Transforming growth factor-β1/Thrombospondin-1/CD47 axis mediates dysfunction in CD34⁺ cells derived from diabetic...

Aging with diabetes is associated with impaired vasoprotective functions and decreased nitric oxide (NO) generation in CD34⁺ cells. Transforming growth factor- β1 (TGF-β1) is known to regulate hematopoietic functions. This study tested the hypothesis that transforming growth factor- β1 (TGF-β1) is upregulated in diabetic CD34⁺ cells and impairs NO generation via thrombospondin-1 (TSP-1)/CD47/NO pathway. CD34⁺ cells from nondiabetic (ND) (n=58) or diabetic older adults (DB) (both type 1 and type 2) (n=62) were isolated from peripheral blood. TGF-β1 was silenced by using an antisense delivered as phosphorodiamidate morpholino oligomer (PMO-TGF-β1). Migration and proliferation in response to stromal-derived factor-1α (SDF-1α) were evaluated. NO generation and eNOS phosphorylation were determined by flow cytometry. CD34⁺ cells from older, but not younger, diabetics have higher expression of TGF-β1 compared to that observed in cells derived from healthy individuals (P<0.05, n=14). TSP-1 expression was higher (n=11) in DB compared to ND cells. TGFβ1-PMO decreased the secretion of TGF-β1, which was accompanied with decreased TSP-1 expression. Impaired proliferation, migration and NO generation in response to SDF-1α in DB cells were reversed by TGF-β1-PMO (n=6). TSP-1 inhibited migration and proliferation of nondiabetic CD34⁺ cells that was reversed by CD47-siRNA, which also restored these responses in diabetic CD34⁺ cells. TSP-1 opposed SDF-1α-induced eNOS phosphorylation at Ser¹¹⁷⁷ that was reversed by CD47-siRNA. These results infer that increased TGF-β1 expression in CD34⁺ cells induces dysfunction in CD34⁺ cells from diabetic older adults via TSP-1/CD47-dependent inhibition of NO generation.