Transforming growth factor-ß1/Thrombospondin-1/CD47 axis mediates dysfunction in CD34+ cells derived from diabetic older adults.

Aging with diabetes is associated with impaired vasoprotective functions and decreased nitric oxide (NO) generation in CD34+ cells. Transforming growth factor- ß1 (TGF-ß1) is known to regulate hematopoietic functions. This study tested the hypothesis that transforming growth factor- ß1 (TGF-ß1) 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-ß1 was silenced by using an antisense delivered as phosphorodiamidate morpholino oligomer (PMO-TGF-ß1). 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-ß1 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. TGFß1-PMO decreased the secretion of TGF-ß1, 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-ß1-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-ß1 expression in CD34+ cells induces dysfunction in CD34+ cells from diabetic older adults via TSP-1/CD47-dependent inhibition of NO generation.

Reversal of Bone Marrow Mobilopathy and Enhanced Vascular Repair by Angiotensin-(1-7) in Diabetes.

The angiotensin (ANG)-(1-7)/Mas receptor (MasR) pathway activates vascular repair-relevant functions of bone marrow progenitor cells. We tested the effects of ANG-(1-7) on mobilization and vasoreparative functions of progenitor cells that are impaired in diabetes. The study was performed in streptozotocin-induced diabetic (db/db) mice. Diabetes resulted in a decreased number of Lineage-Sca-1+c-Kit+ (LSK) cells in the circulation, which was normalized by ANG-(1-7). Diabetes-induced depletion of LSK cells in the bone marrow was reversed by ANG-(1-7). ρ-Kinase (ROCK) activity was increased specifically in bone marrow LSK cells by ANG-(1-7) in diabetes, and the beneficial effects of ANG-(1-7) were prevented by fasudil. ANG-(1-7) increased Slit3 levels in the bone marrow supernatants, which activated ROCK in LSK cells and sensitized them for stromal-derived factor-1α (SDF)-induced migration. Diabetes prevented the mobilization of LSK cells in response to ischemia and impaired the recovery of blood flow, both of which were reversed by ANG-(1-7) in both models of diabetes. Genetic ablation of MasR prevented ischemia-induced mobilization of LSK cells and impaired blood flow recovery, which was associated with decreased proliferation and migration of LSK cells in response to SDF or vascular endothelial growth factor. These results suggest that MasR is a promising target for the treatment of diabetic bone marrow mobilopathy and vascular disease.

Transient inhibition of transforming growth factor-beta1 in human diabetic CD34+ cells enhances vascular reparative functions.

OBJECTIVE: Peripheral blood CD34(+) cells from diabetic patients demonstrate reduced vascular reparative function due to decreased proliferation and diminished migratory prowess, largely resulting from decreased nitric oxide (NO) bioavailability. The level of TGF-beta, a key factor that modulates stem cell quiescence, is increased in the serum of type 2 diabetic patients. We asked whether transient TGF-beta1 inhibition in CD34(+) cells would improve their reparative ability. RESEARCH DESIGN AND METHODS: To inhibit TGF-beta1 protein expression, CD34(+) cells were treated ex vivo with antisense phosphorodiamidate morpholino oligomers (TGF-beta1-PMOs) and analyzed for cell surface CXCR4 expression, cell survival in the absence of added growth factors, SDF-1-induced migration, NO release, and in vivo retinal vascular reparative ability. RESULTS: TGF-beta1-PMO treatment of diabetic CD34(+) cells resulted in increased expression of CXCR4, enhanced survival in the absence of growth factors, and increased migration and NO release as compared with cells treated with control PMO. Using a retinal ischemia reperfusion injury model in mice, we observed that recruitment of diabetic CD34(+) cells to injured acellular retinal capillaries was greater after TGF-beta1-PMO treatment compared with control PMO-treated cells. CONCLUSIONS: Transient inhibition of TGF-beta1 may represent a promising therapeutic strategy for restoring the reparative capacity of dysfunctional diabetic CD34(+) cells.

Autocrine transforming growth factor-beta regulation of hematopoiesis: many outcomes that depend on the context.

Transforming growth factor-beta (TGF-beta) is a pleiotropic regulator of all stages of hematopoieis. The three mammalian isoforms (TGF-beta1, 2 and 3) have distinct but overlapping effects on hematopoiesis. Depending on the differentiation stage of the target cell, the local environment and the concentration and isoform of TGF-beta, in vivo or in vitro, TGF-beta can be pro- or antiproliferative, pro- or antiapoptotic, pro- or antidifferentiative and can inhibit or increase terminally differentiated cell function. TGF-beta is a major regulator of stem cell quiescence, at least in vitro. TGF-beta can act directly or indirectly through effects on the bone marrow microenvironment. In addition, paracrine and autocrine actions of TGF-beta have overlapping but distinct regulatory effects on hematopoietic stem/progenitor cells. Since TGF-beta can act in numerous steps in the hematopoietic cascade, loss of function mutations in hematopoeitic stem cells (HSC) have different effects on hematopoiesis than transient blockade of autocrine TGF-beta1. Transient neutralization of autocrine TGF-beta in HSC has therapeutic potential. In myeloid and erythroid leukemic cells, autocrine TGF-beta1 and/or its Smad signals controls the ability of these cells to respond to various differentiation inducers, suggesting that this pathway plays a role in determining the cell fate of leukemic cells.

Haematopoietic stem cells do not transdifferentiate into cardiac myocytes in myocardial infarcts.

The mammalian heart has a very limited regenerative capacity and, hence, heals by scar formation. Recent reports suggest that haematopoietic stem cells can transdifferentiate into unexpected phenotypes such as skeletal muscle, hepatocytes, epithelial cells, neurons, endothelial cells and cardiomyocytes, in response to tissue injury or placement in a new environment. Furthermore, transplanted human hearts contain myocytes derived from extra-cardiac progenitor cells, which may have originated from bone marrow. Although most studies suggest that transdifferentiation is extremely rare under physiological conditions, extensive regeneration of myocardial infarcts was reported recently after direct stem cell injection, prompting several clinical trials. Here, we used both cardiomyocyte-restricted and ubiquitously expressed reporter transgenes to track the fate of haematopoietic stem cells after 145 transplants into normal and injured adult mouse hearts. No transdifferentiation into cardiomyocytes was detectable when using these genetic techniques to follow cell fate, and stem-cell-engrafted hearts showed no overt increase in cardiomyocytes compared to sham-engrafted hearts. These results indicate that haematopoietic stem cells do not readily acquire a cardiac phenotype, and raise a cautionary note for clinical studies of infarct repair.