| Background and ObjectiveDiabetes is associated with severe atherosclerosis in humans and represents a leading cause of morbility and mortality.Diabetic cardiomyopathy,a prominent cardiovascular complication,has been recognized as one of the most common and specific microvascular complication of diabetes.Cardiac microvascular endothelial cells(CMECs)as the chief basic and initiative cells contribute to the pathogenesis of cardiac microvascular diseases.Furhermore,it is well known that cells from different vascular beds have functional and morphological differences.The heterogeneity of the endothelium is different between the macro-and microvasculature.Therefore,use of cultured rat cardiac microvascular endothelial cell model is beneficial to understanding the pathology of diabetic cardiac microvascular dysfunction and evaluating therapeutic effects of new drugs for diabetic microvascular disease. The recognition that diabetic complication has an important immune and inflammation component.In previous studies,the production of advanced glycation end products(AGEs)formed via a non-enzymatic reaction between high doses of sugars and proteins has been identified as a key determinant in the development and progression of diabetic complications.AGEs binding to its specific receptors RAGE lead to prolonged inflammation,mainly as a result of RAGE-dependent overexpression of proinflammatory cytokines and chemokines and accelerate atherosclerotic progression.AGEs-RAGE has been reported to induce the production of cytokines in microvascular diabetic complications.As a results, blockade of AGEs-RAGE interaction might provide a protent and safe strategy for the prevention of complications that typify long-term diabetes.Statins,3-Hydroxy-3-methylglutaryl coenzyme A(HMG-CoA)reductase inhibitors,have been reported to possess broad immunomodulatory and anti-inflammatory properties.Statins not only effectively reduce cardiovascular events in diabetic patients,but also significantly improve endothelial function for atherosclerosis.Recently,some researchers have found statins can block the AGEs-RAGE signaling in microvascular endothelial cells.But the exact mechanism is not clear.Therefore,we speculate whether statins might be a promising remedy for cardiac microvascular atherosclerosis by acting as a potential inhibitor of the AGEs-RAGE signaling in cardiac microvascular endothelial cell.We observed the impact of AGEs on expression of Monocyte chemoattractant protein-l(MCP-l)and Intercellular adhesion molecule-1 (ICAM-1)in Rat cardiac microvascular endothelial cells(CMECs)and also investigated the effect and mechanism of HMG-COA reductase inhibitor Simvastatin on inhibition of AGEs-induced expression of these proinflammatory cytokines,so as to contribute to the new basis on statins therapy for diabetic cardiac microvascular diseases.MethodsPart one:Isolation,culture and identification of rat CMECs in vitro and preparations for AGEs.1.CMECs were isolated enzymatically from rat left ventricle.Isolated cells were plated onto culture dishes in supplemented with rat tail collagen and passaged after 5~7 days.CMECs were characterized as expression ofⅧfactors and intake of DiI-Ac-LDL under a laser scanning confocal microscope.The cultured cells were calculated positive rate.2.Bovine serum albumin was incubated in the presence of D-glucose under sterile conditions at 37℃for 12 weeks in PBS(pH 7.4)to obtain AGEs.Nonglycosylated bovine serum albumin was subjected to the same conditions AGEs was sterilized and stored at-20℃until used.AGEs content was deter-mined spec-trofluorometrically with excitation set at 365 nm and emission set at 460 nm.Part two:The effects and possible mechanism of expression of MCP-1 and ICAM-1 induced by AGEs in CMECs.The generation CMECs was stimulated with various concentrations of AGEs for 72 hours.The production of MCP-1 was performed by Enzyme-Linked Immunosorbnent Assay(ELISA).ICAM-1 induced by AGEs was determined by Flow cytometry(FCM).Receptor for advanced glycation end products(RAGE)was detected at both protein and mRNA level through Western-blot and Reverse-transcription polymerase chain reaction(RT-PCR)respectively.Part three:Simvastatin inhibits the expression of MCP-1 and ICAM-1 induced by AGEs in CMECs.After pre-incubation of Simvastatin at the concentration of 0.1,lμmol/L for 6 hours,the cultured CMECs was stimulated with AGEs for 72 hours.Thereafter,the production of ICAM-1 was determined by FCM.MCP-1 were performed by ELSA.Surface RAGE protein expression was determined by Western blot and RAGE mRNA levels was assayed by RT-PCR.Results1.Rat CMECs were cultured and identified in vitro successfully.after 2~3 days the cells can form tube-like structure.After 7~9 days,cells reached confluence and always displayed a uniform"cobblestone"morphology.90.68% of the cells areⅧpositive.93.82%of the cells can intake Dil-AC-LDL.Preparations for AGEs is also confirmed successfully.The average fluorescence for AGE-BSA and control were 218 U/mg and 0.65U/mg,respectively。2.Compared with BSA control,AGEs at diverse concentration enhanced the expression of MCP-1,ICAM-1 of CMECs(P＜0.05)in a dose-dependent manner and increased the expression of RAGE at both protein and mRNA level(P＜0.05).3.Compared with only AGEs stimulation,Small dose of simvastatin significantly reduced the AGEs-induced MCP-1 and ICAM-1 expression(P＜0.05),and inhibited RAGE expression at both protein and mRNA level(P＜0.05).Conclusions1.AGEs can upregulate ICAM-1 and MCP-1 expression,and increase expression of RAGE in CMECs.A plausible explanation is that AGEs increase the expression of proinflammatory cytokines through up-regulation of RAGE. AGEs-RAGE system triggered an inflammatory process,suggesting that AGEs-RAGE interaction is also a key contributing factor leading to the de-velopment of microvascular diabetic complications.2.The blockade of AGEs-RAGE system by simvastatin may play a pro- tective role against early diabetic cardiac microvascular complication by attenuating the deleterious effects of AGEs via down-regulation of RAGE.
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