Abstract
Abstract: :
Purpose: PPARs alpha, beta and gamma are a family of ligand dependent transcription factors, belonging to the nuclear hormone receptor superfamily. Here we investigate if PPARs are expressed in retinal endothelial cells (RECs) and study the effect of PPAR ligands on VEGF induced angiogenic responses, in vitro. Methods: Primary cultures of human and bovine RECs were grown in fibronectin-coated flasks. To detect PPAR expression, nuclear extracts or fixed RECs were probed with antibodies to PPARs. For proliferation and cytotoxicity assays, RECs were seeded in 96 well plates, starved in serum free medium prior to incubation in experimental medium with VEGF and vehicle or PPAR ligands. For tube differentiation assay, RECs were grown on collagen matrix in complete medium, then in experimental medium for 48 hrs. Lengths of tube-like structures were measured from digital images using NIH image software. Specific PPARγ ligands, 15dΔ12,14PGJ2 (0.2-100µM) and ciglitazone (5-60µM); PPARα ligand, WY 14643 (1-100µM) and PPARß ligand, SSd (6-120µM) were used in experimental medium. Serum free medium and vehicle alone with VEGF served as control. Results: All three PPAR subtypes are expressed in RECs and mainly localized to the nuclei as detected by immunohistochemistry. The expression levels as detected by western analysis appear in the order PPARγ>PPARß>>PPARα. All PPAR ligands tested, dose dependently inhibited REC proliferation and tube formation except WY 14643. WY 14643 inhibited REC proliferation ~ 90% at 10mM, but the PPARα ligand did not inhibit tube formation effectively even at 10 fold higher dose. The effective optimal dosages of PPAR ligands at which proliferation was inhibited 90-100% were, 2.0µM for 15dΔ12,14 PGJ2 , 10µM for ciglitazone and 120µM for SSd. Tube formation was also inhibited at these optimal doses in both REC types. Vehicle treatment did not inhibit VEGF induced angiogenic responses of RECs. None of the endogenous and synthetic ligands exhibited significant toxicity at the optimal effective doses. Conclusions: We have confirmed that PPARs are expressed in RECs. Our results also demonstrate that PPAR ligands inhibit VEGF induced angiogenic responses of RECs in a receptor subtype specific and dosage dependent manner. The antiangiogenic activity of different classes of PPAR ligands, underscores the potential of PPARs as molecular therapeutic targets for retinopathies.
Keywords: retinal neovascularization • transcription factors • diabetic retinopathy