Pharmacological Manipulation of Endothelial Progenitor Cell Function for Cellular Therapy for the Vasodegenerative Phase of Diabetic Retinopathy

Abstract

Purpose: : Current available treatments for diabetic retinopathy are focused on end-stages of the disease. These therapies do not address the primary pathology of retinal neurovascular degeneration that precedes pathological neovascularization. Intervention at the vasodegenerative phase could prevent disease advancement. Stem cell therapy may represent a therapeutic strategy at this earlier point; however, diabetic EPCs (CD34⁺ cells) are dysfunctional and this severely limits there therapeutic utility. Diabetic CD34⁺ cells show impaired migration and reduced incorporation into the areas of vascular injury. Diabetes is associated with reduced bioavailability of NO due in part to increased production of reactive oxygen species (ROS), resulting in endothelial dysfunction. We hypothesized that pretreatment of diabetic CD34⁺ with pharmacological agents to increase NO bioavailability would correct their migratory defect.

Methods: : Pioglitazone, a PPARgamma agonist and an antidiabetic agent and NADPH oxidase (NOX) inhibitors, Apocynin and gp91ds-tat were used in this study. Peripheral blood was collected from normal and diabetic patients with severe non proliferative DR and CD34⁺ cells were isolated by immunomagnetic selection. NO and ROS productions were measured by DAF-FM and DHE fluorimetry, respectively, using TillPhotonics imaging system. Migration of cells in response to SDF-1 or VEGF was evaluated by Boyden chamber assay.

Results: : Pretreatment of diabetic CD34⁺ cells with pioglitazone increased mRNA and protein levels of endothelial nitric oxide synthase (eNOS), enhanced NO production, decreased ROS production and enhanced migration of cells in vitro. Pretreatment with apocynin increased NO production in diabetic cells. Pretreatment with gp91ds-tat decreased ROS production, enhanced NO release and reversed impaired migration to SDF-1 and VEGF in diabetic cells.

Keywords: nitric oxide • retinal neovascularization • protective mechanisms