Aims/hypothesis We sought to determine the impact of longstanding type 1 diabetes on haematopoietic stem/progenitor cell (HSC) number and function and to examine the impact of modulating glycoprotein (GP)130 receptor in these cells. Methods Wild-type, gp130−/− and GFP chimeric mice were treated with streptozotocin to induce type 1 diabetes. Bone marrow (BM)-derived cells were used for colony-formation assay, quantification of side population (SP) cells, examination of gene expression, nitric oxide measurement and migration studies. Endothelial progenitor cells (EPCs), a population of vascular precursors derived from HSCs, were compared in diabetic and control mice. Cytokines were measured in BM supernatant fractions by ELISA and protein array. Flow cytometry was performed on enzymatically dissociated retina from gfp+ chimeric mice and used to assess BM cell recruitment to the retina, kidney and blood. Results BM cells from the 12-month-diabetic mice showed reduced colony-forming ability, depletion of SP-HSCs with a proportional increase in SP-HSCs residing in hypoxic regions of BM, decreased EPC numbers, and reduced eNos (also known as Nos3) but increased iNos (also known as Nos2) and oxidative stress-related genes. BM supernatant fraction showed increased cytokines, GP130 ligands and monocyte/macrophage stimulating factor. Retina, kidney and peripheral blood showed increased numbers of CD11b+/CD45hi/CCR2+/Ly6Chi inflammatory monocytes. Diabetic gp130−/− mice were protected from development of diabetes-induced changes in their HSCs. Conclusions/interpretation The BM microenvironment of type 1 diabetic mice can lead to changes in haematopoiesis, with generation of more monocytes and fewer EPCs contributing to development of microvascular complications. Inhibition of GP130 activation may serve as a therapeutic strategy to improve the key aspects of this dysfunction.

Autologous CD34+ cells are widely used for vascular repair; however, in individuals with diabetes and microvascular disease these cells are dysfunctional. In this study, we examine expression of the clock genes Clock, Bmal, Per1, Per2, Cry1, and Cry2 in CD34+ cells of diabetic and nondiabetic origin and determine the small encoding RNA (miRNA) profile of these cells. The degree of diabetic retinopathy (DR) was assessed. As CD34+ cells acquired mature endothelial markers, they exhibit robust oscillations of clock genes. siRNA treatment of CD34+ cells revealed Per2 as the only clock gene necessary to maintain the undifferentiated state of CD34+ cells. Twenty-five miRNAs targeting clock genes were identified. Three of the miRNAs (miR-18b, miR-16, and miR-34c) were found only in diabetic progenitors. The expression of the Per2-regulatory miRNA, miR-92a, was markedly reduced in CD34+ cells from individuals with DR compared with control subjects and patients with diabetes with no DR. Restoration of miR-92a levels in CD34+ cells from patients with diabetes with DR reduced the inflammatory phenotype of these cells and the diabetes-induced propensity toward myeloid differentiation. Our studies suggest that restoring levels of miR-92a could enhance the usefulness of CD34+ cells in autologous cell therapy.

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.

4795 We 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.

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.