Shujia Jiang

IGF-1–Overexpressing Mesenchymal Stem Cells Accelerate Bone Marrow Stem Cell Mobilization via Paracrine Activation of SDF-1α/CXCR4 Signaling to Promote Myocardial Repair

We hypothesized that mesenchymal stem cells (MSCs) overexpressing insulin-like growth factor (IGF)-1 showed improved survival and engraftment in the infarcted heart and promoted stem cell recruitment through paracrine release of stromal cell–derived factor (SDF)-1α. Rat bone marrow–derived MSCs were used as nontransduced (NormMSCs) or transduced with adenoviral-null vector (NullMSCs) or vector encoding for IGF-1 (IGF-1MSCs). IGF-1MSCs secreted higher IGF-1 until 12 days of observation (P<0.001 versus NullMSCs). Molecular studies revealed activation of phosphoinositide 3-kinase, Akt, and Bcl.xL and inhibition of glycogen synthase kinase 3β besides release of SDF-1α in parallel with IGF-1 expression in IGF-1MSCs. For in vivo studies, 70 &mgr;L of DMEM without cells (group 1) or containing 1.5×106NullMSCs (group 2) or IGF-1MSCs (group 3) were implanted intramyocardially in a female rat model of permanent coronary artery occlusion. One week later, immunoblot on rat heart tissue (n=4 per group) showed elevated myocardial IGF-1 and phospho-Akt in group 3 and higher survival of IGF-1MSCs (P<0.06 versus NullMSCs) (n=6 per group). SDF-1α was increased in group 3 animal hearts (20-fold versus group 2), with massive mobilization and homing of ckit+, MDR1+, CD31+, and CD34+ cells into the infarcted heart. Infarction size was significantly reduced in cell transplanted groups compared with the control. Confocal imaging after immunostaining for myosin heavy chain, actinin, connexin-43, and von Willebrand factor VIII showed extensive angiomyogenesis in the infarcted heart. Indices of left ventricular function, including ejection fraction and fractional shortening, were improved in group 3 as compared with group 1 (P<0.05). In conclusion, the strategy of IGF-1 transgene expression induced massive stem cell mobilization via SDF-1α signaling and culminated in extensive angiomyogenesis in the infarcted heart.

Ischemic Preconditioning Augments Survival of Stem Cells via miR-210 Expression by Targeting Caspase-8-associated Protein 2

MicroRNAs (miRs) participate in most cellular functions by posttranscriptional regulation of gene expression albeit with little information regarding their role in ischemic preconditioning (IP) of stem cells. We report that IP of bone marrow-derived mesenchymal stem cells (MSCs) with two cycles of 30-min ischemia/reoxygenation (I/R) supported their survival under subsequent longer exposure to anoxia and following engraftment in the infarcted heart. IP significantly reduced apoptosis in MSCs through activation of Akt (Ser473) and ERK1/2 (Thr202/Tyr204) and nuclear translocation of hypoxia-inducible factor-1α (HIF-1α). We observed concomitant induction of miR-210 in the preconditioned MSCs (PCMSCs). Inhibition of HIF-1α or of miR-210 abrogated the cytoprotective effects of preconditioning. Extrapolation of these data to in vivo studies in a rat model of acute myocardial infarction predominantly improved stem cell survival after engraftment with a role for miR-210. Notably, multiple I/R cycles more effectively regulated the miR-210 and hence promoted MSC survival compared with single-cycle hypoxia of an equal duration. Real time PCR array for rat apoptotic genes, computational target gene analyses, and luciferase reporter assay identified FLICE-associated huge protein (FLASH)/caspase-8-associated protein-2 (Casp8ap2) in PCMSCs as the target gene of miR-210. Induction of FLASH/CASP8AP2 in miR-210 knocked-down PCMSCs resulted in increased cell apoptosis. Taken together, these data demonstrated that cytoprotection afforded by IP was regulated by miR-210 induction via FLASH/Casp8ap2 suppression. These results highlighted that IP by multiple short episodes of I/R is a novel strategy to promote stem cell survival.

Pharmacologically Preconditioned Skeletal Myoblasts Are Resistant to Oxidative Stress and Promote Angiomyogenesis via Release of Paracrine Factors in the Infarcted Heart

Strategies to enhance skeletal myoblast (SkM) survival after transplantation in the ischemic heart have achieved little success. We posit that preconditioned (PC) SkMs show improved survival and promote repair of the infarcted myocardium via paracrine signaling after transplantation. SkMs from male Fischer-344 rats (rSkMs) were PC for 30 minutes with 200 &mgr;mol/L diazoxide. Treatment of PC rSkMs with 100 &mgr;mol/L H2O2 for 2 hours resulted in significantly reduced cell injury, as shown by lactate dehydrogenase–release assay, and prevented apoptosis, as demonstrated by cytochrome c translocation, TUNEL, annexin V staining, and preservation of mitochondrial membrane potential. PC rSkMs expressed elevated phospho-Akt (1.85-fold), basic fibroblast growth factor (1.44-fold), hepatocyte growth factor (2.26-fold), and cyclooxygenase-2 (1.33-fold) as compared with non-PC rSkMs. For in vivo studies, female Fischer-344 rats after permanent coronary artery ligation were grouped (n=12/group) to receive 80 &mgr;L of basal medium without rSkMs (group 1) or containing 1.5×106 non-PC (group 2) or PC (group 3) rSkMs. Real-time PCR for sry gene 4 days after transplantation (n=4/group) showed 1.93-fold higher survival of rSkMs in group 3 as compared with group 2. Four weeks later, echocardiography revealed improved indices of left ventricular function, including ejection fraction and fractional shortening in group 3 (P<0.02) as compared with groups 1 and 2. Blood vessel count per surface area (at ×400 magnification) was highest in scar and periscar areas in group 3 as compared with the other groups (P<0.05). We conclude that activation of signaling pathways of preconditioning in SkMs promoted their survival by release of paracrine factors to promote angiomyogenesis in the infarcted heart. Transplantation of PC SkMs for heart cell therapy is an innovative approach in the clinical perspective.

Supportive Interaction Between Cell Survival Signaling and Angiocompetent Factors Enhances Donor Cell Survival and Promotes Angiomyogenesis for Cardiac Repair

Akt is a major cell survival and angiogenic mediator downstream of angiopoietin-1 (Ang-1)/Tie-2 signaling pathway. We hypothesize that transplantation of mesenchymal stem cells (MSCs) co-overexpressing Ang-1 and Akt lead to better prognosis. Ang-1 and Akt genes were adenovirally transduced into MSCs from male Fischer rats. Cytoprotective effects of transgene overexpression in vitro were assessed by exposure of cells to 8 hours of anoxia. TUNEL and measurement of lactate dehydrogenase showed that MSCs co-overexpressing Ang-1 and Akt (MAAs) were more resistant to anoxia as compared with the nontransduced MSCs or those transduced with Ang-1 or Akt alone. For in vivo studies, after permanent coronary artery occlusion, animals were grouped (n=20/group) to receive intramyocardial injections of 70 &mgr;L of basal medium without cells (group 1) or containing 3×106 nontransduced MSCs (group 2) or MAAs (group 3). Four animals per group were euthanized on 4, 7, and 14 days after cell transplantation for molecular studies. Extensive survival of MAAs was observed in group 3, which continued to co-overexpress transgenes in rat heart at 2 weeks after cell transplantation. Immunohistology at 4 weeks revealed myogenic differentiation of donor cells at the site of cell graft. Blood vessel density was highest in the infarct and periinfarct regions in group 3 (P<0.05). Echocardiography at 4 weeks showed that heart function indices were significantly improved in group 3 (P<0.05), including ejection fraction and fractional shortening as compared with groups 1 and 2. We conclude that supportive interaction between Ang-1 and Akt during MSC transplantation gave better prognosis via enhanced cell survival, improved angiomyogenesis, and restored global cardiac function.

Sca-1+ Stem Cell Survival and Engraftment in the Infarcted Heart: Dual Role for Preconditioning-Induced Connexin-43

Background— We report that elevated connexin-43 (Cx-43) in stem cells preconditioned with insulin-like growth factor-1 (IGF-1) is cytoprotective and reprograms the cells for cardiomyogenic differentiation. Methods and Results— Sca-1+ cells were preconditioned with 100 nmol/L IGF-1 for 30 minutes followed by 8 hours of oxygen glucose deprivation to assess the cytoprotective effects of preconditioning. LDH release assay, cytochrome c release, and mitochondrial membrane potential assay showed improved survival of preconditioned Sca-1+ cells under oxygen glucose deprivation compared with nonpreconditioned Sca-1+ cells via PI3K/Akt-dependent caspase-3 downregulation. We observed PI3K/Akt-dependent upregulation of cardiac-specific markers including MEF-2c (2.5-fold), GATA4 (3.1-fold), and Cx-43 (3.5-fold). Cx-43 inhibition with specific RNA interference reduced cell survival under oxygen glucose deprivation and after transplantation. In vivo studies were performed in a female rat model of acute myocardial infarction (n=78). Animals were grouped to receive intramyocardially 70 &mgr;L Dulbecco modified Eagles medium without cells (group 1) or containing male 1×106 nonpreconditioned Sca-1+ cells (group 2) or preconditioned Sca-1+ (group 3) cells labeled with PKH26. Survival of the preconditioned Sca-1+ cells was 5.5-fold higher in group 3 compared with group 2 at 7 days after transplantation. Confocal imaging after actinin and Cx-43 specific immunostaining showed extensive engraftment and myogenic differentiation of preconditioned Sca-1+ cells. Compared with group 2, group 3 showed increased blood vessel density (22.3±1.7 per microscopic field; P<0.0001) and attenuated infarction size (23.3±3.6%; P=0.002). Heart function indices including ejection fraction (56.2±3.5; P=0.029) and fractional shortening (24.3±2.1; P=0.03) were improved in group 3 compared with group 2. Conclusions— Preconditioning with IGF-1 reprograms Sca-1+ for prosurvival signaling and cardiomyogenic differentiation with an important role for Cx-43 in this process.

Transcriptional profiling of young and old mesenchymal stem cells in response to oxygen deprivation and reparability of the infarcted myocardium

Most clinical studies have used autologous bone marrow (BM) stem cells for myocardial regeneration in elderly patients. We hypothesize that aging impairs the survival and differentiation potential of BM stem cells thus limiting their therapeutic efficacy. BM-derived MSCs from young ((Yng)MSCs; 8-12 weeks) and old ((Old)MSCs; 24-26 months) rats were purified and assessed for their responsiveness to anoxia and reparability of infarcted heart. Higher expression of angiogenic growth factors was observed by (Yng)MSCs under anoxia as compared to (Old)MSCs, cultured either alone or in co-culture ((Co-old)MSCs) with (Yng)MSCs. Likewise, (Yng)MSCs were more tolerant to apoptotic stimuli and showed higher ability to form tubular structures during in vitro Matrigel assay as compared to (Old)MSCs and (Co-old)MSCs with a possible role of p21 and p27 as contributory survival factors. For in vivo studies, acute myocardial infarction model was developed in Fischer-344 rats (n=38). The animals were grouped to receive 70 microl basal DMEM without cells (group 1) or containing 2 x 10(6)(Yng)MSCs (PKH67 labeled; group 2) or (Old)MSCs (PKH26 labeled; group 3) and mixture of (Yng)MSCs + (Old)MSCs (1 x 10(6) cells each; group 4). Histological studies revealed that by day 7, (Yng)MSCs showed elongated morphology with orientation similar to the host muscle architecture. Electron microscopy and confocal imaging after fluorescent immunostaining showed superior angiomyogenic potential of (Yng)MSCs. Echocardiography showed significantly preserved heart function indices in the animals transplanted with (Yng)MSCs. Aging impairs the responsiveness of (Old)MSCs to anoxia and their differentiation potential. (Yng)MSCs fail to alter the survival of (Old)MSCs under in vitro as well as in vivo conditions. It is therefore concluded that transplantation of stem cells from young donors would be a better option for heart cell therapy in future clinical studies.

Preconditioning Promotes Survival and Angiomyogenic Potential of Mesenchymal Stem Cells in the Infarcted Heart via NF-κB Signaling

We proposed that pharmacological manipulation of mesenchymal stem cells (MSCs) with diazoxide enhanced their survival and regenerative potential via NFkappaB regulation. MSCs preconditioned ((PC)MSCs) with diazoxide and later subjected to oxidant stress with 100 micromol/L H(2)O(2) either immediately or after 24 h exhibited higher survival (p < 0.01 vs nonpreconditioned MSCs; (Non-PC)MSCs) with concomitantly increased phosphorylation of PI3K, Akt, GSK3beta (cytoplasmic), and NF-kappaB (p65) (nuclear). Akt kinase activity was determined as a function of GSK3beta activity. Pretreatment of (PC)MSCs with Wortmannin (Wt), NEMO-binding domain (NBD), or NF-kappaB (p50) siRNA abolished NF-kappaB (p65) activity. Preconditioning increased NF-kappaB-dependent elevation of secretable growth factors associated with their paracrine effects. Inhibition of PI3K activity with Wt reduced (PC)MSCs viability at both early and 24 h time-points. However, inhibition of NF-kappaB reduced viability of (PC)MSCs only at 24 h time-point. For in vivo studies, DMEM without cells (group-1) or containing 1 x 10(6) male (Non-PC)MSCs (group-2), (PC)MSCs (group-3), (PC)MSCs pretreated with Wortmannin (group-4) or NF-kappaB decoy (group-5) were transplanted in a female rat model of acute myocardial infarction. Group-3 showed highest cell survival and growth factor expression, increased angiomyogenesis, and functional improvement. We conclude that activation of NF-kappaB by preconditioning promoted (PC)MSCs survival and angiomyogenic potential in the infarcted heart.

Reprogramming of skeletal myoblasts for induction of pluripotency for tumor-free cardiomyogenesis in the infarcted heart.

Rationale: Skeletal myoblasts (SMs) with inherent myogenic properties are better candidates for reprogramming to pluripotency. Objective: To reprogram SMs to pluripotency and show that reprogrammed SMs (SiPS) express embryonic gene and microRNA profiles and that transplantation of predifferentiated cardiac progenitors reduce tumor formation. Methods and Results: The pMXs vector containing mouse cDNAs for Yamanakas quartet of stemness factors were used for transduction of SMs purified from male Oct4-GFP+ transgenic mouse. Three weeks later, GFP+ colonies of SiPS were isolated and propagated in vitro. SiPS were positive for alkaline phosphatase, expressed SSEA1, and displayed a panel of embryonic stem (ES) cell–specific pluripotency markers. Embryoid body formation yielded beating cardiomyocyte-like cells, which expressed early and late cardiac-specific markers. SiPS also had an microRNA profile that was altered during their cardiomyogenic differentiation. Noticeable abrogation of let-7 family and significant up-regulation of miR-200a-c was observed in SiPS and SiPS-derived cardiomyocytes, respectively. In vivo studies in an experimental model of acute myocardial infarction showed extensive survival of SiPS and SiPS-derived cardiomyocytes in mouse heart after transplantation. Our results from 4-week studies in DMEM without cells (group 1), SMs (group-2), SiPS (group-3), and SiPS-derived cardiomyocytes (group 4) showed extensive myogenic integration of the transplanted cells in group 4 with attenuated infarct size and improved cardiac function without tumorgenesis. Conclusions: Successful reprogramming was achieved in SMs with ES cell-like microRNA profile. Given the tumorgenic nature of SiPS, their predifferentiation into cardiomyocytes would be important for tumor-free cardiogenesis in the heart.

Improved angiogenic response in pig heart following ischaemic injury using human skeletal myoblast simultaneously expressing VEGF165 and angiopoietin-1.

To achieve angiogenic interaction between VEGF165 and angiopoietin‐1 (Ang‐1) using a novel adenoviral bicistronic vector (Ad‐Bic) encoding the two factors and delivered ex vivo using sex‐mismatched human skeletal myoblasts.

MicroRNA-143 is a critical regulator of cell cycle activity in stem cells with co-overexpression of Akt and angiopoietin-1 via transcriptional regulation of Erk5/cyclin D1 signaling

We report that simultaneous expression of Akt and angiopoietin-1 (Ang-1) transgenes supported mitogenesis in stem cells with a critical role for microRNA-143 (miR-143) downstream of FoxO1 transcription factor. Mesenchymal stem cells (MSC) from young male rats were transduced with Ad-vectors encoding for Akt (AktMSC) and Ang-1 (Ang-1MSC) transgenes for their individual or simultaneous overexpression (AAMSC; > 5-fold gene level and > 4-fold Akt and Ang-1 protein expression in AAMSC vs. Ad-Empty transduced MSC; EmpMSC). AAMSC had higher phosphorylation of FoxO1, which activated Erk5, a distinct mitogen-induced MAPK that drove transcriptional activation of cyclin D1 and Cdk4. Flow cytometry showed > 10% higher S-phase cell population that was confirmed by BrdU assay (15%) and immunohistology for Ki67 (11%) in AAMSC using EmpMSC as controls. miR array supported by real-time PCR showed induction of miR-143 in AAMSC (4.73-fold vs.. EmpMSC). Luciferase assay indicated a dependent relationship between miR-143 and Erk5 in AAMSC. FoxO1-specific siRNA upregulated miR-143, whereas inhibition of miR-143 did not change FoxO1 activation. However, miR-143 inhibition repressed phosphorylation of Erk5 and abrogated cyclin D1 with concomitant reduction in cells entering cell cycle. During in vivo studies, male GFP+ AAMSC transplanted into wild-type female infarcted rat hearts showed significantly higher number of Ki67 expressing cells (p < 0.05 vs. EmpMSC) 7 days after engraftment (n = 4 animals/group). In conclusion, co-overexpression of Akt and Ang-1 in MSC activated cell cycle progression by upregulation of miR-143 and stimulation of FoxO1 and Erk5 signaling.