Inhibitory Effects Of Grape Seed Proanthocyanidin Extracts On Expression Of Cell Adhesion Molecule And RAGE By Advanced Glycation End Productsin Endothelial Cells

Part oneSelective inhibition by Grape seed proanthocyanidin extracts of cell adhesion molecule expression induced by Advanced glycation end products in endothelialcellsBackgroundAlong with social development and population aging, the incidence of diabetes is steadily increasing every year and diabetic vascular complications are the major cause of morbidity and mortality for patients with diabetes. Recent work has demonstrated that strict glucose control can ameliorate certain microvascular diseases, but has few influences on atherosclerosis. Moreover, close control of blood sugar often results in serious hypoglycemic episodes, which is unacceptable for patients. Further studies have indicated this phenomenon of "hyperglycemic memory" is close associated with advanced glycation end products (AGEs) accumulated in patients with diabetes. AGEs directly cause vessel wall thickening, decreased elasticity and compliance, and have an influence on lipid as showed by increased susceptibility of LDL to oxidative modification and diminished role of HDL in reverse cholesterol transport. However, a major means by which AGEs exert their detrimental effects on the vasculature is through interacting with their signal-transduction receptor RAGE. The blockage of AGEs-RAGE interaction by administration of anti-RAGE IgG or sRAGE reversed the enhanced vascular hyperpermeability, suppressed the accelerated early lesions expansion or the progression of established atherosclerotic lesions, inhibited neointimal expansion after vessel injury, improved nerve conduction and mitigated renomegaly, glomerular sclerosis and proteinuria. Hence, in addition to close glucose control, drugs targeting AGEs-RAGE system become an emphasis on the prevention and treatment of patients with diabetic vascular complications accompanying research deepening.ObjectiveA growing body of evidences have demonstrated that interaction of AGEs with RAGE in endothelium triggers intracellular oxidative stress and ROS generation, and the increased ROS, in return, activates a complex cascade of signaling pathways and subsequent transcriptional factor NF-κB, which further promotes many genes expressions, the majority of which are highly relevant for inflammation, immunity and atherosclerosis such as cell adhesion molecules, tissue factor, chemokines and cytokines. Grape seed proanthocyanidin extracts (GSPE), a naturally occurring polyphenolic compounds obtained from grape seeds, have been reported to possess potent radical-scavenging and antioxidant properties. The aim of present study is to investigate whether GSPE can inhibit AGEs-induced cell adhesion molecules expression in cultured human umbilical vein endothelial cells through its antioxidant mechanisms.MethodsAGEs was prepared by incubating BSA with high concentration of glucose for 12 weeks at 37 degree C. Intracellular ROS production was detected by laser scanning confocal microscopy (LSCM) and flow cytometer. Surface expression of VCAM-1 and ICAM-1 was determined by flow cytometer. The levels of VCAM-1 and ICAM-1 mRNA were assayed by reverse-transcription polymerase chain reaction (RT-PCR). Firstly, HUVEC were stimulated with 200ug/ml AGE-BSA for different time. The time-ordered stimulatory effect of AGEs on intracellular ROS generation and expressions of VCAM-1 and ICAM-1 protein and mRNA in endothelial cells was observed. Next, after pre-incubation of GSPE of the concentrations of 5, 15, 25μg/ml for 4 hours, the cultured HUVEC was treated with 200μg/ml AGE-BAS for defined periods. We explored whether GSPE can prevent intracellular ROS production, thereby inhibiting the expression of VCAM-1 and ICAM-1 protein and mRNA.Results1. The protein expression of VCAM-1 and ICAM-1 started to enhance when HUVEC were stimulated with 200μg/ml AGE-BSA for 3 hours, reached the peak for 12 hours, was decreased for 24 hours without approach to basal level. The lower expression of VCAM-1 and ICAM-1 proteins was found in untreated endothelial cells. Both unmodified BSA and 25μg/ml GSPE were without effect on VCAM-1 and ICAM-1 expression. However, pre-incubation of 5, 15, 25μg/ml GSPE for 4 hours, in a dose-dependent manner, markedly prevented the up-regulation of VCAM-1 protein induced by 200μg/ml AGE-BSA, but, the enhanced ICAM-1 proteins were not altered. The levels of VCAM-1 expression were reduced to 79.90%±8.49% for 5μg/ml, to 52.09%±5.19% for 15μg/ml, to 20.53%±5.21% for 25μg/ml respectively (P< 0.01, vs AGE-BSA alone).2. The levels of VCAM-1 and ICAM-1 mRNA started to increase at 2 hours of AGE-BSA stimulation of endothelial cells, reached the peak at 12 hours, were moderately decreased at 24 hours without approach to basal level. After endothelial cells were pre-treated with 5, 15, 25μg/ml GSPE for 4 hours, AGE-BSA (200μg/ml) was added for stimulation for 6 hours. Consistent with protein expression, GSPE, in a concentration-dependent fashion, significantly reduced the VCAM-1 mRNA levels, but did not influence the increase in ICAM-1 mRNA levels. Reduction differed ranging from 0.388±0.035 to 0.275±0.015 for 5μg/ml, to 0.146±0.031 for 15μg/ml, to 0.077±0.009 for 25μg/ml respectively (P<0.01, vs AGE-BSA alone).3. The results from LSCM demonstrated that intracellular fluorescence intensity was very weak and ROS was generated at a lower level at 0 min of incubation of endothelial cells with AGE-BSA. But, intracellular ROS production started to augment at 15 min, reached the peak at 30 min, subsequently, was gradually decreased and approached to basal level at 60 min. The pretreatment of 5, 15, 25μg/ml GSPE ,in a dose dependent manner , apparently prevented intracellular ROS generation stimulated by 200μg/ml AGE-BSA, which was reduced from 3.12±0.31 to 2.36±0.34 for 5 ug/ml, to 1.95±0.41 for 15 ug/ml, to 1.14±0.23 for 25μg/ml respectively (P<0.01,vs AGE-BSA alone). Meanwhile, the similar results were gained using flow cytometer. Conclusions1. Treatment of endothelial cells with AGE-BSA prepared in vitro could induce intracellular oxidative stress and ROS generation, thereby leading to the expression of VCAM-1 and ICAM-1 protein and mRNA.2. Pre-incubation of GSPE of different concentrations selectively inhibited VCAM-1 protein and mRNA expression induced by AGE-BSA in endothelial cells in a dose-dependent manner, but did not influence ICAM-1 protein and mRNA levels.3. Through its antioxidant properties, GSPE dose-dependently prevented intracellular ROS generation, which might involve in selective inhibitory effect of GSPE on AGEs-induced cell adhesion molecules expression.4. GSPE selectively down-regulated AGEs-induced VCAM-1 expression. Hence, GSPE contribute to the treatment of early diabetic vascular lesion. Our results will provide theoretic evidences for application of GSPE for patient with diabetesPart twoEffects of Grape seed proanthocyanidin extracts on RAGE gene expressioninduced by Advanced glycation end products in endothelial cells BackgroundMany studies have confirmed that RAGE, a member of immunoglobulin superfamily, has already been identified to function predominantly as a signal transduction receptor and mediates diabetic vascular lesions caused by AGEs. The ligation of RAGE by AGEs induces intracellular oxidative stress, activates downstream signal transduction and evokes inflammatory responses. A variety of cell types, including endothelial cells, monocytes, smooth muscle cells and lymphocytes can express RAGE on their cell surface. RAGE is normally expressed at a lower level, but induced and lasted for several years under certain pathological conditions and there exists a positive feedback regulation mechanism between AGEs and RAGE, At the sites of accumulated AGEs in the vascular lesions there is increased RAGE expression. This positive feedback activation is thought to augment and prolong RAGE-ligands mediated detrimental effects. Besides, RAGE on endothelial cells may function as an adhesive receptor that interacts with leukocyte p2-integrins, thereby directly being involved in inflammatory cell recruitment. Hence, limiting RAGE expression will contribute to inhibiting AGEs-RAGE caused pathogenic effects, blocking its vicious cycle.ObjectiveGSPE is a naturally occurring antioxidant compound from grape seeds. The present study, taking the cultured HUVEC as study object, examines whether GSPE can inhibit AGEs-induced RAGE expression in endothelial cells.MethodsIntracellular oxidative stress was detected by LSCM or flow cytometry. The RAGE mRNA expression was assayed by RT-PCR. Firstly, endothelial cells were treated with 200μg/ml AGE-BSA for varying time period, and observed AGEs-induced irritant action on RAGE gene expression in endothelial cells. Next, cells were pre-incubated with GSPE of the concentrations of 0, 5, 15, 25μg/ml for 4 hours, and thereafter, stimulated in the presence of 200μg/ml AGE-BSA, and investigated whether GSPE can prevent AGEs-mediated RAGE gene expression in endothelial cells.Results1. RAGE mRNA levels had a tendency to increase at 2 hours of treatment of endothelial cells with 200μg/ml AGE-BSA, were significantly enhanced at 4 hours, reached the peak at 6 hours, were gradually decreased at the following time, but did not approach the normal level at 12hours. Both BSA and GSPE alone could not impact on RAGE mRNA levels. Pretreatment of GSPE of different concentrations for 4 hours, in a dose-dependent manner, inhibited RAGE mRNA expression induced by AGE-BSA in endothelial cells, which differed ranging from 1.12±0.15 to 0.75±0.11 for 5μg/ml, to 0.48±0.08 for 15μg/ml, to 0.27±0.10 for 25μg/ml respectively (P < 0.01, vs AGE-BSA alone).2. The results from LSCM showed that both BSA and GSPE alone did not enhance intracellular ROS generation. Likewise, pre-incubation of GSPE for 4 hours dose-dependently suppressed intracellular ROS production, which was reduced from 3.02±0.29 to 2.49±0.42 for 5μg/ml, to 1.95±0.24 for 15μg/ml, to 1.26±0.41 for 25μg/ml respectively. (P< 0.01, vs AGE-BSA alone) Moreover, similar results from flow cytometer were obtained.Conclusions1. AGE-BSA prepared in vitro could lead to intracellular oxidative stress and ROS generation, and subsequently cause the increase in RAGE mRNA levels. But, both unmodified BSA and GSPE alone did not influence ROS and RAGE mRNA levels.2. The pretreatment of GSPE of different concentrations dose-dependently prevented RAGE mRNA expression stimulated by AGE-BSA in endothelial cells, which contributed to inhibiting the interaction between AGEs and RAGE, interrupted the vicious cycle, further provided theory evidences for application of GSPE for patient with diabetic vascular complications.3. GSPE dose-dependently inhibited AGEs-induced ROS production in endothelial cells as much. Thus, effects of GSPE on RAGE mRNA might have close relation to its inhibitory role in ROS generation.4. We further speculate that selective inhibition by GSPE of cell adhesion molecule expression induced by AGEs in endothelial cells might also involve in down-regulated RAGE expression.