We show that ex vivo transient inhibition of endogenous transforming growth factor-β1 (TGFβ1) in highly enriched and partially enriched murine hematopoietic stem cells (HSC): (1) accelerates engraftment of long-term repopulating HSC ("LTR-HSC"), (2) increases donor stem cell chimeras in competitive bone marrow repopulation studies, (3) permits transplant of as few as sixty LTR-HSC to rescue mice from hematopoietic death after lethal irradiation using direct (non-competitive) transplants and (4) promotes extended survival in vitro of single or multiple LTR-HSC in the absence of growth factors. Our approach to inhibit TGFβ1 involved use of either neutralizing monoclonal antibodies ("TGFβ-MAB") or antisense phosphorodiamidate morpholino oligomers ("TGFβ1-PMO") prior to iv transplant. Our previous studies showed that TGFβ1 is a potent reversible inhibitor of LTR-HSC proliferation. Unexpectedly, LTR-HSC treated with TGFβ-MAB and transplanted 1-2 hr later, or treated with TGFβ1-PMO for 16hr then transplanted, rapidly engrafted and produced relatively high levels of donor chimeras that persisted for >6-10 months. Early post-transplant, donor neutrophils predominated but only if TGFβ1 was inhibited in the LTR-HSC. Finally, we tested the ability of LTR-HSC to rescue mice from hematopoietic death after lethal irradiation: as few as 250 LTR-HSC treated with TGFβ-MAB or TGFβ1-PMO rescued essentially 100% of mice and produced a durably graft. In contrast, control treated LTR-HSC (untreated, isotype control MAB, or control-PMO) essentially could not rescue lethally irradiate mice. In cases where donor stem cell numbers are limiting, these methods could prove to be clinically useful. Our more recent studies have demonstrated that human specific TGF-β1-PMO can reverse the dysfunctions of diabetic lin -CD34 + CD45 + stem cells to be able to repair endothelium in the retina.
Adult hematopoietic stem/progenitor cells (HSPCs) participate in the cardiovascular repair by stimulating regeneration and angiogenic functions of endothelium. Long‐term diabetes is associated with impaired vasoprotective functions of HSPCs. Transforming growth factor β (TGFβ1) is pleiotropic regulator of HSPC functions. Previous studies have shown that transient silencing of TGFβ1 expression improves in vivo migratory functions of diabetic HSPCs partly by restoring nitric oxide (NO) generation. This study tested the hypothesis that restoration of NO by TGFβ1‐silencing is mediated by thrombospondin‐1 (TSP1)/CD47 pathway. TSP1 is known to attenuate NO generation via inhibition of endothelial nitric oxide synthase (eNOS). HSPCs were isolated from peripheral blood samples obtained from either male or female healthy (n=25) or diabetic (both type 1 and type 2) (n=27) individuals (50–80 years of age) by immunomagnetic enrichment. TGFβ1 expression was transiently blocked by using TGFβ1‐antisense delivered in the form of phosphorodiamidate morpholino oligomer (PMO‐TGFβ1). TGFβ1 and TSP1 gene expressions were determined in HSPCs treated with either PMO‐control or PMO‐TGFβ1. NO generation induced by stromal‐derived factor‐1α (SDF) was determined by DAF‐FM flow cytometry. Diabetic cells have higher expression of TGFβ1 or TSP1 compared to that observed in cells derived from healthy individuals (n=10). Treatment with TGFβ1‐PMO decreased the expression of TGFβ1, which was associated with decreased TSP1 expression. CD47 expression is similar in healthy or diabetic individuals. NO generation by SDF is attenuated in diabetic compared to healthy cells (P<0.01, n=5). PMO‐TGFβ1‐treated diabetic cells showed increased generation of NO in response to SDF compared to PMO‐control‐treatment (P<0.01, n=5). Future studies will determine the effect of TSP1 on SDF/NO generation in cells with or without CD47‐siRNA, to further support the hypothesis. Collectively, these studies indicate the involvement of TGFβ1/TSP1/CD47/eNOS pathway in diabetic impairment of NO generation however involvement of other pathways cannot be ruled out. Support or Funding Information This study is partly supported by funding from NIH National Institute of Aging (AG056681).
Circulating CD34+ hematopoietic stem progenitor cells (HSPCs) stimulate vasculogenesis and play an important role in the ischemic vascular repair. Long‐term diabetes is associated with impaired vasculogenic potential of HSPCs, which is at least in part due to decreased nitric oxide (NO) generation. Transforming growth factor‐ β1 (TGF‐β1) has pleiotropic functions in CD34+ HSPCs and is known to stimulate the expression of matrix protein thrombospondin‐1 (TSP‐1). Previous studies have shown that transient silencing of TGF‐β1 restores NO generation and improves migratory functions in diabetic CD34+ HSPCs. In this study, we tested the hypothesis that diabetic dysfunction in NO generation is mediated by activation of TSP‐1/CD47 receptor pathway. CD34+ HSPCs were isolated from peripheral blood mononuclear cells obtained from either male or female nondiabetic (ND, n=63) or diabetic (type 1 and type 2) (DB, n=51) individuals of age 38–85 years. TGF‐β1 expression was transiently blocked by using TGFβ1‐antisense delivered in the form of phosphorodiamidate morpholino oligomer (PMO‐TGFβ1). CD47 expression was blocked by siRNA approach. Migration and proliferation of cells were determined by chemotaxis assay and BrdU‐colorimetric ELISA, respectively. Activation of eNOS was evaluated by determining phosphorylation at Ser1177 and Thr495 by using fluorescent‐conjugated antibodies and flow cytometry. Expression of TGF‐β1 and TSP‐1 mRNA were higher in DB CD34+ cells compared to ND cells, which were decreased by PMO‐TGFβ1 (n=18). SDF‐induced migration and proliferation were impaired in DB cells compared to ND (P<0.05, n=5) that were reversed by PMO‐TGFβ1 (n=5). TSP‐1 decreased SDF‐induced migration and proliferation (P<0.05, n=6) that were reversed by knockdown of CD47 (n=6). CD47 siRNA restored SDF‐induced migration and proliferation in diabetic cells in the absence or in the presence of TSP‐1 (P<0.05, n=5). Diabetic cells showed decreased p‐eNOS‐Ser1177 and higher p‐eNOS‐Thr495 in response to SDF compared to ND cells (P<0.05, n=5). TSP‐1 decreased SDF‐induced changes in pSer1177 and pThr495 in ND cells (n=5) that were reversed by CD47 siRNA (n=5). In summary, diabetic impairment of eNOS activation and NO generation are mediated by TGF‐β1/TSP‐1/CD47 pathway.
Previously, we demonstrated that a transient blockade of endogenous transforming growth factor-beta type 1 (TGF-β1) in murine and human HSC accelerates bone marrow engraftment while dramatically reducing the number of HSC needed for long-term reconstitution. CD34+ cells give rise to endothelial progenitor cells (EPC) and have been shown to participate in the repair of damaged vasculature. CXCR4 is the receptor for stromal derived factor (SDF-1), a chemoattractant released by ischemic tissue that guides EPCs to damaged sites. In this study, we examined levels of TGF- β1 mRNA in CD34+ blood and bone marrow (BM) cells of diabetic and non diabetic individuals. We also treated CD34+ cells of diabetic and nondiabetic origin with antisense phosphorodiamidate morpholino oligomers (PMOs) to TGF-β1 and examined surface expression of CXCR-4, migratory response to SDF-1 as well as in vivo reparative function in a model of retinal ischemic injury. Our results show that CD34+ EPC from the blood of diabetic patients are markedly defective in their ability to repair damaged retinal vessels compared to control cells in contrast to CD34+ cells from the diabetic BM. Diabetic CD34+EPC from blood express elevated levels of TGF-β1 mRNA and have a blunted migratory response to SDF-1 compared to controls (p<0.05 and p<0.01 respectively). Transient (2–4 days) blockade of endogenous TGF-β1 using PMOs to TGF- β1 in diabetic CD34+/EPC increases CXCR-4 expression in these cells, enhances their migratory prowess and restores their ability to repair damaged retinal vessels. This approach is an enhanced autologous stem cell therapy based on a well studied, rapid and reversible modification of bone marrow CD34+EPCs derived from the diabetic patient.
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