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ABSTRACT: Muscle progenitor cells (MPCs) are currently being investigated as cellular vectors to deliver neurotrophic factor (NF) for the promotion of re-innervation after axonal injury. Ideally NF delivery in such a model would enhance axonal regeneration while simultaneously promoting MPC viability. To date, insulin-like growth factor 1 (IGF-1) is one of the few NFs known to promote both re-innervation and MPC viability. We herein identify ciliary neurotrophic factor (CNTF) as a factor that promotes MPC viability in culture, and demonstrate CNTF to impart greater viability effects on MPCs than IGF-1. We demonstrate that pharmacological inhibition via LY294002 results in abrogation of CNTF-mediated viability, suggesting that the CNTF-mediated MPC viability benefit occurs via the PI3-Akt pathway. Finally, we employ a genetic model, establishing MPC cultures from mice deficient in class I(A) PI-3 K (p85α(-/-) ) mice, and demonstrate that the viability benefit imparted by CNTF is completely abrogated in PI-3 K-deficient MPCs compared to wild-type controls. In summary, our investigations define CNTF as a promoter of MPC viability beyond IGF-1, and reveal that the CNTF-mediated MPC viability effects occur via the PI3-Akt pathway. Copyright © 2012 John Wiley & Sons, Ltd.
Journal of Tissue Engineering and Regenerative Medicine 11/2012; · 3.28 Impact Factor
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ABSTRACT: OBJECTIVES/HYPOTHESIS: To determine if the spontaneous reinnervation that characteristically ensues after recurrent laryngeal nerve (RLN) injury could be selectively promoted and directed to certain laryngeal muscles with the use of neurotrophic factor (NF)-secreting muscle stem cell (MSC) vectors while antagonistic reinnervation is inhibited with vincristine (VNC). STUDY DESIGN: Basic science investigation involving primary cell cultures, gene cloning/transfer, and animal experiments. METHODS: MSC survival assays were used to test multiple individual NFs in vitro. Motoneuron outgrowth assays assessed the trophic effects of identified NF on cranial nerve X (CNX)-derived motoneurons in vitro. Therapeutic NF was cloned into a lentiviral vector, and MSCs were tranduced to secrete NF. Sixty rats underwent left RLN transection injury, and at 3 weeks received injections of either MSCs (n = 24), MSCs secreting NF (n = 24), or saline (n = 12) into the left thyroarytenoid muscle complex; half of the animals in the MSC groups simultaneously received left posterior cricoarytenoid injections of VNC, whereas half of the animals received saline. RESULTS: Ciliary neurotrophic factor (CNTF) had the greatest survival-promoting effect on MSCs in culture. The addition of CNTF (50 ng/mL) to CNX motoneuron cultures resulted in enhanced neurite outgrowth and branching. In the animal model, the injected MSCs fused with the denervated myofibers, immunohistochemistry demonstrated enhanced reinnervation based on motor endplate to nerve contact, and reverse transcriptase-polymerase chain reaction confirmed stable CNTF expression at longest follow-up (4 months) in the CNTF-secreting MSC treated groups. CONCLUSIONS: MSC therapy may have a future role in selectively promoting and directing laryngeal reinnervation after RLN injury. Laryngoscope, 2012.
The Laryngoscope 09/2012; · 1.75 Impact Factor
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ABSTRACT: Laryngeal muscle and skeletal muscle stem cells (MSC) have been shown to differ in physiological basal activity and responsiveness to stimuli. Given these differences, it is the purpose of this investigation to characterize the in vitro proliferation and survival of laryngeal and skeletal MSC to determine whether intrinsic differences exist that may account for differences noted in vivo.
Basic science experiment utilizing rat MSC.
Cultures of both laryngeal and skeletal MSC were harvested and equal numbers from both groups were expanded under similar conditions, quantifying cellular population to determine proliferation rate for each population. Increased proliferative potential was confirmed using Western blot analysis of extracellular signal-regulated kinase phosphorylation. As per standard survival assay protocol, cultures were placed in serum-deprived medium and cell survival was assessed by terminal uridine deoxynucleotidyl transferase-mediated dUTP nick end labeling assay at 72 hours.
Our results demonstrated increased proliferation of laryngeal MSC relative to the skeletal MSC when cultured under similar conditions. Western blot demonstrated increased activation of the proliferation pathway, extracellular signal-regulated kinase, in the laryngeal group. There was no detectable difference in the MSC survival between the two groups.
Compared with skeletal MSC, laryngeal MSC demonstrate increased proliferation and regenerative capacity. This may explain some of the differences in physiological role and responses involved in each cell population's tissue of origin.
The Laryngoscope 06/2008; 118(8):1422-6. · 1.75 Impact Factor
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ABSTRACT: Genetic loss of surface Fas antigen expression leads to reduced apoptosis of myeloid and lymphoid progenitor cells, and a propensity to develop autoimmunity and myeloid leukemia in mouse models. Oncogenic p21(ras) decreases surface Fas antigen expression and renders fibroblasts resistant to Fas mediated apoptosis. Neurofibromin, which is encoded by NF1, is a GTPase activating protein that negatively regulates p21(ras) activity. NF1 loss leads to deregulation of p21(ras)-effector pathways, which control myeloid cell survival. Heterozygous inactivation of Nf1 increases mast cell numbers in Nf1 +/- mice, and enhances mast cell survival in response to c-kit ligand (kit-L). Here, we show that Nf1-deficient mast cells have reduced surface Fas antigen expression in response to kit-L and are resistant to Fas ligand-mediated apoptosis. Using genetic intercrosses between Nf1 +/- and class I (A)-PI-3K-deficient mice, we demonstrate that hyperactivation of the p21(ras)-class I(A) PI-3K pathway is the mechanism for this phenotype. Finally, we demonstrate that mast cells from both Fas antigen-deficient mice and Nf1 +/- mice are resistant to apoptosis following kit-L withdrawal in vivo. Thus, therapies designed to decrease p21(ras) activity and up-regulate Fas antigen expression may limit the pathological accumulation of myeloid cells in disease states where p21(ras) is hyperactivated.
American Journal Of Pathology 05/2004; 164(4):1471-9. · 4.89 Impact Factor
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David A Ingram, Kelly Hiatt,
Alastair J. King,
Lucy Fisher,
Rama Shivakumar,
Christina Derstine,
Mary Jo Wenning,
Bruce Diaz,
Jeffrey B. Travers,
Antoinette Hood,
Mark Marshall,
David A Williams,
D Wade Clapp
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ABSTRACT: Mutations in the NF1 tumor suppressor gene cause neurofibromatosis type I (NF1), a disease characterized by the formation of cutaneous neurofibromas infiltrated with a high density of degranulating mast cells. A hallmark of cell lines generated from NF1 patients or Nf1-deficient mice is their propensity to hyperproliferate. Neurofibromin, the protein encoded by NF1, negatively regulates p21ras activity by accelerating the conversion of Ras-GTP to Ras-GDP. However, identification of alterations in specific p21ras effector pathways that control proliferation in NF1-deficient cells is incomplete and critical for understanding disease pathogenesis. Recent studies have suggested that the proliferative effects of p21ras may depend on signaling outputs from the small Rho GTPases, Rac and Rho, but the physiologic importance of these interactions in an animal disease model has not been established. Using a genetic intercross between Nf1+/− and Rac2−/− mice, we now provide genetic evidence to support a biochemical model where hyperactivation of the extracellular signal–regulated kinase (ERK) via the hematopoietic-specific Rho GTPase, Rac2, directly contributes to the hyperproliferation of Nf1-deficient mast cells in vitro and in vivo. Further, we demonstrate that Rac2 functions as mediator of cross-talk between phosphoinositide 3-kinase (PI-3K) and the classical p21ras-Raf-Mek-ERK pathway to confer a distinct proliferative advantage to Nf1+/− mast cells. Thus, these studies identify Rac2 as a novel mediator of cross-talk between PI-3K and the p21ras-ERK pathway which functions to alter the cellular phenotype of a cell lineage involved in the pathologic complications of a common genetic disease.
Journal of Experimental Medicine 08/2001; · 13.85 Impact Factor
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David A. Ingram, Kelly Hiatt,
Alastair J. King,
Lucy Fisher,
Rama Shivakumar,
Christina Derstine,
Mary Jo Wenning,
Bruce Diaz,
Jeffrey B. Travers,
Antoinette Hood,
Mark Marshall,
David A. Williams,
D. Wade Clapp
[show abstract]
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ABSTRACT: Mutations in the NF1 tumor suppressor gene cause neurofibromatosis type I (NF1), a disease characterized by the formation of cutaneous neurofibromas
infiltrated with a high density of degranulating mast cells. A hallmark of cell lines generated from NF1 patients or Nf1-deficient mice is their propensity to hyperproliferate. Neurofibromin, the protein encoded by NF1, negatively regulates p21ras activity by accelerating the conversion of Ras-GTP to Ras-GDP. However, identification of alterations in specific p21ras effector pathways that control proliferation in NF1-deficient cells is incomplete and critical for understanding disease pathogenesis. Recent studies have suggested that the
proliferative effects of p21ras may depend on signaling outputs from the small Rho GTPases, Rac and Rho, but the physiologic importance of these interactions
in an animal disease model has not been established. Using a genetic intercross between Nf1+/− and Rac2−/− mice, we now provide genetic evidence to support a biochemical model where hyperactivation of the extracellular signal–regulated
kinase (ERK) via the hematopoietic-specific Rho GTPase, Rac2, directly contributes to the hyperproliferation of Nf1-deficient mast cells in vitro and in vivo. Further, we demonstrate that Rac2 functions as mediator of cross-talk between
phosphoinositide 3-kinase (PI-3K) and the classical p21ras-Raf-Mek-ERK pathway to confer a distinct proliferative advantage to Nf1+/− mast cells. Thus, these studies identify Rac2 as a novel mediator of cross-talk between PI-3K and the p21ras-ERK pathway which functions to alter the cellular phenotype of a cell lineage involved in the pathologic complications of
a common genetic disease.
Journal of Experimental Medicine 07/2001; 194(1):57-70. · 13.85 Impact Factor
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ABSTRACT: Neurofibromatosis type 1 (NF1) is a common autosomal-dominant disorder characterized by cutaneous neurofibromas infiltrated with large numbers of mast cells, melanocyte hyperplasia, and a predisposition to develop malignant neoplasms. NF1 encodes a GTPase activating protein (GAP) for Ras. Consistent with Knudson's “two hit” model of tumor suppressor genes, leukemias and malignant solid tumors in NF1 patients frequently demonstrate somatic loss of the normal NF1 allele. However, the phenotypic and biochemical consequences of heterozygous inactivation of Nf1 are largely unknown. Recently neurofibromin, the protein encoded by NF1, was shown to negatively regulate Ras activity in Nf1−/− murine myeloid hematopoietic cells in vitro through the c-kit receptor tyrosine kinase (dominant white spotting, W). Since the W and Nf1 locus appear to function along a common developmental pathway, we generated mice with mutations at both loci to examine potential interactions in vivo. Here, we show that haploinsufficiency at Nf1 perturbs cell fates in mast cells in vivo, and partially rescues coat color and mast cell defects in W41 mice. Haploinsufficiency at Nf1 also increased mast cell proliferation, survival, and colony formation in response to Steel factor, the ligand for c-kit. Furthermore, haploinsufficiency was associated with enhanced Ras–mitogen-activated protein kinase activity, a major downstream effector of Ras, via wild-type and mutant (W41) c-kit receptors. These observations identify a novel interaction between c-kit and neurofibromin in vivo, and offer experimental evidence that haploinsufficiency of Nf1 alters both cellular and biochemical phenotypes in two cell lineages that are affected in individuals with NF1. Collectively, these data support the emerging concept that heterozygous inactivation of tumor suppressor genes may have profound biological effects in multiple cell types.
Journal of Experimental Medicine 02/2000; · 13.85 Impact Factor
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ABSTRACT: The literature on hospice care and palliative medicine lacks a focus on the combined and concurrent assessment of services. This deficiency is problematic because research findings are then limited in their scope and applicability to particular stakeholder groups. The current study used the perspective of 72 participants and service delivery personnel of a home-based hospice program to address this perceived problem and limitation in the literature through a program evaluation of the multiple perspectives on a single hospice organization. Interviews were analyzed by stakeholder group and then across stakeholder groups to extract themes. Results include a description of the quality of service delivery and participant satisfaction, areas of current weakness, and ideas for potential growth and development for the program. Findings from this study are compared with previous research on hospice care and the implications of the results to the provision and continued development of hospice care are discussed.
American Journal of Hospice and Palliative Medicine 24(5):376-82. · 1.15 Impact Factor
