Diabetes increases the risk for ischemic vascular diseases, which is further elevated in older adults. Bone marrow-derived hematopoietic CD34+ stem/progenitor cells have the potential of revascularization; however, diabetes attenuates vasoreparative functions. Angiotensin-converting enzyme 2 (ACE2) is the vasoprotective enzyme of renin–angiotensin system in contrast with the canonical angiotensin-converting enzyme (ACE). The present study tested the hypothesis that diabetic dysfunction is associated with ACE2/ACE imbalance in hematopoietic stem/progenitor cells (HSPCs) and that increasing ACE2 expression would restore reparative functions. Blood samples from male and female diabetic (n=71) or nondiabetic (n=62) individuals were obtained and CD34+ cells were enumerated by flow cytometry. ACE and ACE2 enzyme activities were determined in cell lysates. Lentiviral (LV) approach was used to increase the expression of soluble ACE2 protein. Cells from diabetic older adults (DB) or nondiabetic individuals (Control) were evaluated for their ability to stimulate revascularization in a mouse model of hindlimb ischemia (HLI). DB cells attenuated the recovery of blood flow to ischemic areas in nondiabetic mice compared with that observed with Control cells. Administration of DB cells modified with LV-ACE2 resulted in complete restoration of blood flow. HLI in diabetic mice resulted in poor recovery with amputations, which was not reversed by either Control or DB cells. LV-ACE2 modification of Control or DB cells resulted in blood flow recovery in diabetic mice. In vitro treatment with Ang-(1-7) modified paracrine profile in diabetic CD34+ cells. The present study suggests that vasoreparative dysfunction in CD34+ cells from diabetic older adults is associated with ACE2/ACE imbalance and that increased ACE2 expression enhances the revascularization potential.
Bone marrow‐derived hematopoietic stem progenitor cells (HSPCs) have vasoreparative functions and are physiologically mobilized into circulation. Aging or diabetes increases risk for cardiovascular diseases and are known to be associated with impaired HSPC functions. Angiotensin converting enzyme (ACE) and ACE2 are primary enzymes of cardiovascular detrimental and protective axes of renin‐angiotensin system (RAS). Angiotensin‐(1‐7) is generated largely by degradation of Ang II by ACE2 and produces vasoprotective functions by activating Mas receptor. We have shown that Ang‐(1‐7) reverses diabetic dysfunction in murine and human HSPCs. This study tested the hypothesis that aging healthy or with diabetes is associated with imbalance in the vascular protective and detrimental axes of RAS in HSPCs. Peripheral blood samples were obtained from either male or female healthy (n=23) or diabetic (n=21), both type 1 and type 2, individuals of age ranging from 25 to 87 years. Lineage‐negative (Lin−) or CD34+ HSPCs were enriched from mononuclear cells. ACE and ACE2 activities in the cell lysates were determined by using enzyme‐specific fluorogenic substrates. Enzyme activities are expressed as arbitrary fluorescence units that is inhibitable by the enzyme‐selective inhibitors of ACE or ACE2, captopril and MLN‐4760, respectively. ACE2/ACE activity ratios in HSPCs in different individuals are plotted against the respective ages. ACE2/ACE is negatively correlated with the age in healthy (Pearson's r=−0.88, P<0.0001) and in diabetic individuals (Pearson's r=−0.82, P<0.0001). As predicted, a negative correlation was observed with ACE2/ACE vs HbA1C (Pearson's r=−0.899, P<0.0001). These results imply that vascular protective axis of RAS is dysfunctional in HSPCs of aging individuals either healthy or diabetic, which is likely to be an underlying mechanism of the impaired reparative functions in HSPCs. Molecular or pharmacological approaches that enhance ACE2 activity or expression could be of great promise in reversing the age‐ or diabetes‐associated dysfunctions and restore cardiovascular health.Support or Funding InformationNational Institute of General Medical Sciences (NIGMS), and National Institute of Aging (AG056881)This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Hematopoietic stem/progenitor cells (HS/PCs) have the propensity of ischemic vascular repair, and this innate vasoprotective function is impaired in diabetes. Angiotensin-converting enzyme (ACE) and ACE-2 are primary enzymes of the cardiovascular-detrimental and protective axes of the renin-angiotensin system, respectively. In this study, we tested the hypothesis that diabetic dysfunction in HS/PCs is due to ACE2/ACE-imbalance, and that increasing ACE2 expression will restore the vasoreparative potential. Lineage- (Lin - ) or CD34 + cells were isolated from the peripheral blood mononuclear cells (MNCs) of nondiabetic subjects (ND), and type 1 or type 2 diabetic (DB) patients (male or female, age 48-76 years, HbA1C 6.5-11.2). ACE and ACE2 activities were measured in lysates of MNCs, Lin - and CD34 + cells by enzyme-selective fluorogenic substrates and inhibitors. Lentiviral ACE2 (LV-ACE2) gene transfer was carried out by spinoculation. Reparative function of Lin - cells was evaluated in Foxn1 nu mice undergoing hind limb ischemia (HLI). Lin - cells with or without ACE2-overexpression were administered (i.m) in the peri-ischemic region, and the blood flow recovery was monitored. Circulating Lin - or CD34 + cells are lower in DB (Lin - ((2±0.2)х10 5 , P<0.04) and CD34 + ((0.6±0.1)х10 3 , P<0.02), n=8) compared to ND group (Lin - (25±4)х10 5 ; CD34 + (2±0.4)х10 3 per 10 6 MNCs, n=8). This was associated with decrease in ACE2 and increase in ACE activity, resulting in 4.5-fold decrease in ACE2/ACE ratio in DB-CD34 + cells (0.4±0.07 vs ND 1.8±0.2, P<0.01, n=6). This was negatively correlated with HbA1C (r 2 =0.89, P<0.01). Administration of ND-Lin - cells has no effect on the blood flow recovery of mice following HLI. In contrast, administration of DB-Lin - cells decreased this recovery (48±6%, n=5, P<0.001 vs ND-Lin - 105±7%). However, treatment with ACE2-overexpressing DB-Lin - cells robustly enhanced blood flow recovery (112±8%, n=4, P<0.001 vs DB). These observations suggest that ACE2/ACE imbalance is correlated with diabetic vasoreparative dysfunction in HS/PCs, and that increasing ACE2 expression reverses the dysfunction. ACE2 gene transfer is a promising approach for enhancing vascularization outcomes of cell-based therapies in diabetic individuals.
Adult circulating CD34+ stem progenitor cells (HSPCs) participate in vascular repair by stimulating angiogenic functions of the endothelium via paracrine mechanisms. Aging and diabetes are associated with reduced number and impaired vasculogenic potential of CD34+cells. Previous studies showed that diabetic dysfunction was largely due to the paracrine pro‐inflammatory and anti‐angiogenic switch in the phenotype of CD34+ cells. Furthermore, diabetic cells have a higher potential to generate pro‐inflammatory monocyte‐macrophages. We have recently shown that older‐diabetic (DB) cells have increased expression of transforming growth factor β1 (TGF‐β1) compared to the age‐matched nondiabetic (ND) cells. Transient silencing of this pleiotropic regulator restored reparative functions such as migration, proliferation, and nitric oxide generation. The current study tested the hypothesis that TGF‐β1‐silencing reverses myelopoietic bias and paracrine pro‐inflammatory phenotype in CD34+ cells derived from diabetic older adults. Expression of alarmins and receptor for advanced glycation end‐products (RAGE) were evaluated. CD34+ cells were isolated from either male or female non‐diabetic (ND) (n=31) or diabetic (DB), both type 1 and type 2, (n=39) subjects. Phosphorodiamidate morpholino oligomers (PMO) were used for TGF‐β1‐silencing. Circulating CD34+cells and monocyte‐macrophages were enumerated by flow cytometry in both groups. Gene‐expression assays were carried out by qPCR. Myelopoiesis was determined by CFU‐GM assay followed by characterization of monocytes and macrophages by flow cytometry. Circulating CD34+ cells (p<0.05, n=31) and classical monocytes (anti‐inflammatory, CD14++CD16‐) were lower (p<0.05, n=16) whereas non‐classical (pro‐inflammatory, CD14lowCD16++) monocytes (P<0.05, n=16) were higher in DB compared to the ND group. However, no differences were observed in the intermediate monocyte and macrophage populations in circulation between the groups. DB‐CD34+ cells generated a higher number of colonies in the CFU‐GM assay than ND cells. Nonclassical monocytes were higher in DB‐CFUs (p<0.05 vs ND‐CFUs, n=4) with no difference in classical or intermediate monocytes. A higher number of pro‐inflammatory (CCR2+CD68+) (p<0.05, n=4) and lower number of anti‐inflammatory (CX3CR1+CD206+) macrophages (p<0.05, n=4) were detected in DB‐CFUs compared to that observed in ND‐CFUs. This imbalance was not observed in DB‐cells that were modified with TGF β1‐PMO. Gene expression of S100A8/S100A9, S100A10, S100A14, S100P, HMGb1 and RAGE were higher (p<0.05, n=5) in DB CD34+ cells compared to the ND. These changes were reversed by TGFβ1‐PMO. RAGE‐antagonist recapitulated the effects of TGFβ1‐PMO in assays for myelopoiesis. This study suggests that silencing of TGF‐β1 is a promising approach for enhancing the revascularization potential of CD34+cells from diabetic older adults by reversing the paracrine pro‐inflammatory functions that would negatively impact vascular repair.
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