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1.  Rosuvastatin Enhances Angiogenesis via eNOS-Dependent Mobilization of Endothelial Progenitor Cells 
PLoS ONE  2013;8(5):e63126.
Circulating endothelial progenitor cells (circEPCs) of bone marrow (BM) origin contribute to postnatal neovascularization and represent a potential therapeutic target for ischemic disease. Statins are beneficial for ischemia disease and have been implicated to increase neovascularization via mechanisms independent of lipid lowering. However, the effect of Statins on EPC function is not completely understood. Here we sought to investigate the effects of Rosuvastatin (Ros) on EPC mobilization and EPC-mediated neovascularization during ischemic injury. In a mouse model of surgically-induced hindlimb ischemia (HLI), treatment of mice with low dose (0.1 mg/kg) but not high dose (5 mg/kg) significantly increased capillary density and accelerated blood flow recovery, as compared to saline-treated group. When HLI was induced in mice that had received Tie2/LacZ BM transplantation, Ros treatment led a significantly larger amount of endothelial cells (ECs) of BM origin incorporated at ischemic sites than saline. After treatment of mice with a single low dose of Ros, circEPCs significantly increased from 2 h, peaked at 4 h, declined until 8 h. In a growth-factor reduced Matrigel plug-in assay, Ros treatment for 5 d induced endothelial lineage differentiation in vivo. Interestingly, the enhanced circEPCs and post-HLI neovascularization stimulated by Ros were blunted in mice deficient in endothelial nitric oxide synthase (eNOS), and Ros increased p-Akt/p-eNOS levels in EPCs in vitro, indicating these effects of Ros are dependent on eNOS activity. We conclude that Ros increases circEPCs and promotes their de novo differentiation through eNOS pathway.
PMCID: PMC3660394  PMID: 23704894
2.  A novel two-step procedure to expand Sca-1+ cells clonally 
Resident cardiac stem cells (CSCs) are characterized by their capacity to self-renew in culture, and are multi-potent for forming normal cell types in hearts. CSCs were originally isolated directly from enzymatically digested hearts using stem cell markers. However, long exposure to enzymatic digestion can affect the integrity of stem cell markers on the cell surface, and also compromise stem cell function. Alternatively resident CSCs can migrate from tissue explant and form cardiospheres in culture. However, fibroblast contamination can easily occur during CSC culture. To avoid these problems, we developed a two-step procedure by growing the cells before selecting the Sca1+ cells and culturing in cardiac fibroblast conditioned medium, they avoid fibroblast overgrowth.
PMCID: PMC2140194  PMID: 17577582
Stem cells; Differentiation; cell transplantation; myocardial infarction
3.  Contrasting roles of E2F2 and E2F3 in endothelial cell growth and ischemic angiogenesis 
The growth of new blood vessels after ischemic injury requires endothelial cells (ECs) to divide and proliferate, and the E2F transcription factors are key regulators of the genes responsible for cell-cycle progression; however, the specific roles of individual E2Fs in ECs are largely unknown. To determine the roles of E2F2 and E2F3 in EC proliferation and the angiogenic response to ischemic injury, hind-limb ischemia was surgically induced in E2F2−/− mice, endothelial-specific E2F3-knockout (EndoE2F3Δ/Δ) mice, and their littermates with wild-type E2F2 and E2F3 expression. Two weeks later, laser-Doppler perfusion measurements, capillary density, and endothelial proliferation were significantly greater in E2F2−/− mice and significantly lower in EndoE2F3Δ/Δ mice than in their littermates, and EndoE2F3Δ/Δ mice also developed toe and limb necrosis. The loss of E2F2 expression was associated with increases in the proliferation and G1/S-phase gene expression of isolated ECs, while the loss of E2F3 expression led to declines in these parameters. Thus E2F2 impairs, and endothelial E2F3 promotes, the angiogenic response to peripheral ischemic injury through corresponding changes in EC cell-cycle progression.
PMCID: PMC3684263  PMID: 23603666
E2F; Endothelial cells; Proliferation; Angiogenesis; Ischemia
4.  CXCR4 mediated bone marrow progenitor cell maintenance and mobilization are modulated by c-kit activity 
Circulation research  2010;107(9):1083-1093.
The mobilization of bone-marrow (BM) progenitor cells (PCs) is largely governed by interactions between stromal–cell derived factor 1 (SDF-1) and CXC-chemokine receptor 4 (CXCR4). Ischemic injury disrupts the SDF-1–CXCR4 interaction and releases BM PCs into the peripheral circulation, where the mobilized cells are recruited to the injured tissue and contribute to vessel growth. BM PCs can also be mobilized by the pharmacological CXCR4 antagonist AMD3100, but the other components of the SDF-1–CXCR4 signaling pathway are largely unknown. c-kit, a membrane bound tyrosine-kinase and the receptor for stem cell factor, has also been shown to play a critical role in BM PC mobilization and ischemic tissue repair.
To investigate the functional interaction between SDF-1–CXCR4 signaling and c-kit activity in BM PC mobilization.
Methods and Results
AMD3100 administration failed to mobilize BM PCs in mice defective in c-kit kinase activity or in mice transplanted with BM cells that expressed a constitutively active c-kit mutant. Furthermore, BM levels of phosphorylated c-kit (phospho–c-kit) declined after AMD3100 administration and after CXCR4 deletion. In cells adhering to culture plates coated with vascular cell adhesion molecule 1 (VCAM-1), SDF-1 and SCF increased phospho–c-kit levels, and AMD3100 treatment suppressed SDF-1–induced, but not SCF-induced, c-kit phosphorylation. SDF-1–induced c-kit phosphorylation also required the activation of Src non-receptor tyrosine kinase: pre-treatment of cells with a selective Src inhibitor blocked both c-kit phosphorylation and the interaction between c-kit and phosphorylated Src.
These findings indicate that the regulation of BM PC trafficking by SDF-1 and CXCR4 is dependent on Src-mediated c-kit phosphorylation.
PMCID: PMC2966940  PMID: 20847314
CXCR4; c-kit; Integrin; Stem cells; Bone marrow; Niche; Mobilization; Homing
5.  Regulation of Vascular Contractility and Blood Pressure by the E2F2 Transcription Factor 
Circulation  2009;120(13):1213-1221.
Recent studies have identified a polymorphism in the ECE-1b promoter (−338C/A) that is strongly associated with hypertension in women, and the polymorphism is located in a consensus binding sequence for the E2F family of transcription factors. E2F proteins are crucially involved in cell-cycle regulation, but their roles in cardiovascular function are poorly understood. Here, we investigated the potential role of E2F2 in blood pressure (BP) regulation.
Methods and Results
Tail-cuff measurements of systolic and diastolic BP were significantly higher in E2F2-null (E2F2−/−) mice than in their wild-type (WT) littermates, and in ex vivo ring assays, aortas from the E2F2−/− mice exhibited significantly greater contractility in response to big endothelin-1 (BigET-1). BigET-1 is activated by endothelin converting enzyme-1 (ECE-1), and mRNA levels of ECE-1b, the repressive ECE-1 isoform, were significantly lower in E2F2−/− mice than in WT mice. In endothelial cells, chromatin-immunoprecipitation (ChIP) assays confirmed that E2F2 binds the ECE-1b promoter, and promoter-reporter assays indicated that E2F2 activates ECE-1b transcription. Furthermore, loss or downregulation of E2F2 led to a decline in ECE-1b levels, to higher levels of the membranous ECE-1 isoforms (i.e., ECE-1a, -1c, and -1d), and to deregulated ECE-1 activity. Lastly, Sam68 co-immunopreciptated with E2F2, occupied the ECE-1b promoter (ChIP), and repressed E2F2-mediated ECE-1b promoter activity (promoter-reporter assays).
Our results identify a cell cycle-independent mechanism by which E2F2 regulates endothelial function, arterial contractility, and BP.
PMCID: PMC2785027  PMID: 19752322
E2F; Sam68; Endothelium; Endothelin; Blood pressure
6.  Hypoxic Preconditioning Enhances the Benefit of Cardiac Progenitor-Cell Therapy for Treatment of Myocardial Infarction by Inducing CXCR4 Expression 
Circulation research  2009;104(10):1209-1216.
Myocardial infarction (MI) rapidly depletes the endogenous cardiac progenitor-cell pool, and the inefficient recruitment of exogenously administered progenitor cells limits the effectiveness of cardiac-cell therapy. Recent reports indicate that interactions between the CXC chemokine stromal-cell–derived factor 1 (SDF-1) and its receptor CXC chemokine receptor 4 (CXCR4) critically mediate the ischemia-induced recruitment of bone-marrow—derived circulating stem/progenitor cells, but the expression of CXCR4 in cardiac progenitor cells is very low. Here, we studied the influence of hypoxia on CXCR4 expression in cardiac progenitor cells, on the recruitment of intravenously administered cells to ischemic heart tissue, and on the preservation of heart function in a murine MI model. We found that hypoxic preconditioning increased CXCR4 expression in cardiosphere-derived, Lin−/c-kit+ progenitor (CLK) cells and markedly augmented CLK-cell migration (in vitro) and recruitment (in vivo) to the ischemic myocardium. Four weeks after surgically induced MI, infarct size and heart function were significantly better in mice administered hypoxia-preconditioned CLK cells than in mice treated with cells cultured under normoxic conditions. Furthermore, these effects were largely abolished by the addition of a CXCR4 inhibitor, indicating that the benefits of hypoxic preconditioning are mediated by the SDF-1/CXCR4 axis, and that therapies targeting this axis may enhance cardiac-progenitor-cell—based regenerative therapy.
PMCID: PMC2756190  PMID: 19407239
Cardiac progenitor cells; Hypoxia; CXCR4; Cell migration; Myocardial infarction
7.  Genetic Modification of Stem Cells for Transplantation 
Advanced drug delivery reviews  2007;60(2):160-172.
Gene modification of cells for prior to their transplantation, especially stem cells, enhances their survival and increases their function in cell therapy. Like the Trojan horse, the gene modified cell has to gain entrance inside the host’s walls and survive and deliver its transgene products Using cellular, molecular and gene manipulation techniques the transplanted cell can be protected in a hostile environment from immune rejection, inflammation, hypoxia and apoptosis. Genetic engineering to modify cells involves constructing modules of functional gene sequences. They can be simple reporter genes or complex cassettes with gene switches, cell specific promoters and multiple transgenes. We discuss methods to deliver and construct gene cassettes with viral and non viral delivery, siRNA, and conditional Cre/Lox P. We review the current uses of gene modified stem cells in cardiovascular disease, diabetes, neurological diseases,( including Parkinson’s, Alzheimer’s and spinal cord injury repair), bone defects, hemophilia, and cancer.
PMCID: PMC2734411  PMID: 18031863
vigilant vector; stem cells; microRNA; Cre/LoxP; heart failure; cancer; diabetes; neurological diseases

Results 1-7 (7)