly increased compared to drunk AG(P＜0.05).However, there is no significant difference between diabetic rats that drunk AG and non-diabetic rats(P＞0.05).Conclusion1.Upregulation of RAGE expression has a close relationship with intimal hyperplasia and vascular smooth muscle cell proliferation in autogenous vein graft of diabetic rats.2.The engagement of RAGE by AGE activated the NF-κB,which increased the release of adhesion molecule;the next consequence is the proliferation of vascular smooth muscle cell.It maybe the mechanism of vein graft intimal hyperplasia increasing of diabetic rats.3.Aminoguanidine can inhibit the intimal hyperplasia of diabetic rats by blocking the engagement of RAGE by AGE.4.RAGE can become an effective target for preventing the development of intimal hyperplasia.