Effect of hypoxia upon the growth and sprouting activity of cultured aortic endothelium from the rat.

Endothelial cells were obtained from the aortas of Wistar rats by collagenase digestion. Cells were grown to confluence in medium 199 enriched with L-glutamine but without specific growth factors. Cells were subcultured into 35 mm dishes or 25 cm2 flasks coated with fibronectin. For cell growth studies, cells were seeded onto multiwell plates or 35 mm dishes. In two experiments the cells were grown in an hypoxic atmosphere of 5.3% O2 and in a third the level of oxygen was 2.5%. Control cultures for each experiment were grown in 5% CO2 and air. Cell populations were counted at 2-day intervals and at the termination of each experiment the cells were fixed and the area of each plate or flask occupied by sprouting cells was assessed by point counting. Endothelial cells grown in 5.3% O2 grew more rapidly and attained confluence earlier than in the controls. An atmosphere of 2.5% O2 did not accelerate growth but neither did it inhibit it, so after 9 days there were as many hypoxic cells as there were controls. Hypoxia also stimulated sprouting activity to occur earlier and to become much more extensive than in control cultures. Under the influence of hypoxia, sprouting consisted of complex anastomotic or arborizing patterns forming syncytium-like masses beneath the monolayer of oval cells. This process appeared to originate from foci of altered endothelial cells that had become retracted, smaller, elongated and migratory, and which displayed increased immunoreactivity for factor VIII antigen. It was concluded that a level of hypoxia, similar to that in systemic veins, stimulates growth of arterial endothelium and provokes enhanced sprouting activity.(ABSTRACT TRUNCATED AT 250 WORDS)