Regulates Receptor For Advanced Glycation Endproducts Expression And Promote Rat Endothelial Progenitor Cells Aging

Diabetes associated cardiovascular complications are one of the major causes of patient mortality. Numerous epidemiological studies suggest that diabetes can accelerate atherosclerosis and increase the incidence of heart attacks and strokes. However, the underlying mechanisms behind this relationship have not been fully elucidated. Diabetes and its observed ability to accelerate the progression of atherosclerosis cannot simply be explained by the traditional cardiac risk factors, such as smoking, hypertension, and hyperlipidemia. Recently, the receptor of advanced glycation end products(RAGE) was identified as one of the underlying mechanisms that may assist in explaining this observation. The interaction of AGEs with specific cell receptors leads to cellular activation, increased expression of extracellular matrix proteins and the release of proinflammatory cytokines and ROS. AGE-binding receptors include AGE-R1, AGE-R2, AGER3, the scavenger receptorⅡand receptor for AGEs(RAGE). AGE-R1, formerly termed oligosaccharyltransferase 48 (OST48), is a 48 kDa endocytosis-mediating protein. AGE-R2 is involved in signal transduction via protein kinase C. AGE-R3 (galectin-3) can influence cell growth, adhesion, differentiation, apoptosis and malignant transformation. The scavenger receptorⅡare probably implicated in the removal of AGEs. RAGE is the best characterized AGE receptor and responsible for most of the deleterious effects of AGEs. As a multiligand receptor on vascular cells, RAGE has been shown to promote atherogenesis. Recent studies suggest that RAGE may play an important role in promoting inflammatory processes and endothelial activation, which accelerate atherosclerotic development. RAGE, a member of the immunoglobulin superfamily of cell surface molecules, was initially isolated from bovine lung, vascular endothelial and smooth muscle cells; however, subsequent studies from several laboratories demonstrated that RAGE is also expressed on the surface of endothelial progenitor cells (EPCs). EPCs are bone-marrow-derived stem cells that have the ability to proliferate, migrate, and differentiate into functional, mature endothelial cells. EPCs circulate in the blood and seem to preferentially localize to sites of vascular or tissue injury, contributing significantly to both re-endothelialization and neovascularization, which are functionally important in vascular repair. Part of the regenerative capacity of EPCs seems to be due to their low baseline ROS levels and reduced sensitivity to ROS-induced cell apoptosis.As a multiligand cell surface receptor, the endogenous ligands of RAGE are variable and include advanced glycation endproducts (AGEs), S100/calgranulins, and high mobility group box 1 protein. Diabetes associated hyperglycemia and oxidative stress are the main sources of AGEs, which subsequently activate the RAGE pathway and initiate the inflammatory response. Furthermore, RAGE is highly expressed in atherosclerotic lesions and colocalizes with pro-inflammatory and pro-oxidative mediators.Therefore, we hypothesized that regulate RAGE expression in endothelial progenitor cells and, via this pathway can alter antioxidant defenses, promotes apoptosis in EPCs and accelerating the process of EPCs aging.Methods:1,ALT711 (1mg/kg.d) were feed to 3-4 month, 10-12 month and 18-20 month old rats. After 4 weeks, rats were killed and mononuclear cells were obtained from bone marrow. Mononuclear cells were cultured with medium DMEM for 7 days. EPCs were identified as adherent cells double positive stained for FITC-UEA-I and DiI-acLDL under laser confocal microscopey. A modified Boyden's chamber was used to assess the migration of EPCs and the number of recultured EPCs was counted to measure the adhesion function. A spectrophotometer was used to determine the proliferation function. Serum from different aging rats was collected and used to culture EPCs from 3-4month old rats in the absence or presence of ALT711 (20μg/ml).After 7 days culture, the migration, adhesion and proliferation functions of EPCs were detected.2,EPCs, isolated from bone marrow, were cultured in the absence or presence of AGEs (50, 100, and 200μg/ml). ROS were analyzed using the ROS assay kit. A spectrophotometer was used to assess superoxide dismutase (SOD) and glutathione peroxidase expression, and the PCR was also used to determine the mRNA expression. A modified Boyden's chamber was used to assess the migration of EPCs and the number of recultured EPCs was counted to measure the adhesiveness function. A spectrophotometer was used to determine the proliferation function.3,Mononuclearcells were got from bone marrow. Chemical analysis was used after 7 days culture. EPCs were characterized as DiI-acLDL / FITC-UEA-I double positive cell detected by laser confocal microscopy. EPCs were cultured with different concentrations of CRP (5,10,15,20μg/ml) for 12h,24h and 48h.The biological functions of EPCs were determined after culture.4,EPCs, isolated from bone marrow, were cultured in the absence or presence of lipopolysaccharide (LPS)-free CRP (5, 10, 15, 20and50μg/ml). RAGE protein expression was measured by flow cytometric analysis and Western blot. RAGE mRNA expression was detected by PCR. ROS were analyzed using the ROS assay kit. A spectrophotometer was used to assess superoxide dismutase (SOD) and glutathione peroxidase(GSH-PX) activity,and PCR was used to test mRNA expression of SOD and GSH-PX. Apoptosis was evaluated by Annexin V immunostaining.5,The carotid arteries intima injury model were made by balloon damage. One group received EPCs with high RAGE expression, another received EPCs with low RAGE expression,the rest were as control group. After 14 days transplanted EPCs, rats were killed and carotid arteries were sliced. The effects of EPCs with different RAGE expression on endothelium repair and neointima formation were observed.Results:1,The function of EPCs was significantly reduced with aging increased(P<0.01). ALT711 feeding released aging impaired proliferation, migration and adhesion of EPCs. Aged rat serum partially inhibits the activity of young rat EPCs.And ALT711 intervention can block this effect.2,Co-culturing with AGEs increases ROS production, alters antioxidant defenses inEPCs and inhibits the proliferation, migration and adhesion of EPCs(P<0.01).3,C-reactive protein at concentrations and time dependent to inhibit EPCs proliferation, migration and adhesive functions(P<0.01).4,Co-culturing with CRP caused a significant up-regulate expression of RAGE in EPCs, increases ROS production, alters antioxidant defenses and induces EPCs apoptosis (P<0.01). In addition, these effects were attenuated during blocking RAGE protein expression by siRNA.5,Neointima formation in transplant group were less than the untransplant group (P<0.01). Received EPCs with with low RAGE expression group neointima formation were less than the received EPCs with high RAGE expression group (p<0.01).Conclusions:1,It is suggested that the function of EPCs are correlated with ages. And treat with ALT711 can mediate aging impairment of progenitor cell function.2,AGEs increase oxidative stress and mediate impairment of progenitor cell function, which indicates a new pathophysiological mechanism of disturbed vascular adaptation in atherosclerosis and suggests that lower levels of AGEs might improve the success of progenitor cell therapy.3,CRP may serve to inhibit the biological characteristics of EPCs. And these changes were positive correlate to dose and action time of CRP.4,CRP at concentrations known to predict cardiovascular event, may serve to impair EPCs antioxidant defenses for up-regulating the expression of RAGE, and promote EPCs sensitivity toward oxidative stress mediated apoptosis. These data further support a direct role of CRP in the development and/or progression of atherosclerosis.5,Transplanting bone marrow derived EPCs can substitute endothelial cells in carotid arteries and take part in neointima formation. The function is correlated with RAGE expression, the higher of RAGE expression,the worse of EPCs functional activity.