Cell proliferation of human endothelial cells under hypoxia (3% O2)

[Speaker] Kazuhisa Ikemoto:1
[Co-author] Yui Suganuma:1, Taiki Kano:1, Chiho S Ichinose:1, Kazunao Kondo:1
1:Pharmacology, Fujita Health University School of Medicine, Japan

[Background] Hypoxia is considered to play important roles in several diseases such as cancer and atherosclerosis. We investigated how hypoxia (3% O2 concentration) affect the proliferation of human endothelial cells.
[Methods] HUVEC (human umbilical vein endothelial cell) were isolated from normal umbilical cord and used in accordance with the institutional review board of Fujita Health University. The cells were cultivated in an incubator in which O2 concentration was adjusted to 3% with N2 gas. The proliferation was assessed by colorimetric methods with WST-8 (Dojindo Labolatories) or by direct cell count after staining with Hoechst 33342. (1) Effect of hypoxia (3% O2) on cell proliferation up to 72h: HUVECs were incubated for various incubation time (14, 48, and 72h). Reagents were added at the beginning of incubation under the hypoxic condition. (2) Effect of hypoxia (3% O2) on cell proliferation after 72h: In order to examine whether the maintenance condition affects on the stimulated proliferation, we changed O2 concentration after 72h. HUVECSs were pre-incubated under 3% O2 for 72h, and one group continued to grow under the same condition (group A), while the other was changed into normoxic (20% O2) condition (group B).
[Results] (1) 3% O2 significantly promoted the cell proliferation at 72h, whereas not significantly at 24 or 48h. Supplementation of tetrahydrobiopterin (BH4), an essential cofactor of nitric oxide synthase, with sepiapterin did not affect the promotion. Chrysin, an inhibitor of hypoxia inducible factor-1α (HIF-1α), inhibited the promotion. Quercetin, a promoting drug of HIF-1α, weakly inhibited the promotion. (2) The cell numbers of group A and B (indicated in Methods) were not significantly different.
[Conclusion] Two characteristics were found over HUVEC proliferation promotion triggered by hypoxia: 1) relatively long time as 72 hrs was necessary to observe the promoted proliferation, 2) the promoted proliferation was maintained even after cells were reversed into normoxic condition.
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