Abstract
Purpose: :
Hypoxia inducible factor-1α is a fragile protein under normal physiological conditions. In response to hypoxia, HIF-1α becomes stable and induces transcription of angiogenic factors such as VEGF. Disruption of regulated HIF-1 activity causes induction of pathologic angiogenesis observed in diabetic retinopathy. However, the detailed mechanism of angiogenesis induced by hypoxia is not clear because no appropriate culture system exists. Thus, the purposes of the present experiment were to 1) establish an in vitro model for retinal angiogenesis induced by hypoxia, 2) delineate a molecular mechanism for in vitro angiogenesis, and 3) compare our in vitro model to an in vivo rat model of retinopathy of prematurity.
Methods: :
Human retinal pigment epithelial (RPE) cells (ARPE-19) were cultured under hypoxic conditions. HIF-1α protein in RPE cells was detected by immunoblotting. Expression of mRNAs for angiogenic factors were analyzed by PCR arrays. VEGF protein in the conditioned medium secreted from RPE cells cultured under hypoxia was measured using ELISA. The conditioned medium was used to induce tube formation in human vein endothelial cells co-cultured with human fibroblasts. A rat model for retinopathy of prematurity was produced by standard methods. HIF-1α protein in retina was analyzed by immunoblotting. mRNA for VEGF in retina was detected by RT-PCR. VEGF protein in vitreous humor was measured by ELISA.
Results: :
HIF-1α protein and expression of mRNA for angiogenic factors such as VEGF, αFGF, FGFR3 and TGF-α increased in hypoxic RPE cells. The conditioned medium secreted from hypoxia-treated RPE cells was rich in VEGF protein and induced tube formation in endothelial cells. HIF inhibitor, chetomin, significantly inhibited mRNA expressions in RPE cells, VEGF secretion into the conditioned medium, and the increased tube formation. Similar to our in vitro model, mRNA for VEGF and HIF-1α protein increased in retina from the in vivo rat model. VEGF protein in vitreous humor, which may be released from retina, increased before development of angiogenesis.
Conclusions: :
We established an in vitro model of angiogenesis that mimicked the mechanism of rat angiogenesis in vivo. HIF-1α was a key molecule for inducing angiogenesis. Along with inhibition of VEGF, inhibition of HIF-1 activity may be a possible therapy against retinal angiogenesis in conditions such as diabetic retinopathy.
Keywords: neovascularization • retinal neovascularization • hypoxia