| Atherosclerosis (AS) is a kind of inflammatory disease. The infiltration of inflammatory cells in tunica intima of artery, proliferation of smooth muscular cells , increase of extracellular matrix and thrombosis all exist during the whole process of Atherosclerosis. Diabetes mellitus is established independent risk factor for cardiovascular disease. There is an increased frequency and more rapid progression of coronary artery disease in diabetic patients , and the mortality of acute thromboembolic events , such as myocardial infarction , is higher than in nondiabetic subjects. Several lines of evidence suggest that a common mechanism for the impairment of endothelial function may be operative in diabetes . In addition to disturbed endotheliumdependent relaxation , activation of coagulation pathways , platelet hypersensitivity, and depressed fibrinolysis cause a prothrom-botic state, as do the changes in vascular architecture and matrix composition , o-verall, these and other abnormalities in hemostasis and hemodynamics in diabetes point to an adaptive/maladaptive replicative and biosynthetic program of vascular cells that endures the pathological process triggered by the metabolic perturbation. Vascular Endothelial Growth Factor (VEGF) is the most important growth factor in atherosclerosis, it promotes endothelial proliferation and vascular angiogenesis.Endothelial cells are first activated during the process of atherosclerosis. After Endothelial cells are stimulated by many factors , they can cause and amplify the inflammatory reaction of arterial wall by excreting inflammatory medium or regulating the adhesiveness of leucocytes on the surface of endothelial cells. Hy-perglycemia, advanced glycocation end products ( AGEs) and free fatty acid(FFA) are the major metabolic abnormalities in diadetes. Advanced glycocation end products (AGEs) are products of nonenzymatic reaction between glucose and various extracellular and intracellular macromolecules. The terminal adducts are irreversibly bound to vascular matrix molecules, most notably subendothelial collagen, and accumulate with age and the duration of the disease. Free fatty acid (FFA) is the important part of blood lipid and pahnitate(PA) is the main part of free fatty acid. Glucose N AGEs and FFA have been shown to participate in AS, stimulate smooth muscular cells ( SMCs ) , macrophages and other to upregu-late VEGF expression. It is reported that plasm levels of glucose >, AGEs and FFA in diabetes are high, but the mechanism and the expression of VEGFmRNA and protein are not clear completely at present. In this study we used the methods of and Western blot to detect the expression of VEGFmRNA and protein in Human umbilical vein cells ( HUVECs) and tried to find out the effects of different metabolic situations( Glucose ? AGEs and FFA) on the formation and development of atherosclerosis in diabetes.Methods1. Cell cultureHuman umbilical vein cell line(ECV304) was purchased from Shanghai Cell Institute. ECV304 were cultured with RMPI1640 culture media containing 10% inactivated newborn bovine serum at 37*0 and 5% C02. Culture media were changed every two days and 0. 25% trypsin was used to digest and transfer of culture. Cells were cultivated on 6 pore culture boards with coverslips placed in advance and 100ml culture bottles. Endothelial cells were cultured with serum -free medium for 24h before endothelial cells were used.2. in situ hybridizationEndothelial cells were fixed for 20 minutes in 4% paraformaldehyde at room temprature( containing 1/1000DEPC). Cells were digested by pepsin at 37 X. for 60 seconds. After addition of pre - hybridization solution , the fixed cells were incubated at 37 X, for 4h. The pre - hybridization solution was discarded, then 20fxl hybridization solution was added. The sections were put into a wet box andincubated at 37^ for 18h. After being washed with 2 x SSCN0.5 x SSC and 0.2 x SSC, the sections were stained with DAB for 3 - 10 minutes. Relative quantative analysis of VEGFmRNA was carried out by using Metamorph/DPlO/image analyzer. Probe was replaced by pre - hybridization solution in the negative control group.3. Western blotEridothelial cells were cultured in 100ml culture bottles and collected at required time , then protein were extracted. Equal amounts of protein in every exponent were subjected to electrophorosis on a 15% SDS - PAGE and transferred onto nitrocellulose membrane. After washing membrane >, blockings hybridization, the protein were revealed . Quantative analysis of VEGF protein was carried out by using GDS - 8000 electrophoresis gel image analyzer.Result1. With the increase from 5. 6mmol/L to 22. 2 mmol/L of glucose concentration, VEGFmRNA expression increased obviously. VEGFmRNA expression was the most when glucose concentration was 22. 2 mmol/L;when endothelial cells were cultured with 22.2 mmol/L glucose, VEGFmRNA expression also increased gradually with the elongation of time, and VEGFmRNA expression was the most when it reached 15d ( P < 0. 05 ). The results of in situ hybridization showed that glucose could increase VEGFmRNA expression in endothelial cells in a dose - dependent and time - dependent manner. By analyzing the expression of VEGF protein in each group was 1.37 - fold^l. 84 - fold and 2.64 - fold compared with 5. 6mmol/L glucose group(P <0.05) , respectively;when endothelial cells were cultured with 22.2 mmol/L glucose, VEGF protein expression also increased gradually with the elongation of time, and VEGF protein expression was the most when it reached 15d(P<0.05);.By analyzing the expression of VEGF protein in each group was 1. 82 - fold^3. 13 - fold and 4. 07 - fold compared with Od glucose group ( P < 0. 05 ) , respectively. The results of Western blot showed that glucose could increase VEGF protein expression in endothelial cells in a dose - dependent and time - dependent manner. 2. After endothe-lial cells were cultured with 100mg/LN200mg/L and 400mg/L AGEs for 24h, VEGFmRNA expression increased significantly;. After endothelial cells were cultured with400mg/L AGEs for0hJ2h^24h and 36h, VEGFmRNA expression also increased obviously. The results of in situ hybridization showed that AGEs could increase VEGFmRNA expression in endothelial cells in a dose - dependent and time - dependent manner. By analyzing the expression of VEGF protein, After endothelial cells were cultured with 100mg/LN200mg/L and 400mg/ L AGEs for 24h, the expression of VEGF protein in each group was 1.41 - fold,, 1.96 - fold and 2.55 - fold compared with BSA group( P < 0.05 ) ,respectively;After endothelial cells were cultured with 400mg/L AGEs for 0hN12h>24h and 36h, the expression of VEGF protein in each group was 1.91 - fold^2.82 - fold and 3.47 - fold compared with Oh group( P <0.05 ) respectively. The results of Western blot showed that AGEs could increase VEGF protein expression in endothelial cells in a dose - dependent and time - dependent manner.3. After endothelial cells were cultured with lOOjxmmol /L^200|xmmol /L and 300|xmmol /L PA for 24h, VEGFmRNA expression increased significantly;. After endothelial cells were cultured with 300|xmmol /L PA for 0h^l2h^24h and 36h, VEGFmRNA expression also increased obviously. The results of in situ hybridization showed that PA could increase VEGFmRNA expression in endothelial cells in a dose - dependent and time - dependent manner. By analyzing the expression of VEGF protein, After endothelial cells were cultured withl00(xmmol /L^OOjjummol /L and 300jxmmol /L PA for 24h, the expression of VEGF protein in each group was 1. 52 - fold N 2. 07 - fold and 3. 24 - fold compared with Ojxmmol /L group ( P < 0. 05 ) , respectively;After endothelial cells were cultured with 300|xmmol /L PA for 0h^l2h>l24h and 36h, the expression of VEGF protein in each group was 1.72 - fold N2.63 - fold and 3.51- fold compared with Oh group ( P < 0. 05 ) , respectively. The results of Western blot showed that PA could increase VEGF protein expression in endothelial cells in a dose - dependent and time - dependent manner.Conclusion1. High glucose could increase the expression of VEGFmRNA and protein in endothelial cells and there was a positive correlation between the role and glucose concentration Nlasting time of glucose. Glucose could promote the formation and development of atherosclerosis in diabetes.2. AGEs could increase the expression of VEGFmRNA and protein in endothelial cells in a dose - dependent and time - dependent manner. AGEs may be one of major mechanisms that diabetes are easily troubled by atherosclerosis.3. PA could increase the expression of VEGFmRNA and protein in endothelial cells and there was a positive correlation between the role and PA concentration ^lasting time of PA. FFA could enhance the formation and development of atherosclerosis in diabetes. |
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