Browsing by Author "Ingram, David A"

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    Methods and mechanisms to improve endothelial colony forming cell (ECFC) survival and promote ECFC vasculogenesis in three dimensional (3D) collagen matrices in vitro and in vivo
    (2015-06-30) Kim, Hyojin; Yoder, Mervin C.; Ingram, David A; Quilliam, Lawrence A.; Harrington, Maureen A.
    Human cord blood (CB) derived circulating endothelial colony forming cells (ECFCs) display a hierarchy of clonogenic proliferative potential and possess de novo vessel forming ability upon implantation in immunodeficient mice. Since survival of ECFC post-implantation is a critical variable that limits in vivo vasculogenesis, we tested the hypothesis that activation of Notch signaling or co-implantation of ECFC with human platelet lysate (HPL) would enhance cultured ECFC vasculogenic abilities in vitro and in vivo. Co-implantation of ECFCs with Notch ligand Delta-like 1 (DL1) expressing OP9 stromal cells (OP9-DL1) decreased apoptosis of ECFC in vitro and increased vasculogenesis of ECFC in vivo. The co-culture of ECFC with HPL diminished apoptosis of ECFC by altering the expression of pro-survival molecules (pAkt, pBad and Bcl-xL) in vitro and increased vasculogenesis of human EC-derived vessels both in vitro and in vivo. Thus, activation of the Notch pathway by OP9-DL1 stromal cells or co-implantation of ECFC with HPL enhances vasculogenesis and augments blood vessel formation by diminishing apoptosis of the implanted ECFC. The results from this study will provide critical information for the development of a cell therapy for limb and organ re-vascularization that can be applied to recovery of ischemic tissues in human subjects.
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    Neurofibromin-deficient Schwann cells secrete a potent migratory stimulus for Nf1+/– mast cells
    (2003-12) Yang, Feng-Chun; Ingram, David A; Chen, Shi; Hingtgen, Cynthia M; Ratner, Nancy; Monk, Kelly R; Clegg, Travis; White, Hilary; Mead, Laura; Wenning, Mary Jo; Williams, David A; Kapur, Reuben; Atkinson, Simon J; Clapp, D Wade
    The NF1 tumor suppressor gene encodes a GTPase-activating protein called neurofibromin that negatively regulates Ras signaling. Mutations in NF1 cause neurofibromatosis type 1 (NF1). The development of neurofibromas, which are complex tumors composed of multiple cell types, is a hallmark of NF1. Somatic inactivation of murine Nf1 in Schwann cells is necessary, but not sufficient, to initiate neurofibroma formation. Neurofibromas occur with high penetrance in mice in which Nf1 is ablated in Schwann cells in the context of a heterozygous mutant (Nf1+/–) microenvironment. Mast cells infiltrate neurofibromas, where they secrete proteins that can remodel the ECM and initiate angiogenesis. Thus, identification of mechanisms responsible for mast cell migration to tumor microenvironments is important for understanding tumorigenesis and for designing potential therapies. Here, we show that homozygous Nf1 mutant (Nf1–/–) Schwann cells secrete Kit ligand (KitL), which stimulates mast cell migration, and that Nf1+/– mast cells are hypermotile in response to KitL. Furthermore, we link hyperactivation of the Ras-class IA-PI3K-Rac2 pathway to increased Nf1+/– mast cell migration. Thus, these studies identify a novel interaction between Nf1–/– Schwann cells and Nf1+/– mast cells that is likely to be important in neurofibroma formation.
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    p21-activated kinase regulates mast cell degranulation via effects on calcium mobilization and cytoskeletal dynamics
    (2009-03) Allen, Jayme D; Jaffer, Zahara M; Park, Su-Jung; Burgin, Sarah; Hofmann, Clemens; Sells, Mary A; Chen, Shi; Derr-Yellin, Ethel; Michels, Elizabeth G; McDaniel, Andrew; Bessler, Waylan K; Ingram, David A; Atkinson, Simon J; Travers, Jeffrey B; Chemoff, Jonathan; Clapp, D Wade
    Mast cells are key participants in allergic diseases via activation of high-affinity IgE receptors (FcϵRI) resulting in release of proinflammatory mediators. The biochemical pathways linking IgE activation to calcium influx and cytoskeletal changes required for intracellular granule release are incompletely understood. We demonstrate, genetically, that Pak1 is required for this process. In a passive cutaneous anaphylaxis experiment, Wsh/Wsh mast cell–deficient mice locally reconstituted with Pak1−/− bone marrow–derived mast cells (BMMCs) experienced strikingly decreased allergen-induced vascular permeability compared with controls. Consistent with the in vivo phenotype, Pak1−/− BMMCs exhibited a reduction in FcϵRI-induced degranulation. Further, Pak1−/− BMMCs demonstrated diminished calcium mobilization and altered depolymerization of cortical filamentous actin (F-actin) in response to FcϵRI stimulation. These data implicate Pak1 as an essential molecular target for modulating acute mast cell responses that contribute to allergic diseases.