Developmental Expression of VEGF Isoforms and Receptors in the Retina and their Regulation by PEDF

Abstract

Abstract: : Purpose: The vascular endothelial growth factors (VEGFs) and their receptors are endothelial cell mitogens and play an important role in ocular angiogenesis. PEDF is one of the most potent anti-angiogenic factors in the eye. This study was designed to determine the expression of the VEGF family and their receptors in fetal and adult retina and to analyze their regulation by PEDF. Methods: Specific PCR primers were designed to analyze expression of each of the members of the VEGF gene family (VEGF-A,B,C & D), the isoforms of VEGF-A (121,165,189 & 206), the VEGF receptors VEGF-R1 (flt-1), VEGF-R2 (flk-1), VEGF-R3 (flt-4), and other receptors that VEGF interacts with neuropilin 1 and neuropilin 2. PCR was performed using RNA from human and mouse fetal and adult retina, human ARPE19 and Y79 retinoblastoma cell lines, and primary cultures of mouse Müller glial cells. To test the effects of PEDF on the expression of VEGF and VEGF-R, cells or tissues were incubated for 48 hrs in 100ng/ml PEDF prior to RNA extraction. RNA was reversed transcribed and 50 ng of cDNA used in PCR reactions. cDNA probes were synthesized from the two cell lines and used to hybridize human 8K gene arrays and mouse 9K gene arrays. Results: VEGF-A isoforms showed a characteristic pattern of developmental expression in the retina. There is varying expression of the VEGF isoforms in fetal and adult retinal tissue, ARPE19, and Y79 cells. The most abundantly expressed isoforms are 121 and 165. VEGF 189 is weakly expressed in the RPE. VEGF receptors are expressed in adult and fetal RPE, ARPE19, and Y79 cells but were not detected in the neural retina. PEDF downregulated expresssion of VEGF-R3 but had minimal effect on the basal levels of expression of other receptors and isoforms of VEGF. The expression profile of VEGF and their receptors correlate with the gene array data. Conclusions: VEGF levels are potentiated in response to hypoxia in the eye. High intraocular level of VEGF is associated with diabetic retinopathy and age related macular degeneration. Interference with VEGF function is, therefore, of major interest in ophthalmic therapy. PEDF may be an important factor in selectively modulating VEGF-receptor mediated function.

Keywords: retinal degenerations: cell biology • neuroprotection • gene microarray