Detecting the expression changes of hypoxia inducible factor-1α(HIF-1α), vascular endothelial growth factor(VEGF)and angiopoietin-1(Ang-1) under anoxic environment, to investigate the effect of cobalt chloride induced hypoxia on expression of Ang-1 in pericytes.

Human brain vascular pericytes cultured in vitro, with different concentrations of cobalt chloride induced by hypoxia. The cultured cells were divided into control group and experimental groups containing different concentrations of cobalt chloride. The control group used the pericyte medium without cobalt chloride. In the experimental groups, cobalt chloride was dissolved by pericyte medium and diluted to the final concentration of 50 μmol/L (50 μmol/L cobalt chloride group), 100 μmol/L (100 μmol/L cobalt chloride group), 200 μmol/L (200 μmol/L cobalt chloride group), 300 μmol/L (300 μmol/L cobalt chloride group) and 400 μmol/L (400 μmol/L cobalt chloride group). The expression of HIF-1α protein was detected by Western-blotting, the mRNA synthesis of HIF-1α and Ang-1 were detected by reverse transcription-polymerase chain reaction (RT-PCR), and the secretion of VEGF and Ang-1 were detected by enzyme-linked immuno sorbent assay (ELISA). Data were processed with one way analysis of variance, Dunnett-t test and t test.

The expression of HIF-1α protein in control group and experimental groups (50, 100, 200, 300, 400 μmol/L cobalt chloride group) was significantly different(F=215.7, P<0.05). The expression of HIF-1α protein increased with the concentration of cobalt chloride first and then decreased. The relative gray value of the relative gray level reached 8.6140±0.3445 at 200 μmol/L. The relative gray value of 200 μmol/L cobalt chloride group was significantly different from those of other groups (t=38.28, 22.18, 10.16, 5.60, 25.47; with P values below 0.05). The expression of HIF-1α mRNA in control group and experimental groups was significantly different, the difference was statistically significant(F=195.5, P<0.05). The expression of HIF-1α mRNA decreased as the concentration of cobalt chloride increased, and the HIF-1α mRNA expression in the 400 μmol/L cobalt chloride group reached the lowest value 5.107×10^-3±8.138×10^-5. The expression of HIF-1α mRNA in 400 μmol/L cobalt chloride group were significantly different from control group and 50, 100, 200 μmol/L cobalt chloride group, the difference was statistically significant (t=21.40, 17.75, 14.96, 5.36; with P values below 0.05). The expression of VEGF protein in control group and experimental groups was significantly different, the difference was statistically significant (F=93.34, P<0.05). The expression of VEGF protein first increased and then decreased with the concentration of cobalt chloride. The count reached a peak value of (901.000±6.798) pg/mL at 200 μmol/L. There were statistically significant differences in 200 μmol/L cobalt chloride group with those of other groups (t=27.70, 10.56, 4.65, 10.49, 17.97; with P values below 0.05). The expression of Ang-1 protein in control group and experimental groups was significantly different, the difference was statistically significant (F=279.3, P<0.05). The expression of Ang-1 protein first increased and then decreased with the concentration of cobalt chloride. The count reached a peak value of (4 364.0±117.3) ng/mL at 100 μmol/L. There were statistically significant differences between 100 μmol/L cobalt chloride group and control group, 50, 200, 300, 400 μmol/L cobalt chloride group(t=8.57, 4.33, 17.03, 19.48, 23.30, with P values below 0.05). The expression of Ang-1 mRNA in control group and experimental groups was significantly different, the difference was statistically significant (F=172.0, P<0.05). The expression of Ang-1 mRNA first increased and then decreased with the concentration of cobalt chloride. The count reached a peak value of 6.732×10^-4±7.140×10^-6 at 100 μmol/L. There were statistically significant differences between 100 μmol/L cobalt chloride group and control group, 200, 300, 400 μmol/L cobalt chloride group(t=10.05, 20.21, 110.20, 34.25; with P values below 0.05).

Under mild hypoxia environment, the expression of Ang-1 in pericytes was promoted with the increasing level of hypoxia, while under severe hypoxia environment, the expression of Ang-1 in pericytes was inhibited with the increasing level of hypoxia.