Hypoxia Induces the Gene Expression of VEGF and its Receptors, FLK-1 and NP-1 in Monkey Choroid-retinal Endothelial Cells

Abstract

Abstract: : Purpose: Hypoxia selectively induces the transcription of MT1-MMP and MMP-2 metalloproteinases and of the inhibitor TIMP-2 in monkey choroid-retinal endothelial cells (RF/6A) (ARVO, 2000, No 2752). Since MMPs modulate extracellular matrix (ECM) remodeling, here we have studied the expression of VEGF isoform-165, its specific VEGFR-1 (FLT1) and VEGFR-2 (FLK1) tyrosine kinase receptors, as well as its co-receptor neuropilin-1 (NP1) in RF/6A cells undergoing hypoxia. Methods: RF/6A cells (5 x 10⁶, CRL-1780 from ATCC) were seeded in collagen type-I coated Petri dishes (60 mm diameter) and cultured in Hams F12-K medium containing 5% fetal bovine serum (FBS). When the cells reached confluence, the medium was replaced with hypoxic (degassed) medium containing 1% FBS and cells were exposed to 95% N₂ and 5% CO₂ at 37 ^°C for 1, 2, 4 and 8 hours. mRNA was extracted from endothelial cells and the levels of gene expression for VEGF-165 and its receptors FLT1, FLK1, and NP1 were determined by RT-PCR. All quantitations were normalized to the 18s rRNA used as endogenous control. For tube formation, RF/6A cells were embedded in collagen containing 8 vol of vitrogen, 5µg/ml of fibronectin and 5µg/ml laminin, overlaid with hypoxic medium, and exposed to hypoxia for 4 hours followed by additional 72-hour incubation under normoxic conditions. Results: VEGF-165 and its FLK1 and NP1 receptors were constitutively expressed in the endothelial cells, while the appearance of the FLT1 receptor was dependent upon cell-passage number. VEGF-165 was the only VEGF-A isoform detected in these cells and was markedly upregulated at 1 and 2 hours of hypoxia, followed by a return to control levels at longer time points. FLK1 induction was detected at 4 hours of hypoxia. Cells exposed to hypoxia for 4 hours, followed by 72 hours under normoxic conditions, produced a markedly higher number of tubules as compared to normoxic cells. Conclusions: In hypoxia-induced tube formation in monkey choroid-retinal endothelial cell cultures, we have identified selective induction of the VEGF-165 isoform and of its receptors FLK1 and NP-1. NP-1 acts as a co-receptor for VEGF-165 activation of FLK1. These events may be critical during the process of vascular development involving ECM remodeling.

Keywords: gene/expression • growth factors/growth factor receptors • hypoxia