Thymosin beta-4 improves endothelial function and reparative potency of diabetic endothelial cells differentiated from patient induced pluripotent stem cells

Background Prior studies show that signature phenotypes of diabetic human induced pluripotent stem cells derived endothelial cells (dia-hiPSC-ECs) are disrupted glycine homeostasis, increased senescence, impaired mitochondrial function and angiogenic potential as compared with healthy hiPSC-ECs. In the current study, we aimed to assess the role of thymosin β-4 (Tb-4) on endothelial function using dia-hiPSC-ECs as disease model of endothelial dysfunction. Methods and results Using dia-hiPSC-ECs as models of endothelial dysfunction, we determined the effect of Tb-4 on cell proliferation, senescence, cyto-protection, protein expression of intercellular adhesion molecule-1 (ICAM-1), secretion of endothelin-1 and MMP-1, mitochondrial membrane potential, and cyto-protection in vitro and angiogenic potential for treatment of ischemic limb disease in a mouse model of type 2 diabetes mellitus (T2DM) in vivo. We found that 600 ng/mL Tb4 significantly up-regulated AKT activity and Bcl-XL protein expression, enhanced dia-hiPSC-EC viability and proliferation, limited senescence, reduced endothelin-1 and MMP-1 secretion, and improved reparative potency of dia-hiPSC-ECs for treatment of ischemic limb disease in mice with T2DM. However, Tb4 had no effect on improving mitochondrial membrane potential and glycine homeostasis and reducing intercellular adhesion molecule-1 protein expression in dia-hiPSC-ECs. Conclusions Tb-4 improves endothelial dysfunction through enhancing hiPSC-EC viability, reducing senescence and endothelin-1 production, and improves angiogenic potency in diabetes.

Combination of Antioxidant Enzyme Overexpression and N‐Acetylcysteine Treatment Enhances the Survival of Bone Marrow Mesenchymal Stromal Cells in Ischemic Limb in Mice With Type 2 Diabetes

Background Therapy with mesenchymal stem cells remains a promising but challenging approach to critical limb ischemia in diabetes because of the dismal cell survival. Methods and Results Critical limb ischemia in type 2 diabetes mouse model was used to explore the impact of diabetic limb ischemia on the survival of bone marrow mesenchymal stromal cells (bMSCs). Inhibition of intracellular reactive oxygen species was achieved with concomitant overexpression of superoxide dismutase (SOD)‐1 and glutathione peroxidase‐1 in the transplanted bMSCs, and extracellular reactive oxygen species was attenuated using SOD‐3 overexpression and N‐acetylcysteine treatment. In vivo optical fluorescence imaging and laser Doppler perfusion imaging were used to track cell retention and determine blood flow in diabetic ischemic limb, respectively. Survival of the transplanted bMSCs was significantly decreased in diabetic ischemic limb compared with the control. In vitro study indicated that advanced glycation end products, not high glucose, significantly decreased the proliferation of bMSCs and increased their apoptosis associated with increased reactive oxygen species production and selective reduction of SOD‐1 and SOD‐3. In vivo study demonstrated that concomitant overexpression of SOD‐1, SOD‐3, and glutathione peroxidase‐1, or host treatment with N‐acetylcysteine, significantly enhanced in vivo survival of transplanted bMSCs, and improved critical limb ischemia in diabetic mice. Combination of triple antioxidant enzyme overexpression in bMSCs with host N‐acetylcysteine treatment further improved bMSC survival with enhanced circulatory and functional recovery from diabetic critical limb ischemia. Conclusions Simultaneous suppression of reactive oxygen species from transplanted bMSCs and host tissue could additively enhance bMSC survival in diabetic ischemic limb with increased therapeutic efficacy in diabetes.

O6 Analysis of angiogenic gene profiling after E-selectin + stem cell therapy in murine ischemic limb

Introduction There remains a paucity of novel therapeutics for limb salvage in patients with critical limb ischemia (CLI) for whom revascularization procedures have failed and amputation is imminent. We have shown that E-selectin+/Mesenchymal Stem Cell (MSC) injections into the ischemic limb tissue of a CLI mouse model improves revascularization and limb function. Thus, we sought to determine a mechanism of action for E-selectin+/MSC’s pro-angiogenic and tissue salvage properties. Methods MSC were extracted from donor mice bone marrow and subsequently engineered via viral transduction with E-selectin-ires-GFP/AAV and GFP/AAV (control) to create E-selectin-GFP+/MSC vs GFP+/MSC. Intramuscular injections of E-selectin-GFP+/MSC, GFP+/MSC, or PBS were performed in a mouse model of hindlimb ischemia. Laser doppler imaging (LDI), confocal laser microscopy, and treadmill exhaustion test were utilized to determine neovascularization and limb function. RNA extraction from engineered MSC (E-selectin-GFP+/MSC vs GFP+/MSC) and ischemic hindlimb tissues treated with E-selectin-GFP+/MSC vs GFP+/MSC was performed, followed by RT2 Profiler PCR Array analysis of 84 genes involved in angiogenesis. GFP+/MSC treated hindlimb tissue served as control. Student’s t-test or ANOVA was utilized to compare means and significance set at P < 0.05. Results Compared with GFP+/MSC and PBS, treatment with E-selectin-GFP+/MSC increased ischemic leg LDI reperfusion (54% vs. 39% vs. 22%, P < 0.001), treadmill distance traversed (162 m vs. 111 m vs. 110 m, P < 0.01) and ischemic mouse footpad vessel density (23% vs. 14% vs. 14%, P < 0.01). RT2 Profiler PCR Array demonstrated pro-angiogenic gene upregulation occurred in 7 genes (Csf3, Cxcl2, Cxcl5, Serpine1, F2, Lep, Tbx1, Table I.) in E-selectin-GFP+/MSC treated ischemic leg tissue while tumour necrosis factor (TNF) was found to be downregulated, when compared with GFP+/MSC treated tissues. Of these 7 upregulated genes, CXCl2, F2, Leptin and T-box1 (Table I.) are likely produced by E-selectin-GFP+/MSC, as analysis of cellular gene expression profiles of E-selectin-GFP+/MSC also revealed upregulation by 2-fold or more in these factors when compared to GFP+/MSC. Validation of gene functions in-vivo are under investigation. Conclusion Stem cell therapy using E-selectin-GFP+/MSC, in a murine model of CLI, confers both augmented postnatal neovascularization and increased limb function. The pro-angiogenic and pro-repair effects are likely mediated by upregulation of a panel of chemokines/cytokines and down-regulation of TNF in ischemic tissues treated with E-selectin-GFP+/MSC.

TRANSIENT ISCHEMIC ATTACK AS THE INITIAL PRESENTATION OF TAKAYASU ARTERITIS IN A YOUNG WOMAN: A CASE REPORT

Takayasu arteritis (TA) is a challenging chronic inflammatory vasculitis of large vessels with unknown etiology. It usually affects the aorta and its main branches and in few cases it affects other arteries like pulmonary and coronary. We report a case of a young woman with transient ischemic attack (TIA) as the initial presentation followed later by ischemic limb syndrome and then ischemic stroke. Lack of specific diagnostic biomarkers for this disease makes early diagnosis and treatment challenging.

Imaging Hydrogen Sulfide in Hypoxic Tissue with [99mTc]Tc-Gluconate

Hydrogen sulfide (H2S) is the third gasotransmitter and is generated endogenously in hypoxic or inflammatory tissues and various cancers. We have recently demonstrated that endogenous H2S can be imaged with [99mTc]Tc-gluconate. In the present study, we detected H2S generated in hypoxic tissue, both in vitro and in vivo, using [99mTc]Tc-gluconate. In vitro uptake of [99mTc]Tc-gluconate was measured under hypoxic and normoxic conditions, using the colon carcinoma cell line CT26, and was higher in hypoxic cells than that in normoxic cells. An acute hindlimb ischemia-reperfusion model was established in BALB/c mice by exposing the animals to 3 h of ischemia and 3 h of reperfusion prior to in vivo imaging. [99mTc]Tc-gluconate (12.5 MBq) was intravenously injected through the tail vein, and uptake in the lower limb was analyzed by single-photon emission computed tomography/computed tomography (SPECT/CT). SPECT/CT images showed five times higher uptake in the ischemic limb than that in the normal limb. The standard uptake value (SUVmean) of the ischemic limb was 0.39 ± 0.03, while that of the normal limb was 0.07 ± 0.01. [99mTc]Tc-gluconate is a novel imaging agent that can be used both in vitro and in vivo for the detection of endogenous H2S generated in hypoxic tissue.