The beneficial effects of stem cells in clinical applications to date have been modest, and studies have reported that poor engraftment might be an important reason. As a strategy to overcome such a hurdle, we developed the spheroid three dimensional (3D) bullet as a delivery method for human umbilical cord blood-derived mesenchymal stem cells (hUCB-MSCs) through the maintenance of cell-cell interactions without additional xenofactors, cytokines, or matrix. We made spheroid 3D-bullets from hUCB-MSCs at 24 hours' anchorage-deprived suspension culture. To investigate the in vivo therapeutic efficacy of 3D-bullets, we used rat myocardial infarction (MI) model. Transplantation of 3D-bullet was better than that of single cells from monolayer culture or from 3D-bullet in improving left ventricular (LV) contractility [LV ejection fraction (LVEF) or LV fractional shortening (LVFS)] and preventing pathologic LV dilatation [LV end-systolic diameter (LVESD) or LV end-diastolic diameter (LVEDD)] at 8 weeks. In the mechanism study of 3D-bullet formation, we found that calcium-dependent cell-cell interaction was essential and that E-cadherin is a key inducer mediating hUCB-MSC 3D-bullet formation among several calcium-dependent adhesion molecules which were nominated as candidates after cDNA array analysis. In more specific experiments with E-cadherin overexpression using adenoviral vector or with E-cadherin neutralization using blocking antibody, we found that E-cadherin regulates vascular endothelial growth factor (VEGF) secretion via extracellular signal-regulated kinase (ERK)/v-akt murine thymoma viral oncogene homolog1 (AKT) pathways. During formation of spheroid 3D-bullets, activation of E-cadherin in association with cell-cell interaction turns on ERK/AKT signaling pathway that are essential to proliferative and paracrine activity of MSCs leading to the enhanced therapeutic efficacy.

Capsaicin (trans-8-methyl-N-vanillyl-6-nonenamide), a natural product of Capsicum species, is known to induce excitation of nociceptive terminals involved in pain perception. Recent studies have also shown that capsaicin not only has chemopreventive properties against certain carcinogens and mutagens but also exerts anticancer activity. Here, we demonstrated the antiangiogenic activity of capsaicin using in vitro and in vivo assay systems. In vitro, capsaicin inhibited vascular endothelial growth factor (VEGF) -induced proliferation, DNA synthesis, chemotactic motility, and capillary-like tube formation of primary cultured human endothelial cells. Capsaicin inhibited both VEGF-induced vessel sprouting in rat aortic ring assay and VEGF-induced vessel formation in the mouse Matrigel plug assay. Moreover, capsaicin was able to suppress tumorinduced angiogenesis in chick chorioallantoic membrane assay. Capsaicin caused G 1 arrest in endothelial cells. This effect correlated with the down-regulation of the expression of cyclin D1 that led to inhibition of cyclin-dependent kinase 4-mediated phosphorylation of retinoblastoma protein. Signaling experiments show that capsaicin inhibits VEGFinduced p38 mitogen-activated protein kinase, p125 FAK , and AKT activation, but its molecular target is distinct from the VEGF receptor KDR/ Flk-1. Taken together, these results demonstrate that capsaicin is a novel inhibitor of angiogenesis and suggest that it may be valuable to develop pharmaceutical drugs for treatment of angiogenesis-dependent human diseases such as tumors.

Ischemia/reperfusion (I/R) injury to myocardium induces death of cardiomyocytes and destroys the vasculature, leading to cardiac fibrosis that is mainly mediated by the transdifferentiation of fibroblasts to myofibroblasts and the collagen deposition. Snail involvement in fibrosis is well known; however, the contribution of Snail to cardiac fibrosis during I/R injury and its underlying mechanisms have not been defined. We showed that I/R injury to mouse hearts significantly increases the expression of Snail. An in vitro hypoxia/reoxygenation (Hy/Reoxy) experiment showed that the cell source of Snail induction is endothelial cells rather than cardiac fibroblasts (cFibroblasts) or cardiomyoblasts. When Snail was overexpressed in endothelial cells, they underwent endothelial-to-mesenchymal transition (EndMT) but showed very poor capacity for collagen synthesis. Instead, reoxygenation- or Snail overexpression-mediated EndMT-like cells noticeably stimulated transdifferentiation of fibroblasts to myofibroblasts via secretion of connective tissue growth factor (CTGF). The injection of a peroxisome proliferator-activated receptor-γ (PPAR-γ) agonist, a selective Snail inhibitor, remarkably suppressed collagen deposition and cardiac fibrosis in mouse I/R injury, and significantly improved cardiac function and reduced Snail and CTGF expression in vivo. Our findings suggested a new mechanism of cell-to-cell communication between EndMT-like cells and fibroblasts for fibrosis induction and implicated Snail as a potential target molecule in cardiac fibrosis after I/R injury.

IntroductionNeovascularization is necessary for repairing the damaged ischemic tissue such as myocardial infarct and ischemic limb. 1,2 Bone marrow (BM)-derived stem/progenitor cells play a pivotal role in repairing the destroyed vasculature in ischemic tissue by multistep processes of mobilization, recruitment to ischemic tissue, and adhesion to hypoxic endothelial cells. [3][4][5] Clinical reports demonstrated that intracoronary infusion of BM-derived cells may improve the outcome of patients with acute myocardial infarction. 6,7 However, therapeutic efficacy was limited because the efficiency of targeting BM-derived cells to ischemic area is low. Thus, the recruitment of BM-derived cells into injured foci should be improved for repairing ischemic tissues efficiently.Hypoxia-inducible factor-1 (HIF-1) is a master transcription factor for adaptive responses to hypoxia and ischemia. 8,9 HIF-1 regulates more than 60 genes affecting cell survival, metabolism, and vessel formation such as vascular endothelial growth factor (VEGF), placental growth factor, and basic fibroblast growth factor. 8 HIF-1 is a heterodimer consisting of a constitutive HIF-1␤ and a hypoxia-induced HIF-1␣, which is mainly regulated by ubiquitination and proteasomal degradation. HIF-1␣ protein synthesis, however, is also up-regulated by growth factors such as insulin, epidermal growth factor, and insulin-like growth factor-1 and is inhibited by tumor suppressors. 9,10 Ang-1 is an endothelial growth factor binding to the Tie2 receptor and induces Tie2 phosphorylation. 11 Ang-1 plays essential roles in regulating vascular development, maintenance of endothelial integrity, and vessel maturation. [12][13][14][15] We recently reported the new role of Ang-1 as a cell primer. 16 Priming of progenitor cells means the short-term stimulation of the cells with cytokine to improve the therapeutic potential. Primed BM-derived progenitor cells (BMPCs) with Ang-1 increased the expression of adhesion molecules and commitment to endothelial lineage leading to improved engraftment into ischemic tissue and vasculogenesis. 16 In this study, we investigated the effect of locally administered Ang-1 on the recruitment of BMPCs to the ischemic tissue. We showed that Ang-1 is a critical stimulator of HIF-1␣ and stromal cell-derived factor 1 (SDF-1), and thus enhances the recruitment of the BMPCs to ischemic foci. We found a new action mechanism of Ang-1 in vasculogenesis, which modulates mammalian target of rapamycin (mTOR) activation on hypoxia, leading to HIF-1␣ protein synthesis and SDF-1 accumulation in hypoxic endothelium. The intermediate mechanisms that transmit the Ang-1 signal down to the mTOR/HIF-1␣/SDF-1 axis were the induction of colocalization of Tie2 receptor and integrin-linked kinase (ILK) at the cell membrane and the enhanced binding between them. We also demonstrated in vivo that Ang-1 facilitated functional incorporation of BMPCs into ischemic endothelium and increased For personal use only. on May 12, 2018. by guest www.bloodjournal.org From neova...