Effect of hypoxia on the proliferation of retinal microvessel endothelial cells in culture

Background To determine if hypoxia stimulates the proliferation of retinal microvessel endothelial cells in culture. Methods Bovine retinal microvessel endothelial cells were cultured in normoxic (95% air, 5% CO2) and hypoxic (2% O2, 5% CO2, 93% N2) conditions. Endothelial cells were identified by acetylated LDL and Factor VIII‐related antigen immunocytochemical staining. Cells from passages three to eight were used in these experiments. Proliferation assays included cell counts by hemocytometer and autoradiographic analysis of incorporated 3H‐thymidine (3H‐TdR). Results At day 4, cell counts of endothelial cells in hypoxia showed a 133% increase over those grown in normoxic conditions (N = 25, P < 0.01). Cell counts per day for 5 days were 121‐181% greater in hypoxia. Autoradiography of endothelial cells exposed to 3H‐TdR and counted every 12 hours for 60 hours exhibited labeling indices 112‐118% higher in hypoxic conditions (P < 0.0001). Endothelial cells cultured under hypoxic conditions were smaller and spindle‐shaped, whereas those grown under normoxic conditions were larger and more polygonal. Conclusions Hypoxia increases DNA synthesis and stimulates proliferation of retinal microvessel endothelial cells in vitro and induces alterations in morphology. These results may be relevant to microvessel angiogenesis, which occurs in vivo under ischemic conditions. Anat. Rec. 248:366‐373, 1997. © 1997 Wiley‐Liss, Inc.