Impact Of High Glucose Condition On The Expression Of Vascular Endothelial Growth Factor And Pigmente Epithelium Derived Factor And The Role Of P42/44 MAPK Signal Transduction Pathway In Inducing Expression Of Vascular Endothelial Growth Factor By Elevate

Purpose To study the impact of high glucose concentration on the expression of vascular endothelial growth factor and pigment epithelium derived factor and the role of p42/44 MAPK signal transduction pathway inhibitor PD98059 in inducing expression of vascular endothelial growth factor by elevated glucose concentration in cultured human retinal pigment epithelial cells.Methods In the first part ,human retinal pigment epithelial cells were divided into groups according to the concentration of glucose in the culture media: 1.normal glucose group(NG) (5.6 mmol/L); 2.high glucose group(HG1)(15.0 mmol/L D-glucose); high glucose group(HG2)(20.0 mmol/L D-glucose); high glucose group(HG3)(30.0 mmol/L D-glucose); 3.high osmolality group NG(HM)(24.4 mmol/L mannitol +NG); In the second part,hRPE cells cultured in elevated glucose concentration were dealed with p42/44MAPK signal transduction pathway inhibitor PD98059 and DMSO. 1. PD98059 group: 20.0umol/L PD98059+HG1(HG1+P),HG2(HG2+P),HG3(HG3+P);2. DMSO group: 0umol/L PD98059+HG1(HG1+D). The expression of VEGF and PEDF mRNA in hRPE cells was detected by RT-PCR, and the amount of VEGF in the hRPE-conditioned supernatants by enzyme linked immumosorbent assay(ELISA).Results (1)VEGF activity in NG,HG1,HG2,HG3,HM group cells was assayed by casein zymography, the value was 0.69±0.0351,0.77±0.0208,0.84±0.0153, 1.06±0.0902, 0.80±0.0351 ,respectively. The difference between group HG1,HG2,HG3,HM and that in normal glucose group(NG) was statistically significant(all P<0.05). the amount of VEGF in the hRPE-conditioned supernatants in group NG,HG1,HG2,HG3,HM cells was also assayed by enzyme linked immumosorbent assay(ELISA). the results accorded with that of RT-PCR. (2) PEDF activity in group NG,HG1,HG2,HG3 cells was assayed by casein zymography, the value was 2.77±0.1159,1.96±0.1050, 1.63±0.1253, 1.11±0.0902,respectively. The difference between group HG1,HG2,HG3 and that in normal glucose group(NG) was statistically significant(P<0.05). (3) VEGF activity in cells treated with 15.0 mmol/L D-glucose for 0,12,24,36,48,60h was assayed by casein zymography, the valuse was 0.33±0.0764,0.57±0.0839,0.77±0.0208,1.91±0.0902,1.76±0.0777,1.82±0.1015,respectively. The difference of the value between the time and the time of 0 hour was statistically significant(P<0.05). the results of ELISA accorded with that of RT-PCR. (4) VEGF activity in PD98059(NG+P,HG1+P,HG2+P,HG3+P),DMSO(HG1+D) group cells was assayed by casein zymography, the value was 0.50±0.0451,0.40±0.0433,0.67±0.0458,0.71±0.0503,0.73±0.1060,respectively. The difference between the PD98059 group and that of the normal glucose group(NG) was statistically significant(P<0.05). the amount of VEGF in the hRPE-conditioned supernatants in PD98059(NG+P,HG1+P,HG2+P,HG3+P),DMSO(HG1+D) group cells was also assayed by enzyme linked Immumosorbent assay(ELISA). the results accorded with that of RT-PCR. (5) The difference between (VEGF/β-actine)/(PEDF/β-actine) activity of NG,HG1+P groups and that of HG1 were statistically significant(P<0.05).Conclusion High glucose condition induces expression of VEGF mRNA,protein and suppress PEDF mRNA in cultured human retinal pigment epithelial cells. P42/44 MAPK signal transduction pathway might play a role in the VEGF expression induced by elevated glucose concentration in cultured hRPE cells.