Diabetic CD34 Research Articles

The role of telomerase reverse transcriptase in the mitochondrial protective functions of Angiotensin-(1–7) in diabetic CD34+ cells

Angiotensin (Ang)-(1–7) stimulates vasoprotective functions of diabetic (DB) CD34+ hematopoietic stem/progenitor cells partly by decreasing reactive oxygen species (ROS), increasing nitric oxide (NO) levels and decreasing TGFβ1 secretion. Telomerase reverse transcriptase (TERT) translocates to mitochondria and regulates ROS generation. Alternative splicing of TERT results in variants α-, β- and α-β-TERT, which may oppose functions of full-length (FL) TERT. This study tested if the protective functions of Ang-(1–7) or TGFβ1-silencing are mediated by mitoTERT and that diabetes decreases FL-TERT expression by inducing splicing.CD34+ cells were isolated from the peripheral blood mononuclear cells of nondiabetic (ND, n = 68) or DB (n = 74) subjects. NO and mitoROS levels were evaluated by flow cytometry. TERT splice variants and mitoDNA-lesions were characterized by qPCR. TRAP assay was used for telomerase activity. Decoy peptide was used to block mitochondrial translocation (mitoXTERT).TERT inhibitor or mitoXTERT prevented the effects of Ang-(1–7) on NO or mitoROS levels in DB-CD34+ cells. FL-TERT expression and telomerase activity were lower and mitoDNA-lesions were higher in DB cells compared to ND and were reversed by Ang-(1–7) or TGFβ1-silencing. The prevalence of TERT splice variants, with predominant β-TERT expression, was higher and the expression of FL-TERT was lower in DB cells (n = 25) compared to ND (n = 30). Ang-(1–7) or TGFβ1-silencing decreased TERT-splicing and increased FL-TERT. Blocking of β-splicing increased FL-TERT and protected mitoDNA in DB-cells.The findings suggest that diabetes induces TERT-splicing in CD34+ cells and that β-TERT splice variant largely contributes to the mitoDNA oxidative damage.

Transient silencing of TGF‐β1 reverses myelopoietic bias in older diabetic CD34+ cells

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.

Transforming growth factor-β1/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- 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.

Abstract P141: Downregulation Of Deletion Variants Of Telomerase Reverse Transcriptase (TERT) By Transforming Growth Factor-β1-silencing Decreases Mitochondrial ROS In Diabetic CD34 + Cells

Telomerase reverse transcriptase (TERT) modulates mitochondrial levels of reactive oxygen species (mitoROS). Deletion variants, α-, β- and αβ, are negative regulators of TERT. Increased ROS levels in diabetic CD34 + cells impair vasoreparative functions. Our recent studies showed that silencing of transforming growth factor-β1 (TGF-β1) decreased ROS levels and stimulated vasoreparative functions. This study tested the hypothesis that prevalence of TERT deletion variants is higher in diabetic CD34 + cells and that TGF-β1-silencing decreases mitoROS by downregulation of the variants. CD34 + cells derived from either male or female nondiabetic (ND) (n=38) or diabetic (DB), both type 1 and type 2, (n=43) subjects were studied. Phosphorodiamidate morpholino oligomers (PMO) were used for TGF-β1-silencing. TERT variants were characterized by qPCR and agarose gel electrophoresis. MitoSOX-flow cytometry and ELISA were used for determining mitoROS levels and telomerase activity, respectively. TGF-β1-silencing decreased mitoROS levels in DB-CD34+ cells (P&lt;0.01 vs ND-cells, n=6). This effect was reversed by TERT inhibitor, BIBR1532 (1μM) (n=6) or mito-X-TERT, a decoy peptide that prevents mitochondrial translocation of TERT (n=5). TERT expression was higher in DB compared to ND cells (P&lt;0.05, n=18) but telomerase activity was lower (P&lt;0.05 vs ND, n=10). Prevalence of TERT deletion variants was higher among DB cells compared to ND (DB - 8/10 vs ND - 2/10 subjects have one or more of α, β, or αβ variants). TGF-β1-silencing downregulated the expression of TERT variants and increased full-length TERT that was accompanied by increased telomerase activity in DB-CD34+ cells (P&lt;0.05 vs untreated, n=5). This study suggests that diabetes is associated with higher prevalence of TERT variants that would impede mitochondrial functions of TERT. TGF-β1-silencing decreases mitoROS levels at least in part by downregulation of TERT variants.

Diabetic Impairment of Nitric Oxide Generation in CD34 + Hematopoietic Stem/ Progenitor Cells is mediated by TGF‐β1/TSP‐1/CD47 pathway

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.

ACE2 gene transfer ameliorates vasoreparative dysfunction in CD34+ cells derived from diabetic older adults.

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.

Abstract P241: Involvement of Mitochondrial Telomerase Reverse Transcriptase in the Restoration of Nitric Oxide Levels by Angiotensin-(1-7) in the Dysfunctional Diabetic CD34 + Cells

Telomerase reverse transcriptase (TERT) has been shown to translocate to mitochondria (mtTERT), and decrease the generation of mitochondrial reactive oxygen species (mtROS). Angiotensin (Ang)-(1-7) stimulates vasoreparative functions in diabetic CD34 + stem/progenitor cells in part by decreasing ROS levels and increasing nitric oxide (NO) bioavailability. This study tested the involvement of mtTERT in mediating the effect of Ang-(1-7) on NO levels in diabetic CD34 + cells. 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) (DB: HbA1C 6.5-11.2). Cells were treated with Ang-(1-7) (100nM) or Stromal derived factor-1α (SDF) and evaluated for NO, and mtROS levels by flow cytometry by using DAF-FAM and mitoSOX, respectively. Number of cells used was ≥5x10 4 per treatment. The Mean Fluorescence Intensity is expressed in arbitrary fluorescence units (AFIx10 5 ). Inhibitors of Mas receptor and TERT, A779 and BIBR-1532, respectively, and decoy peptides that inhibit mitochondrial translocation or nuclear transport of TERT, mtXTERT and nucXTERT, respectively, were used. SDF (100nM) failed to stimulate NO generation in DB-cells (1.2±0.07 vs basal 0.8±0.05, n=10) compared to ND-cells (3.5±0.2 vs basal 1.6±0.2) (n=10, P&lt;0.0001). Ang-(1-7) induced NO levels in ND- (2.9±0.2) and restored NO generation in DB-cells (2.2±0.1) (n=10). DB-cells have increased mitoROS (3.6±0.4) compared to ND-cells (1.7±0.1, P&lt;0.001, n=7) and Ang-(1-7) normalized the mitoROS in DB-cells (1.8±0.3, n=7). The effects of Ang-(1-7) on NO and mitoROS levels were reversed by A-779 or BIBR-1532 (n=5). Decoy peptide mtXTERT not nucXTERT increased mitoROS in ND- (4.4±0.7 vs basal 1.8±0.2, P&lt;0.01) and in DB-cells (12±1 vs basal 6±0.4, P&lt;0.001) (n=5). The decreasing effect of Ang-(1-7) on mitoROS (1.6±0.3) was reversed by mtXTERT (10±0.9, P&lt;0.001, n=5) but not by nucXTERT. Along similar lines, restoration of NO levels by Ang-(1-7) in DB-cells was reversed by mtXTERT but not by nucXTERT. These observations provide compelling evidence for the involvement of TERT in stimulating the vasoreparative functions of diabetic stem/progenitor cells by Ang-(1-7)/Mas pathway.

Quality-Quantity Control Culture Enhances Vasculogenesis and Wound Healing Efficacy of Human Diabetic Peripheral Blood CD34+ Cells.

Autologous endothelial progenitor cell (EPC) therapy is commonly used to stimulate angiogenesis in ischemic repair and wound healing. However, low total numbers and functional deficits of EPCs make autologous EPC therapy ineffective in diabetes. Currently, no known ex vivo culture techniques can expand and/or ameliorate the functional deficits of EPCs for clinical usage. Recently, we showed that a quality‐quantity culture (QQc) system restores the vasculogenic and wound‐healing efficacy of murine diabetic EPCs. To validate these results and elucidate the mechanism in a translational study, we evaluated the efficacy of this QQc system to restore the vasculogenic potential of diabetic human peripheral blood (PB) CD34+ cells. CD34+ cells purified from PB of diabetic and healthy patients were subjected to QQc. Gene expression, vascular regeneration, and expression of cytokines and paracrine mediators were analyzed. Pre‐ or post‐QQc diabetic human PB‐CD34+ cells were transplanted into wounded BALB/c nude mice and streptozotocin‐induced diabetic mice to assess functional efficacy. Post‐QQc diabetic human PB‐CD34+ cell therapy significantly accelerated wound closure, re‐epithelialization, and angiogenesis. The higher therapeutic efficacy of post‐QQc diabetic human PB‐CD34+ cells was attributed to increased differentiation ability of diabetic CD34+ cells, direct vasculogenesis, and enhanced expression of angiogenic factors and wound‐healing genes. Thus, QQc can significantly enhance the therapeutic efficacy of human PB‐CD34+ cells in diabetic wounds, overcoming the inherent limitation of autologous cell therapy in diabetic patients, and could be useful for treatment of not only wounds but also other ischemic diseases. Stem Cells Translational Medicine 2018;7:428–438

Increase in acid sphingomyelinase level in human retinal endothelial cells and CD34+ circulating angiogenic cells isolated from diabetic individuals is associated with dysfunctional retinal vasculature and vascular repair process in diabetes

Diabetic retinopathy is a microvascular disease that results from retinal vascular degeneration and defective repair due to diabetes-induced endothelial progenitor dysfunction. Understanding key molecular factors involved in vascular degeneration and repair is paramount for developing effective diabetic retinopathy treatment strategies. We propose that diabetes-induced activation of acid sphingomyelinase (ASM) plays essential role in retinal endothelial and CD34+ circulating angiogenic cell (CAC) dysfunction in diabetes. Human retinal endothelial cells (HRECs) isolated from control and diabetic donor tissue and human CD34+ CACs from control and diabetic patients were used in this study. ASM messenger RNA and protein expression were assessed by quantitative polymerase chain reaction and enzyme-linked immunosorbent assay, respectively. To evaluate the effect of diabetes-induced ASM on HRECs and CD34+ CACs function, tube formation, CAC incorporation into endothelial tubes, and diurnal release of CD34+ CACs in diabetic individuals were determined. ASM expression level was significantly increased in HRECs isolated from diabetic compared with control donor tissue, as well as CD34+ CACs and plasma of diabetic patients. A significant decrease in tube area was observed in HRECs from diabetic donors compared with control HRECs. The tube formation deficiency was associated with increased expression of ASM in diabetic HRECs. Moreover, diabetic CD34+ CACs with high ASM showed defective incorporation into endothelial tubes. Diurnal release of CD34+ CACs was disrupted with the rhythmicity lost in diabetic patients. Collectively, these findings support that diabetes-induced ASM upregulation has a marked detrimental effect on both retinal endothelial cells and CACs.

Editorial: Cell-Based Therapies for Diabetic Microvascular Complications.

Editorial: Cell-Based Therapies for Diabetic Microvascular Complications.

Vasoreparative Dysfunction of CD34+ Cells in Diabetic Individuals Involves Hypoxic Desensitization and Impaired Autocrine/Paracrine Mechanisms

We hypothesized that endothelial progenitor cells derived from individuals with diabetes would exhibit functional defects including inability to respond to hypoxia and altered paracrine/autocrine function that would impair the angiogenic potential of these cells. Circulating mononuclear cells isolated from diabetic (n = 69) and nondiabetic (n = 46) individuals were used to grow endothelial colony forming cells (ECFC), early endothelial progenitor cells (eEPCs) and isolate CD34+ cells. ECFCs and eEPCs were established from only 15% of the diabetic individuals tested thus directing our main effort toward examination of CD34+ cells. CD34+ cells were plated in basal medium to obtain cell-free conditioned medium (CM). In CM derived from CD34+ cells of diabetic individuals (diabetic-CM), the levels of stem cell factor, hepatocyte growth factor, and thrombopoietin were lower, and IL-1β and tumor necrosis factor (TNFα) levels were higher than CM derived from nondiabetic individuals (nondiabetic-CM). Hypoxia did not upregulate HIF1α in CD34+ cells of diabetic origin. Migration and proliferation of nondiabetic CD34+ cells toward diabetic-CM were lower compared to nondiabetic-CM. Attenuation of pressure-induced constriction, potentiation of bradykinin relaxation, and generation of cGMP and cAMP in arterioles were observed with nondiabetic-CM, but not with diabetic-CM. Diabetic-CM failed to induce endothelial tube formation from vascular tissue. These results suggest that diabetic subjects with microvascular complications exhibit severely limited capacity to generate ex-vivo expanded endothelial progenitor populations and that the vasoreparative dysfunction observed in diabetic CD34+ cells is due to impaired autocrine/paracrine function and reduced sensitivity to hypoxia.

Enhancing the Function of CD34+ Cells by Targeting Plasminogen Activator Inhibitor-1

Previously, we showed that transient inhibition of TGF- β1 resulted in correction of key aspects of diabetes-induced CD34+ cell dysfunction. In this report, we examine the effect of transient inhibition of plasminogen activator inhibitor-1 (PAI-1), a major gene target of TGF-β1 activation. Using gene array studies, we examined CD34+ cells isolated from a cohort of longstanding diabetic individuals, free of microvascular complications despite suboptimal glycemic control, and found that the cells exhibited reduced transcripts of both TGF-β1 and PAI-1 compared to age, sex, and degree of glycemic control-matched diabetic individuals with microvascular complications. CD34+ cells from diabetic subjects with microvascular complications consistently exhibited higher PAI-1 mRNA than age-matched non-diabetic controls. TGF- β1 phosphorodiamidate morpholino oligo (PMO) reduced PAI-1 mRNA in diabetic (p<0.01) and non-diabetic (p=0.05) CD34+ cells. To reduce PAI-1 in human CD34+ cells, we utilized PAI-1 siRNA, lentivirus expressing PAI-1 shRNA or PAI-1 PMO. We found that inhibition of PAI-1 promoted CD34+ cell proliferation and migration in vitro, likely through increased PI3(K) activity and increased cGMP production. 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 PAI-1-PMO treatment compared with control PMO-treated cells. Targeting PAI-1 offers a promising therapeutic strategy for restoring vascular reparative function in defective diabetic progenitors.

Circulating Mononuclear Progenitor Cells: Differential Roles for Subpopulations in Repair of Retinal Vascular Injury

We examined effect on retinal vascular homing of exogenous CD34(+) and CD14(+) progenitor cells using mouse models of chronic (streptozotocin [STZ]-induced diabetes) and acute (ischemia-reperfusion [I/R]) ocular vascular injury. STZ-treated mice of short or long duration (≤4, ≥11 months) diabetes, along with age- and sex-matched controls, were given intravitreous injections of human CD34(+) and CD14(+) cells isolated from healthy or diabetic donors alone or in combination. I/R injured mice were given diabetic or nondiabetic CD34(+) cells with mesenchymal stem cells (MSCs) or diabetic CD34(+) cells manipulated by ex vivo fucosylation with ASC-101. Injected cells were localized by fluorescent immunocytochemistry, and the degree of retinal vascular colocalization quantified morphometrically. Permeability was assessed by fluorescent albumin leakage. Diabetic CD14(+) cells associated with vessels to a greater degree than diabetic CD34(+) cells. Vascular permeability was reduced only by nondiabetic cells and only at the highest number of cells tested. Diabetic CD34(+) cells consistently demonstrated reduced migration. There was a 2-fold or 4-fold increase over control in the specific localization of diabetic CD34(+) cells within the vasculature when these cells were co-administered with MSCs or ex vivo fucosylated prior to injection, respectively. Diabetic CD14(+) cells, unlike diabetic CD34(+) cells, retain robust homing characteristics. CD34(+) or CD14(+) subsets rather than whole bone marrow or peripheral blood cells may prove more beneficial in autologous cell therapy for diabetics. Co-administration with MSCs or ex vivo fucosylation may enhance utility of CD34(+) cells in cell therapy for diabetic ocular conditions like macular ischemia and retinal nonperfusion.

Blockade of NADPH Oxidase Restores Vasoreparative Function in Diabetic CD34+Cells

The vasodegenerative phase of diabetic retinopathy is likely caused by endothelial dysfunction and reduced endothelial repair. Migration of endothelial progenitor cells (EPCs) into areas of vascular injury is critical to vascular repair. This key function, often defective in diabetes, is largely mediated by nitric oxide (NO), which is known to be inactivated by superoxide produced by NADPH oxidase. The authors tested the hypothesis that either increasing eNOS expression or inhibiting NADPH oxidase would restore the reparative function in diabetic EPCs. Peripheral blood was obtained from healthy (n = 27) and diabetic (n = 31) persons, and CD34(+) cells were isolated. Expression and activation of eNOS and NADPH oxidase and intracellular levels of NO, superoxide, and peroxynitrite were evaluated. cGMP production and migration to SDF-1α were also determined. Reparative function was evaluated in a mouse model of retinal ischemia-reperfusion injury. Diabetic EPCs demonstrate reduced eNOS expression and decreased NO bioavailability and migration in response to SDF-1α. Increasing eNOS expression in diabetic cells by AVE3085 resulted in increased peroxynitrite levels and, therefore, did not enhance NO-mediated functions in vitro and in vivo. Expression of Nox2, NADPH oxidase activity, and superoxide levels were higher in diabetic than in nondiabetic EPCs. Pretreatment with apocynin or gp91ds-tat increased NO bioavailability without increasing eNOS activity in response to SDF-1α. Ex vivo NADPH oxidase inhibition in diabetic cells restored migratory function in vitro and enhanced their homing to ischemic retinal vasculature in vivo. The NADPH oxidase system is a promising target for correcting vasoreparative dysfunction in diabetic EPCs.

Differential Expression of Transforming Growth Factor Beta Receptor 2 (TGFβR2) In Diabetic CD34+ Cells: Implications for Vascular Repair

AbstractAbstract 4795We recently reported that the vascular reparative ability of diabetic CD34+ cells can be restored by transient inhibition of transforming growth factor β1 (TGF-b1) using antisense phosphorodiamidate morpholino oligomers (TGF-β1-PMO) (Bhatwadekar 2010). TGF-β1 mediates its action by binding to TGF-β-R2 receptor homodimers, which then form heterotetrameric complexes with two TGF-βR1 subunits. We investigated whether healthy or diabetic CD34+ cells differentially express TGFβR2 receptor and whether inhibition or knock-out of endogenous TGF-β1 expression would affect the surface expression of TGF-βR2.TGF-βR2 expression of murine bone marrow (BM) lin- Sca-1+/− cells from TGF-β1 knockout mice or wild type mice was studied using flow cytometry. Unexpectedly, TGF-β-R2 expression was essentially absent in BM lin- Sca-1 +and - cells from TGF-β1 knockout mice compared to wild type cells. Because the absence of endogenous and exogenous TGF-β1 resulted in complete down regulation of TGF-βR2 in primitive murine hematopoietic cells, we explored the relationship of endogenous TGF-β1in human mature and immature CD34+ cells in blood, bone marrow and cord blood.Human CD34+CD45+ CD38+ or - cells from healthy and diabetic individuals were FACS sorted based on surface expression the analysed for TGF-βR2 expression. At the same time, TGF-βR2 and TGF-β1 mRNA expression was determined for sorted populations using quantitative real time RT-PCR.A comparison of TGF-βR2 surface expression between mature progenitors (lin- CD34+ CD45+ CD38+) from diabetic and healthy cord blood revealed that TGF-βR2 expression did not differ between these groups. In contrast, TGF-βR2 surface expression in immature cells (CD34+ CD45+ CD38-) was very low in diabetic patients compared to healthy controls (p = 0.001).Next, we inhibited endogenous TGF-β1 in healthy CD34+ cells which resulted in a significant (p<0.01) reduction in TGF-β-R2 mRNA expression. However, in diabetic CD34 cells, TGF β1-PMO treatment abolished TGFβR2 expression.Our study suggests that healthy and diabetic CD34+ cells differentially regulate TGF-βR2 expression which may be the result of the elevated intracellular and extracellular levels of TGFb1 in diabetic individuals. A reduction in TGF-βR2 expression in diabetic CD34+ CD38- cells may physiologically enhance proliferation of the primitive CD34+ CD38- cell compartment, an outcome that may be necessary to keep up with increased demand for CD34+ cells in the diabetic individual. Disclosures:Stepps: BetaStem Therapuetics: Employment. Bartelmez: BetaStem Therapeutics: Employment, Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties, Research Funding.

Activation of the Protective Arm of Renin Angiotensin System (RAS) Corrects the Reparative Dysfunction of Diabetic CD34+ Cells.

AbstractAbstract 2637 Purpose:RAS plays a vital role in regulating many physiological processes of the vascular system. Angiotensin II (Ang II), a product of angiotensin converting enzyme (ACE), mediates its effects through activation of either the AT1 receptor – to induce vasoconstriction, proliferation, fibrosis, and inflammation – or the AT2 receptor to promote NO generation. The protective arm of RAS involves ACE2, which produces angiotensin-(1-7) [Ang-(1-7)]. Ang-(1-7) activates the MAS receptor to promote vascular health. Because diabetic endothelial progenitor cells are dysfunctional and this limits their utility in autologous cell therapy, we asked whether angiotensin (Ang)-(1-7) could restore the vasoreparative function of diabetic CD34+cells. Methods:Healthy nondiabetic (ND) and diabetic (D) Lin−CD45midCD34+ cells were obtained from peripheral blood mononuclear cells (PB-MNCs) by FACS. The effect of Ang-(1-7) on migration, proliferation, NO bioavailability, reactive oxygen species (ROS) levels and NADPH oxidase activity were evaluated in ND- and D-CD34+ cells. The effect of Ang-(1-7) on the formation of ECFCs from ND- and D-MNCs was evaluated. Ang-(1-7) production by cells was analyzed and the expression of ACE2 and Mas-receptor were assessed by real-time PCR and flow cytometry. Effects of ACE2 activators XNT and DIZE in CD34+ cells were also evaluated. D-CD34+ cells were genetically modified to overexpress Ang-(1-7) by lentiviral Ang-(1-7)-fusion transgene and their function was evaluated in vitro. The effect of transduction on the surface expression of CD133, CD34 and CD309 was assessed. In vivo homing function was assessed in a mouse model of ischemia-reperfusion (I/R). After one week of I/R insult, when retinal capillary damage was appreciable, CD34 cells were intravitreally injected. Neural retinas were harvested after 48 hours and human cells within the mouse vasculature were localized by immunohistochemistry. Results:Migration to SDF1- and VEGF-were impaired in D-CD34+ cells. In contrast, Ang-(1-7)-induced migration in both D-CD34+ and ND-CD34+cells was dependent on Mas receptor expression and eNOS. ROS levels and NADPH oxidase activity were reduced and proliferation and NO bioavailability were restored in D-CD34+ cells by Ang-(1-7). ECFCs from D-MNCs were appeared only in the presence of Ang-(1-7). Migration, NO release and Ang-(1-7) release by ACE2 activators, XNT and DIZE, were significantly decreased in D-CD34+ cells. Ang-(1-7) release and ACE2 expression were decreased in D-cells while Mas-receptor expression was similar that in ND-cells. Ang-(1-7) gene-modified cells showed reduced ROS levels, increased NO bioavailability, enhanced migration to SDF-1 and proliferation. Lentiviral transduction did not alter surface expression of CD133, CD34 and CD309. Ang-(1-7)-overexpression restored the homing efficiency of D-CD34+ cells similar to that of ND-CD34+ cells in vivo. Conclusions:Ang-(1-7) stimulates the vasoreparative functions in CD34+ cells. Ang-(1-7) bypasses the reduced ACE2 seen in diabetic CD34+ cells and restores the vasoreparative potential of these cells by decreasing oxidative stress and normalizing NO bioavailability. Pharmacological strategies that either increase ACE2 or Ang-(1-7) in diabetic CD34+ cells will improve their therapeutic utility for autologous cell therapy in treatment of diabetic complications. Disclosures:No relevant conflicts of interest to declare

Inhibition of Plasminogen Activator Inhibitor (PAI)-1 Corrects Diabetic CD34+ Dysfunction.

AbstractAbstract 1601 Objective:The dysfunction of human diabetic CD34+ endothelial progenitor cells limits their utility in autologous cell therapy for vascular complications. Previously, we showed that transient inhibition of transforming growth factor-beta 1 (TGF-β1) enhances vascular reparative function of human CD34+ cells isolated from diabetics (Bhatwadekar et al, 2010). Expression of PAI-1, the major gene product of TGF-β1 activation, is increased by high glucose and insulin exposure in endothelial cells and PAI-1 has been shown to be increased in the serum of diabetics. We asked whether the beneficial effects of TGF-β1 blockade on CD34+ cells function were mediated by inhibition of PAI-1 and whether blocking of PAI-1 could correct diabetes associated dysfunction of these cells. Research Design and Methods:Plasma determinations of PAI-1 and TGF-β1 (both measured by ELISA) were compared in type 2 (n=17) and type 1 (n=7) diabetic patients. CD34+ cells from these individuals were isolated and analyzed for cell survival (in the presence and absence of growth factors), cell proliferation, cell cycle analysis and migration. The effect of TGF-β1 phosphorodiamidate morpholino oligomers (PMO) treatment on PAI-1 level was determined in CD34+ cells. In CD34+ cells, PAI-1 was blocked using either lentivirus expressing PAI-1 shRNA or PAI-1 siRNA. In vivo homing ability of PAI-1 inhibited CD34+ cells was assessed using an ocular model of ischemia/reperfusion (I/R) Injury. Results:Plasma PAI-1 level was increased in type 2 diabetic patients compared to type 1 (p<0.05) and directly correlated with TGF-β1 plasma levels (r= 0.44). TGF-β1 PMO treatment resulted in a reduction of PAI-1 mRNA expression (p=0.0018 in diabetic, p=0.05 in non-diabetic). PAI-1 blockade promoted EPC proliferation in vitro and bypassed the inhibitory effect of TGF-β1 on cell survival (p<0.001) even in the absence of growth factors. PAI-1 blockade enhanced the migration of these cells in response to SDF-1α in (p<0.01) compared to cells treated with scrambled siRNA and improved the in vivo re-endothelialization by CD34+ cells in the I/R model. Conclusions:Our results suggest that the cytostatic activity of TGF-β1 in CD34+ cells is mediated largely through PAI-1. Blocking PAI-1 corrects multiple defects in CD34+ cells from type 2 diabetic patients. This approach may offer a promising therapeutic strategy for restoring vascular reparative function in diabetic cells and facilitate their use in autologous cell therapy. Disclosures:No relevant conflicts of interest to declare.

Transient Inhibition of Transforming Growth Factor-β1 in Human Diabetic CD34+ Cells Enhances Vascular Reparative Functions

OBJECTIVEPeripheral 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-β, a key factor that modulates stem cell quiescence, is increased in the serum of type 2 diabetic patients. We asked whether transient TGF-β1 inhibition in CD34+ cells would improve their reparative ability.RESEARCH DESIGN AND METHODSTo inhibit TGF-β1 protein expression, CD34+ cells were treated ex vivo with antisense phosphorodiamidate morpholino oligomers (TGF-β1-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.RESULTSTGF-β1-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-β1-PMO treatment compared with control PMO–treated cells.CONCLUSIONSTransient inhibition of TGF-β1 may represent a promising therapeutic strategy for restoring the reparative capacity of dysfunctional diabetic CD34+ cells.

Inhibition of NADPH oxidase restores vasoreparative function in diabetic CD34+ cells

We have previously showed that inhibition of NADPH oxidase corrected diabetic migratory defect in circulating putative endothelial progenitor cells (CD34+) by decreasing the production of reactive oxygen species (ROS) thereby increasing intracellular nitric oxide (NO). In this study, we evaluated the effect of NADPH oxidase blockade on the vascular engraftment potential of diabetic CD34+ cells. Peripheral blood CD34+ cells were isolated from healthy and Type 1‐diabetic individuals by immunomagnetic selection. Endothelial nitric oxide synthase (eNOS) activity, intracellular NO and cGMP production were quantified. Vascular engraftment potential of CD34+ cells was evaluated in a mouse model of retinal ischemia/reperfusion injury. Activation of eNOS in response to stromal derived factor‐1 (SDF1, 100 nM) was lower in diabetic compared to non‐diabetic cells. Treatment with apocynin or gp91ds‐tat has no effect on eNOS activity but increased intracellular NO levels and cGMP production in response to SDF1. In mouse retinal I/R injury model, vascular incorporation of diabetic CD34+ cells followed by intravitreal injection was significantly lower compared to that with non‐diabetic cells and was significantly increased after ex vivo treatment with apocynin or gp91ds‐tat. These results suggest that ex vivo NADPH oxidase inhibition increases the vascular engraftment potential of diabetic CD34+ cells.

Transient Inhibition of Transforming Growth Factor-β1 in Human Blood-Derived CD34+ Cells Enhances Survival and Proliferation in Vitro and Vascular Reparative functions In Vivo.

Abstract 3546Poster Board III-483CD34+ cells from diabetic patients demonstrate reduced vascular reparative function due to decreased proliferation as well as diminished migration prowess which is largely due to lower levels of bioavailable nitric oxide (NO). We asked whether a transient TGF-β1 blockade in CD34+ cells from diabetics would improve their reparative ability given that TGF-β is a key factor modulating stem cell quiescence. Peripheral blood lin-CD34+ cells or lin-CD34+CD38+/− cells were treated ex vivo with antisense phosphorodiamidate morpholino oligomers (TGF-β1 -PMO), demonstrated to inhibit TGF-β1 protein expression in stem cells. Cells were then analyzed for cell surface TGF-β Receptor 2 (TGF-β R2) and CXCR4 expression, their ability to generate NO, their ability to migrate toward SDF-1, their ability to survive in the absence of added growth factors, and tested in vivo for their vascular reparative ability. After TGF-β1-PMO treatment, healthy and diabetic CD34+CD38+ and - cells downregulated TGF-βR2, upregulated CXCR4 expression, survived in the absence of added growth factors ex vivo and migrated more efficiently to SDF-1 compared to controls. TGF-β1-PMO treated diabetic CD34+ cells restored NO production to non-diabetic levels. In contrast, TGF-β1-PMO did not enhance NO generation in CD34+ cells from healthy subjects. Using an in vivo retinal ischemia reperfusion model, we observed that TGF-β1-PMO treatment increased the ability of both healthy and diabetic CD34+ cells to home to injured capillaries compared to control PMO treated cells. As also observed in our current study, a reduction of TGF-β1 levels in murine hematopoietic stem cells correlates with a reduction in TGF-βR2 expression which may induce proliferation in vivo. We also show that both diabetic and healthy lin-CD34+CD38+ cells express TGF-βR2 by FACS. In contrast, only healthy lin-CD34+CD38- cells expressTGF-βR2 while diabetic lin-CD34+CD38 - cells express essentially no cell surface TGF-βR2 (<5 % of cells are TGF-βR2+). Our results suggest that a transient blockade of TGF-β1 may represent a promising therapeutic strategy in restoring vascular reparative function in diabetic CD34+ cells. Disclosures:Bartelmez:BetaStem Therapeutics: Employment, Equity Ownership, Head, SRB, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties, Research Funding. Iversen:AVI-Biopharma: Employment, Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties, Research Funding.