Involvement Of Profilin-1in AGEs-induced Vascular Endothelial Cell Injury And Mechanism

Chapter1Effects of Profilin-1in AGEs-mediated endothelial injuryAbstractBackgroundAs the augmentation of the incidence of diabetes (diabetes mellitus, DM) year by year, the complication of diabetes caused by diabetic vascular disease disability is responsible for one of the main death. For patients who are hyperglycemia with a long time, a powerful integrated treatment including, e.g., strict control of blood glucose and lipid could not prevent the aggravation of diabetic vascular complications,the phenomenon is called "metabolic memory" or "hyperglycemic memory"A lot of studies have shown that the blood sugar of diabetes patients is long-term uncontrolled, the generation of AGEs (advanced glycation end products) will increase after a series of enzyme saccharification, lipid oxidation, which might be the main factor of the generation with "metabolic memory". The mechanism of AGEs mediated "metabolic memory" caused by vascular disease has not been fully elucidated.Profilin-1, a micro-molecular actin-binding protein, is widely present in other organism except skeletal muscle tissues, which could regulate actin polymerization and depolymerization process and involve in regulating cell proliferation, differentiation and movement as well as signal transduction. In vitro studies have indicated that AGEs could motivate the redistribution and recombinant of endothelial actin cytoskeleton proteins, which resulted in increasing permeability of endothelial cells. It is suggested that profilin-1may be the target molecule of AGEs-inducing endothelial cell damage. This chapter is mainly focused on the cultivation of human umbilical vein endothelial cells in vitro, which were stimulated by exogenous AGEs to investigate the injury effects of AGEs in human umbilical vein endothelial cells and observe the expression of profilin-1in human umbilical vein endothelial cells with AGEs stimulation.MethodsThe human umbilical vein endothelial cell lines were cultured with the treatment at different concentrations (100mg/L,200mg/L,400mg/L) of AGEs for24hours. Subsequently, the human umbilical vein endothelial cells were treated with AGEs (200mg/L) at different time (6h,12h,24h,48h). The distribution of NO, ADMA and ICAM-1cytokine levels in cell supernatants were measured after the human umbilical vein endothelial cells treated with AGEs (200mg/L,24hours). Immunofluorescence staining was used to measure the distribution of F-actin and expression of profilin-1and Western blot detection was used for the expression of profilin-1. The confocal scanning microscope was employed to directly observe changes in reactive oxygen species.Results1. Profilin-1protein expression could be up-regulated by the incubation of endothelial cells with different concentrations of AGEs (100mg/L,200mg/L,400mg/L) for24h, but significantly with200mg/L.2. The expression of Profilin-1in endothelial cells showed a time-dependent increase when treated with200mg/L of AGEs, and AGEs at the concentration of200mg/L were chosen for endothelial cells with the treatment of24hours in the following experiments. 3. profilin-1induced by AGEs made the endothelial cell structure abnormal.4. The ADMA and ICAM cytokine levels in cell supernatants could be significantly up-regulated with the treatment of AGEs (200mg/L) for24h, while the NO cytokine level could be decreased.5. The endothelial cells’shape became very irregular after the treatment with AGEs (200mg/L) for24h, while the obviously increased green fluorescence suggested that AGEs significantly increased intracellular levels of reactive oxygen species.ConclusionProfilin-1had critical efficacy in the process of AGEs-mediated endothelial injury. Chapter2The expression of endothelial cells induced by AGEs with profiling-1silencingBackgroundPathogenesis of diabetic vascular disease is complex, which has not yet been elucidated. The prevailing view is as follow:a long-term high blood sugar induced mitochondrial dysfunction and resulted in an excess of superoxide, cellular proteins and nucleic acids, while nucleic acids are glycated to generate a lot of AGEs, which could directly modify the mitochondrial protein to generate more superoxide ions to inhibit its repair. As this damage accumulates along with prolonged high blood sugar, AGEs damage will persist even after the regulation of hyperglycemia at this moment and the vascular injury will continue to accelerate vascular disease. In recent years, some scientists have proposed "The irreversible phenomenon of mitochondrial AGEs formation could explain the principle of by long-term "metabolic memory". Indirect effects of AGEs were mainly mediated by its receptor (receptor for advanced glycation end products, RAGE), and intracellular oxygen free radicals could be generated after AGEs and RAGE binding together, while RAGE as a signal molecule activates protein kinase C (protein kinase C, PKC) and nuclear factor-B (nuclear factor kappa B, NF-) by intracellular signaling cascades, which final activates the pathway of diabetes-related complications.Accordingly, we conducted a critical preliminary experiment, which indicated that expression of profllin-1increased in STZ diabetes rats’ aorta which had not been affected by high glucose (30mM,24h) stimulation in cultured endothelial cells, but the expression of profilin-1was significantly up-regulated after the treatment of AGEs (50g/mL,24h). The phenomenon coincided with AGEs-mediated "metabolic memory", which was formulated with long-term high sugar stimulation. Therefore, we hypothesized that profilin-1had relation with AGEs-mediated "metabolic memory", so it is hopeful to clearly elucidate the mechanism of "metabolic memory" using profilin-1as the target and block the intracellular pathway from AGEs induced vascular injury.In previous studies, the effects of profiling-1had been confirmed in AGEs-mediated endothelial cell injury. The experiments in this chapter had been designed on AGEs dependent up-regulation of profilin-1mechanism and signal transduction pathway. With gene silencing technology, it could be directly demonstrated the pivotal role profilin-1in AGEs-inducing endothelial damage and vascular lesions and provide a theoretical basis for research and development of new drugs by trying to find new target for vascular lesions.MethodsThe human umbilical vein endothelial cell line was cultured, and the profilin-1siRNA plasmid was synthesized by Shanghai Ji Kaiji Gene Chemical Technology Co., which was successfully transfected into human umbilical vein endothelial cell line. The effects of profilin-1gene silencing were assayed by Real-Time PCR and Werstern-blot. The expression of proliflin-1, ADMA, ICAM-1and NO cytokine levels in cell supernatant were assayed in AGEs+Profilin-1interference group, AGEs+DPI (10μmol/L) group, AGEs+BAY-117802(5μmol/L) group and AGEs+GF109203X (10μmol/L) group. In order to prove that the possible mechanisms of AGEs signaling pathway induced endothelial cell damage, PKC and NF-kB signal transduction pathway inhibitors were used to pretreat human umbilical vein cell lines.Results1. The expression of Profilin-1in AGEs-inducing group was significantly higher (P<0.05) as well as AGEs+Profilin-1interference group (P<0.01)(Figure2-1).2. The ADMA and ICAM-1levels induced by AGEs could be obviously decreased in endothelial cells with interference of pLNCX2-Profilin-1plasmid and incubated with various inhibitors with the increase of NO.3. The expression of Profilin-1in AGEs-induced group was significantly higher than that of each inhibitor group and AGEs+Profilin-1interference group; the expression of profilin-1mRNA in each group and AGEs+cells interference group was significantly decreased compared AGEs group.4. Compared with other groups, the endothelial cells’morphology became quite irregular in AGEs group after24h, and green fluorescence was significantly increased, which suggested that AGEs significantly increased intracellular levels of reactive oxygen species.5. Immunofluorescence staining showed that the levels of green fluorescent in AGEs group were significantly higher than that of control, which further suggested that its expression levels of profilin-1were significantly up-regulated with AGEs (200g/mL) after24h incubation. The green fluorescence of profilin-1was significantly lower after with the treatment of DPI, GF109203x and BAY-117082and other inhibitors, indicating that profilin-1, which implied the profiling-1was reduced by the use of inhibitors, such as DPI, GF109203x and BAY.6. Immunofluorescence staining showed that the expression of profiling-1could be significantly reduced by profiling-1gene silencing, which improved F-actin reorganization and distribution induced by AGEs. Overall, these results confirmed that AGEs improved the levels of profilin-1in the cytoplasm to induce the actin cytoskeleton reorganization and. redistribution in endothelial cells. And, AGEs-mediated profiling-1was activated by PKC and NF-κB pathway.ConclusionSilence profilin-1gene can significantly improve the endothelial cell injury by AGEs induced, profilin-1may be the ultimate and common downstream expression of endothelial injury. Chapter3AGEs mediated expression of profiling-1in rat Specific backgroundBackgroundIn2010, according to statistical data from the ADA (American Diabetes Association), we could see that the ratio of diabetic complications is50percent in the long-term (over3years) diabetes patients, while the ratio of diabetic complications even reached more than60percent in the longer-term (over5years) diabetes patients and raised to100percent the ultra-long (over10years) diabetes patients, the chances of complications chances are100%. With deep understanding of diabetes, more diabetes (diabetes mellitus, DM) patients were found, and vascular complication caused by diabetes disease is one of the main factors to make disability and death when most of diabetic patients were diagnosed. To the long-term hyperglycemia patients, a powerful integrated treatment including strict control of blood glucose and lipid could not prevent the aggravation of diabetic vascular complications. Therefore, It is critical and vital to explore many aspects of the pathogenesis of diabetic vascular disease.This project intends to demonstrate the AGEs still expressed by profilin-1in animanl with the AGEs-induced vascular lesions rats’model, which resulted in damage to the blood vessels and led to diabetes complications. The project aims to discover new targets of anti-vascular lesions occurring and provides a theoretical basis for research and development of new drugs.Methods SD rats’vascular injury model was induced by using intravenous injection of AGEs-BSA (25mg/kg, iM-qd,2consecutive months), Western blot and real-time PCR were employed to assay the expression of profiling-1while HE staining and immunohistochemistry (Immunohistochemistry, IHC) was used to detect the morphological changes and relevant parameters.Results1. There is no significant difference in rats’body weights in each group before employment.2. Compared with the control group, HE staining showed that their endothelial cells and vascular morphology were changed when extending the incubating time of AGEs. Immune kidney tissue staining showed the AGEs rats’glomerular injured as well as the distribution of visible inflammation and lymphocyte infiltration along with extending the cultivating time.3. Compared with normal control group, the expression of profilin-1was significantly increased when administered intravenously with prolonging time, and the profilin-1mRNA expression levels were significantly increased.4. Compared with the control group, the levels of ICAM-1and ADMA increased along with extending intravenous injection time of AGEs, while the levels of NO reduced.ConclusionAGEs-inducing profilin-1was responsible for rat endothelial cell and organs injury.