Kento Tateishi

Erythropoietin-Mobilized Endothelial Progenitors Enhance Reendothelialization via Akt-Endothelial Nitric Oxide Synthase Activation and Prevent Neointimal Hyperplasia

We investigated whether the mobilization of endothelial progenitor cells (EPCs) by exogenous erythropoietin (Epo) promotes the repair of injured endothelium. Recombinant human Epo was injected (1000 IU/kg for the initial 3 days) after wire injury of the femoral artery of mice. Neointimal formation was inhibited by Epo to 48% of the control (P<0.05) in an NO-dependent manner. Epo induced a 1.4-fold increase in reendothelialized area of day 14 denuded vessels, 55% of which was derived from bone marrow (BM) cells. Epo increased the circulating Sca-1+/Flk-1+ EPCs (2.0-fold, P<0.05) with endothelial properties NO dependently. BM replacement by GFP- or β-galactosidase-overexpressing cells showed that Epo stimulated both differentiation of BM-derived EPCs and proliferation of resident ECs. BM-derived ECs increased 2.2- to 2.7-fold (P<0.05) in the Epo-induced neoendothelium, where the expression of Epo receptor was upregulated. Epo induced Akt/eNOS phosphorylation and NO synthesis on EPCs and exerted an antiapoptotic action on wire-injured arteries. In conclusion, Epo treatment inhibits the neointimal hyperplasia after arterial injury in an NO-dependent manner by acting on the injured vessels and mobilizing EPCs to the neo-endothelium.

Controlled Delivery of Basic Fibroblast Growth Factor Promotes Human Cardiosphere-Derived Cell Engraftment to Enhance Cardiac Repair for Chronic Myocardial Infarction

OBJECTIVES This study was designed to determine whether controlled release of basic fibroblast growth factor (bFGF) might improve human cardiosphere-derived cell (hCDC) therapy in a pig model of chronic myocardial infarction. BACKGROUND Current cell therapies for cardiac repair are limited by loss of the transplanted cells and poor differentiation. METHODS We conducted 2 randomized, placebo-controlled studies in immunosuppressed pigs with anterior myocardial infarctions. Four weeks after coronary reperfusion, 14 pigs were randomly assigned to receive an intramyocardial injection of placebo medium with or without bFGF-incorporating hydrogel implantation. As a second study, 26 pigs were randomized to receive controlled release of bFGF combined with or without hCDCs or bone marrow-derived mesenchymal stem cell transplantation 4 weeks after reperfusion. RESULTS Controlled release of bFGF in ischemic myocardium significantly augmented the formation of microvascular networks to enhance myocardial perfusion and contractile function. When combined with cell transplantation, the additive effects of bFGF were confined to hCDC-injected animals, but were not observed in animals receiving human bone marrow-derived mesenchymal stem cell transplantation. This was shown by increased donor-cell engraftment and enhanced cardiomyocyte differentiation in the transplanted hearts, resulting in synergistically improved ventricular function and regional wall motion and reduced infarct size. CONCLUSIONS Controlled delivery of bFGF modulates the post-ischemic microenvironment to enhance hCDC engraftment and differentiation. This novel strategy demonstrates significant functional improvements after myocardial infarction and may potentially represent a therapeutic approach to be studied in a clinical trial in human heart failure.

Clonally amplified cardiac stem cells are regulated by Sca-1 signaling for efficient cardiovascular regeneration.

Recent studies have shown that cardiac stem cells (CSCs) from the adult mammalian heart can give rise to functional cardiomyocytes; however, the definite surface markers to identify a definitive single entity of CSCs and the molecular mechanisms regulating their growth are so far unknown. Here, we demonstrate a single-cell deposition analysis to isolate individually selected CSCs from adult murine hearts and investigate the signals required for their proliferation and survival. Clonally proliferated CSCs express stem cell antigen-1 (Sca-1) with embryonic stem (ES) cell-like and mesenchymal cell-like characteristics and are associated with telomerase reverse transcriptase (TERT). Using a transgene that expresses a GFP reporter under the control of the TERT promoter, we demonstrated that TERTGFP-positive fractions from the heart were enriched for cells expressing Sca-1. Knockdown of Sca-1 transcripts in CSCs led to retarded ex vivo expansion and apoptosis through Akt inactivation. We also show that ongoing CSC proliferation and survival after direct cell-grafting into ischemic myocardium require Sca-1 to upregulate the secreted paracrine effectors that augment neoangiogenesis and limit cardiac apoptosis. Thus, Sca-1 might be an essential component to promote CSC proliferation and survival to directly facilitate early engraftment, and might indirectly exert the effects on late cardiovascular differentiation after CSC transplantation.

Central Role of Calcium-Dependent Tyrosine Kinase PYK2 in Endothelial Nitric Oxide Synthase–Mediated Angiogenic Response and Vascular Function

Background— The involvement of Ca2+-dependent tyrosine kinase PYK2 in the Akt/endothelial NO synthase pathway remains to be determined. Methods and Results— Blood flow recovery and neovessel formation after hind-limb ischemia were impaired in PYK2−/− mice with reduced mobilization of endothelial progenitors. Vascular endothelial growth factor (VEGF)–mediated cytoplasmic Ca2+ mobilization and Ca2+-independent Akt activation were markedly decreased in the PYK2-deficient aortic endothelial cells, whereas the Ca2+-independent AMP-activated protein kinase/protein kinase-A pathway that phosphorylates endothelial NO synthase was not impaired. Acetylcholine-mediated aortic vasorelaxation and cGMP production were significantly decreased. Vascular endothelial growth factor–dependent migration, tube formation, and actin cytoskeletal reorganization associated with Rac1 activation were inhibited in PYK2-deficient endothelial cells. PI3-kinase is associated with vascular endothelial growth factor–induced PYK2/Src complex, and inhibition of Src blocked Akt activation. The vascular endothelial growth factor–mediated Src association with PLC&ggr;1 and phosphorylation of 783Tyr-PLC&ggr;1 also were abolished by PYK2 deficiency. Conclusion— These findings demonstrate that PYK2 is closely involved in receptor- or ischemia-activated signaling events via Src/PLC&ggr;1 and Src/PI3-kinase/Akt pathways, leading to endothelial NO synthase phosphorylation, and thus modulates endothelial NO synthase–mediated vasoactive function and angiogenic response.

Stemming heart failure with cardiac‐ or reprogrammed‐stem cells

Despite extensive efforts to control myocyte growth by genetic targeting of the cell cycle machinery and small molecules for cardiac repair, adult myocytes themselves appeared to divide a limited number of times in response to a variety of cardiac muscle stresses. Rare tissue‐resident stem cells are thought to exist in many adult organs that are capable of self‐renewal and differentiation and possess a range of actions that are potentially therapeutic. Recent studies suggest that a population of cardiac stem cells (CSCs) is maintained after cardiac development in the adult heart in mammals including human beings; however, homeostatic cardiomyocyte replacement might be stem cell‐dependent, and functional myocardial regeneration after cardiac muscle damage is not yet considered as sufficient to fully maintain or reconstitute the cardiovascular system and function. Although it is clear that adult CSCs have limitations in their capabilities to proliferate extensively and differentiate in response to injury in vivo for replenishing mature car‐diomyocytes and potentially function as resident stem cells. Transplantation of CSCs expanded ex vivo seems to require an integrated strategy of cell growth‐enhancing factor(s) and tissue engineering technologies to support the donor cell survival and subsequent proliferation and differentiation in the host microenvironment. There has been substantial interest regarding the evidence that mammalian fibroblasts can be genetically reprogrammed to induced pluripotent stem (iPS) cells, which closely resemble embryonic stem (ES) cell properties capable of differentiating into functional cardiomyocytes, and these cells may provide an alternative cell source for generating patient‐specific CSCs for therapeutic applications.

Therapeutic Potential of Stem/Progenitor Cells in Human Skeletal Muscle for Cardiovascular Regeneration

Although myoblast transplantation in patients with ischemic heart failure results in a significant improvement of cardiac function, subsequent studies have consistently shown the myotubes formation in the absence of electromechanical coupling with the neighboring host myocardium, accompanied with the short-term release of paracrine effectors from implanted cells. One major pitfall of using myoblasts is that transplanted cells do not differentiate into cardiomyocytes, which may cause the inherent proarrhythmogenic events. Therefore, whether a discrete subpopulation in heterogeneous muscle-cell cultures is responsible for substantial cardiovascular regeneration has yet to be investigated. We describe here the isolation of progenitor cells from human skeletal muscle. These cells proliferated as non-adherent myospheres in suspension and displayed early embryonic factors and mesenchymal cell-like characteristics. Flow cytometric analyses demonstrated that CD56/N-CAM/Leu-19, a neural cell adhesion molecule abundantly present in myoblasts, was absent in myospheres but was expressed in an adherent cell population containing myogenic precursors. Myosphere-derived progenitor cells (MDPCs) differentiated in culture to produce cardiac, smooth muscle, and endothelial cells. Transplantation of MDPCs into ischemic hearts in NOD/scid mice promoted angiogenesis with substantial cardiovascular regeneration. Our results provide a foundation to further study the cell and biological function of human MDPCs which may have potential therapeutic implications.