Abstract
Purpose: :
CD34+ EPCs from diabetics demonstrate reduced vascular reparative function and cellular senescence which may contribute to the vasodegenerative phase of diabetic retinopathy. Diabetic EPC dysfunction (reduced proliferation, migration and bioavailable NO) is associated with elevated TGF- β1 expression by these cells which can be corrected by transient inhibition of TGF-β using phosphorodiamidate morpholino oligomers (TGF-β1-PMO). We asked whether the cytostatic effects of TGF-β requires PAI-1 in diabetic EPCs and assessed whether the reparative ability of diabetic CD34+ cells is improved by transient blockade of PAI-1.
Methods: :
Peripheral blood CD34+ cells from diabetics (n=10) were treated ex vivo with TGF-β1-PMO and were analyzed for PAI-1 expression. CD34+ cells were stabily infected with retroviral constructs expressing PAI-1 shRNA or nonfunctional shRNA. To assess knockdown efficiency we determined mRNA levels of PAI-1. Effect on EPC function was assessed by cell proliferation (cell number and colony forming ability), cell migration (Boyden chamber assay) and NO levels (DAF-FM fluorescence). SMAD2 phosphorylation was assessed by western blot.
Results: :
Treatment of diabetic cells with TGF-β1-PMO resulted in a 80% reduction in PAI-1 mRNA levels. PAI-1 levels were suppressed by more than 90% in cells expressing retroviral constructs of PAI-1 shRNA. Retroviral treatment of cells with the PAI-1 shRNA resulted in a 2-fold increase in cell numbers at day 3 (p<0.05) and a 6-fold increase in colony formation by day 12 (p<0.001) compared to cells treated with nonfunctional shRNA. SDF-1-induced migration of PAI-1 shRNA treated cells was enhanced compared to control treated cells (p<0.05). In the diabetic shPAI-1 treated CD34+ cells, NO production was restored to non-diabetic levels. TGF-β1 treatment of nondiabetic cells resulted in growth inhibition while knockdown of PAI-1 using viral PAI-1 shRNA in these cells resulted in cell proliferation. SMAD activation by TGF-β1 (200 pM) was not affected by PAI-1 knockdown in either diabetic or controls.
Conclusions: :
Our results suggest that the cytostatic activity of TGF β1 in diabetic CD34+ cells requires PAI-1 since knockdown PAI-1 resulted in bypass of the growth inhibition by TGF-β1. Blockade of PAI-1 may offer a promising therapeutic strategy for restoring vascular reparative function in senescent diabetic CD34+ cells.
Keywords: nitric oxide • diabetic retinopathy • signal transduction