Kelly Hiatt

Indiana University-Purdue University Indianapolis, Indianapolis, IN, United States

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Publications (13)63.87 Total impact

  • [show abstract] [hide abstract]
    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; · 2.83 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; · 1.98 Impact Factor
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    ABSTRACT: Persistent vocal fold motion impairment after recurrent laryngeal nerve (RLN) injury is not characteristically due to absent reinnervation, but often results from spontaneous aberrant reinnervation (synkinesis). We administered local neurotoxins to selected laryngeal muscles after RLN injury to determine whether aberrant reinnervation could be selectively inhibited. Unilateral RLN transection was performed in 24 male rats. Three weeks later, the denervated laryngeal adductor complex was injected with phenol, high- or low-dose vincristine sulfate (VNC), or saline solution. One month later, rat larynges were evaluated via videolaryngoscopy and laryngeal electromyography (LEMG). Larynges from euthanized animals were analyzed via immunofluorescent staining for the presence of reinnervation. One animal that received phenol and 3 animals that received high-dose VNC died of toxicity-related complications. In the surviving neurotoxin-treated animals, videolaryngoscopy showed increased lateralization of the immobile vocal fold. Only 1 phenol-injected rat had adductor complex motor recruitment (score of 3+) with LEMG. The other neurotoxin-treated animals demonstrated an absence of adductor complex reinnervation, with only insertional activity and fibrillations (no motor units/recruitment). Spontaneous ipsilateral abductor reinnervation was not affected by the adductor injections. Low-dose VNC injections appear to be relatively safe and effective in selectively inhibiting spontaneous aberrant reinnervation after RLN injury in an animal model.
    The Annals of otology, rhinology, and laryngology 12/2009; 118(12):887-93. · 1.21 Impact Factor
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    ABSTRACT: Current treatments for vocal fold paralysis are suboptimal in that they fail to restore dynamic function. Autologous muscle stem cell (MSC) therapy is a promising potential therapy for vocal fold paralysis in that it can attenuate denervation-induced muscle atrophy and provide a vehicle for delivery of neurotrophic factors, thereby potentially selectively guiding reinnervation. The goal of this project was to characterize optimal conditions for injected autologous MSC survival in the thyroarytenoid (TA) muscle following recurrent laryngeal nerve (RLN) injury by local administration of adjuvant factors. Animal experiment. Unilateral RLN transection and sternocleidomastoid muscle (approximately 1 g) biopsies were performed in 20 male Wistar rats. One month later, 10 autologous MSCs labeled via retroviral-enhanced green fluorescent protein (EGFP) transduction were injected into the denervated hemilarynx of each animal with one of four adjuvant therapies: cardiotoxin [(CTX) 10 M], insulin-like growth factor-1 [(IGF- 1) 100 microg/mL], ciliary neurotrophic factor [(CNTF) 50 microg/mL], or saline. Animals were euthanized 1 month later and larynges harvested, sectioned, and analyzed for MSC survival. All specimens demonstrate extensive MSC survival, with fusion of the MSCs with the denervated myofibers. Based on mean fluorescent intensity of the laryngeal specimens, IGF-1 and CNTF had the greatest positive influence on MSC survival. Myofiber diameters demonstrated myofiber atrophy to be inversely related to MSC survival, with the least atrophy in the groups having the greatest MSC survival. Autologous MSC therapy may be a future treatment for vocal fold paralysis. These findings support a model whereby MSCs genetically engineered to secrete CNTF and/or IGF-1 may not only promote neural regeneration, but also enhance MSC survival in an autocrine fashion.
    The Laryngoscope 08/2008; 118(7):1308-12. · 1.98 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. · 1.98 Impact Factor
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    ABSTRACT: Schwann cells derived from peripheral nerve sheath tumors from individuals with Neurofibromatosis Type 1 (NF1) are deficient for the protein neurofibromin, which contains a GAP-related domain (NF1-GRD). Neurofibromin-deficient Schwann cells have increased Ras activation, increased proliferation in response to certain growth stimuli, increased angiogenic potential, and altered cell morphology. This study examined whether expression of functional NF1-GRD can reverse the transformed phenotype of neurofibromin-deficient Schwann cells from both benign and malignant peripheral nerve sheath tumors. We reconstituted the NF1-GRD using retroviral transduction and examined the effects on cell morphology, growth potential, and angiogenic potential. NF1-GRD reconstitution resulted in morphologic changes, a 16-33% reduction in Ras activation, and a 53% decrease in proliferation in neurofibromin-deficient Schwann cells. However, NF1-GRD reconstitution was not sufficient to decrease the in vitro angiogenic potential of the cells. This study demonstrates that reconstitution of the NF1-GRD can at least partially reverse the transformation of human NF1 tumor-derived Schwann cells.
    Biochemical and Biophysical Research Communications 10/2006; 348(3):971-80. · 2.41 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. · 4.52 Impact Factor
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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; · 13.21 Impact Factor
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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. · 13.21 Impact Factor
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    ABSTRACT: Members of the Ras superfamily of signaling proteins modulate fundamental cellular processes by cycling between an active GTP-bound conformation and an inactive GDP-bound form. Neurofibromin, the protein product of the NF1 tumor suppressor gene, and p120GAP are GTPase-activating proteins (GAPs) for p21(Ras) (Ras) and negatively regulate output by accelerating GTP hydrolysis on Ras. Neurofibromin and p120GAP differ markedly outside of their conserved GAP-related domains (GRDs), and it is therefore unknown if the respective GRDs contribute functional specificity. To address this question, we expressed the GRDs of neurofibromin and p120GAP in primary cells from Nf1 mutant mice in vitro and in vivo. Here we show that expression of neurofibromin GRD, but not the p120GAP GRD, restores normal growth and cytokine signaling in three lineages of primary Nf1-deficient cells that have been implicated in the pathogenesis of neurofibromatosis type 1 (NF1). Furthermore, utilizing a GAP-inactive mutant NF1 GRD identified in a family with NF1, we demonstrate that growth restoration is a function of NF1 GRD GAP activity on p21(Ras). Thus, the GRDs of neurofibromin and p120GAP specify nonoverlapping functions in multiple primary cell types.
    Journal of Biological Chemistry 04/2001; 276(10):7240-5. · 4.65 Impact Factor
  • Journal of Biological Chemistry. 11/2000;
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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; · 13.21 Impact Factor
  • Pediatric Research 01/1999; 45. · 2.67 Impact Factor