Relationship of human microvascular endothelial cell activation and adenosine level under the hypoxia

Abstract

Background Retinal hypoxia is one of primary causes of retinal neovascularization, and its mechanism is research hot topic.Studies showed that hypoxia stimulates the activation of many trancripation factors and vascular endothelial cells, which leads to angiogenesis.Besides to the vascular endothelial growth factor, the effect of adenosine on angiogenesis is increasingly concerned. Objective This study was to invastigate the relationship of biological behaviour of human microvascular endothelial cells (HMEC-1) under the hypoxia and adenosine, and to explore the effect of adenosine on angiogenesis under hypoxia. Methods HMEC-1 cell line was cultured in vitro, and the cells were divided into normoxia group and hypoxia group.The cells in the normoxia group were cultured under the 5%CO2 environment, and those in the hypoxia group were cultured under the 1% O2, 94%N2 and 5%CO2 environment.The proliferation ability and percentage of the cells were assayed by cell counting kit-8 (CCK8) and EDU.The migration number and invasive number of the cells were detected by transwell chamber.The expressions of CD39 and CD73 proteins in the cells were tested by Western blot and immunofluorescence technique, and the level of adenosine was measured by high performance liquid chromatography (HPLC). Results The proliferation values (absobancy) were 0.715±0.067 and 0.821±0.056 in the normoxia group and the hypoxia group in 12 hours after culture, and those in 24 hours were 0.946±0.028 and 0.998±0.028, showing significant increase in the hypoxia group compared with the normoxia group (t12h= 3.805, t24h= 3.222, all at P<0.01). The precentage of the proliferation in the hypoxia group was evidently higher than that in the normoxia group (t=-6.868, P<0.01). The number of the cell migration and invasion in 24 hours after culture was 185.3±10.594 and 74.2±10.741 respectively in the normoxia group, and that in the hypoxia group was 300.7±22.853 and 107.5±7.007, with significant differences between the two groups (t=-12.124, -6.367, both at P<0.01). The expression levels of CD39 and CD73 proteins in the hypoxia group were significantly higher than those in the normoxia group in both12 hours and 24 hours after culture(all at P<0.05), and the adenosine content in the cells is gradually increased in 2, 6, 12, 24 and 36 hours after culture, with the highest content in 36 hours.The adenosine content was significantly higher in the hypoxia group than in the normoxia group at various time points (t2h=2.469, P=0.017; t6h=5.442, P<0.001; t12h=3.841, P<0.001; t24h=4.458, P<0.001; t36h=2.757, P=0.008; t48h=3.319, P=0.002). Conclusions Compared with the normoxia group, the proliferation, migration and invasion abilities of HMEC-1 are stronger, meanwhile, the expression of CD39 and CD73 as well as the adenosine level in the cells are all increased under the hypoxic condition.It is suggested that the activation of human microvascular endothelial cells might be significantly related to the level of adenosine and its key enzymes. Key words: Neovascularization; Retina; Microvascular endothelial cells; Human; Angiogenesis; adenosine