Targeting TGF-Beta1 to Restore Vascular Reparative Function of Diabetic Endothelial Progenitor Cells

Abstract

Purpose: : Previously, we showed that endothelial precursors (EPCs) from diabetics with nonproliferative diabetic retinopathy (DR) were dysfunctional and exhibited decreased migratory and proliferative capacity; moreover when injected into the vitreous of diabetic animals, these cells could not repair acellular capillaries as could non-diabetic EPCs. Transforming growth factor-beta1 (TGF-beta1) inhibits proliferation of hematopoietic stem cells (HSC), the precursors of EPC, and is expressed at high levels in diabetic CD34⁺ cells. We asked whether reducing TGF-beta1 would improve the reparative function of diabetic EPCs.

Methods: : Peripheral blood from diabetics and healthy subjects was obtained and CD34⁺ cells isolated. CD34⁺ were exposed to antisense phosphorodiamidate morpholino oligomers (PMOs) to TGF-beta1 or scrambled PMOs and then injected intravitreally into mice that underwent retinal ischemia/reperfusion (I/R) injury, which results in generation of acellular capillaries similar to the vasodegenerative phase of DR. Effects of different PMO treatments on EPC functions, including nitric oxide generation and migration to the main EPC chemokine, stromal derived factor-1 (SDF-1) were also tested.

Results: : Elevated levels of TGF-beta1 mRNA were observed in all diabetic EPCs. Transient (2-4 days) blockade of endogenous TGF-beta1 using PMOs in diabetic CD34⁺ cells resulted in a reduction of TGF-beta1 expression and increased CXCR-4 expression, the receptor for SDF-1, in these cells. TGF-beta1 PMO treatment also enhanced the migratory prowess of diabetic CD34⁺ cells and restored their ability to repair damaged retinal vessels in the I/R model. This treatment also restored NO release by SDF1 in diabetic cells and it was sensitive to pretreatment with the CXCR-4 antagonist, AMD3100 and pertussis toxin.

Keywords: gene/expression • retinal neovascularization • diabetic retinopathy