171 Ashley Ave.
Charleston, SC 29425
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November 2007
2007 MD/PhD Projects
DeAnna A. Baker
Mentor: Dr. Gary Gilkeson
Role of Sphingosine Kinase 1 in a Mouse Model of Chronic Inflammation
DeAnna Baker, Lina Obeid, MD, Gary Gilkeson, MD
Ralph H. Johnson VA Medical Center, Charles P. Darby Children's Research Institute, Medical University of South Carolina, Charleston, SC
Sphingosine kinase 1 (SphK1) is one of the two enzymes that phosphorylates sphingosine to create sphingosine 1 phosphate (S1P) and has an established relationship with cell signaling molecules such as ERK and p38 MAPK. Abnormatlities in sphingolipids are implicated in a variety of disease states, especially in the pathogenesis of certain cancers. However, their role in other disease states is poorly understood. Based on previous in vitro results, S1P has an apparent role in inflammation. Recent data demonstrated a relationship between TNF alpha, a contributor to inflammation, and S1P produced by SphK1. Fibroblast cell lines stimulated with TNF alpha lead to an increase in S1P following SphK1 activation. Removal of SphK1 by siRNA in fibroblast cell lines stimulated with human TNF alpha (hTNF) lead to a decrease in the formation of the inflammatory mediator prostaglandin E2 (PGE2) (Pettus et al., 2003). This in vitro data lead us to further experiment in an in vivo model. We have obtained transgenic mice that cosntitutively express a modified copy of the hTNF alpha gene leading to chronic, progressive synovitis detectable as swelling and deformity in the fore and hind paws at around 20 weeks of age. These mice were crossed with mice lacking functional copies of the SphK1 gene (SphK1 KO). Thus, the absence of the SphK1 gene will allow us to study its direct effects on hTNF-induced chronic synovial inflammation. The mice were genotyped to determine the presence/absence of SphK1 and the transgene and monitored weekly for evidence of inflammation. In preliminary observations of a limited number of mice, hTNF transgenic SphK1 KO mice develop less joint swelling and deformity than hTNF transgenic mice with functioning copies of SphK1 and transgenic mice heterozygous for SphK1 of comparable age. Also, TNF transgenic, Sphk1KO mice had less detectable activated ERK than transgenic mice heterozygous for SphK1 or Sphk1 heterozygous mice. These preliminary data suggest that SphK1 induced S1P production is a key factor in the inflammatory arthritis present in hTNF mice.
Pettus, B.J., Bielawski, J., Porcelli, A.M., Reames, D.L., Johnson, K.R., Morrow, J., Chalfant, C.E., Obeid, L.M., and Hannun, Y.A. (2003). The sphingosine kinase 1/sphingosine-1-phosphate pathway mediates COX-2 induction and PGE2 production in response to TNF-alpha. FASEB J 17, 1411-1421.
Loretta L. Hoover
Mentor: Dr. Steve Kubalak
Regulation of TGF-beta signaling by retinoid receptor ligands occurs in a time frame that is independent of transcriptional events
Loretta L. Hoover, M. Elizabeth G. Burton, Bonnie A. Brooks, and Steven W. Kubalak, Department of Cell Biology and Anatomy, Cardiovascular Developmental Biology Center, Charles P. Darby Children's Research Institute, Medical University of South Carolina, Charleston, SC
The molecular events responsible for most congenital heart defects are poorly understood. We have previously reported increased transforming growth factor beta 2 (TGF-beta-2) as well as increased apoptosis in the developing heart of the midgestational (E11.5-13.5) retinoid x receptor alpha knockout (RXR alpha -/-) mouse model of congenital heart disease. Interestingly, Smad2, a signaling factor downstream of TGF-beta-2, is perturbed in the E12.5 RXR alpha -/- such that outflow tract mesenchymal cells show less nuclear-localized activated (i.e. phosphorylated) Smad2 (pSmad2) when compared to similar cells in the wild type. This finding led us to investigate the temporal expression of pSmad2, and preliminary evidence suggests a futile negative feedback mechanism is at work in this mouse. Our in vitro work is focused on whether activation of retinoic acid signaling affects pSmad2 accumulation. We treated wild type E12.5 dispersed heart cells with combinations of TGF-beta-2 and the RXR alpha agonist, 9-cis-retinoic acid (9-cis-RA) and evaluated activation of Smad2, which occurs within minutes of treatment. As expected, treating cells with TGF-beta-2 for 1 hour resulted in an increase in pSmad2 as detected by Western blot when compared to treating with 9-cis-RA. When cells were treated with the combination of TGF-beta-2 and 9-cis-RA, we detected a statistically significant increase in the amount of pSmad2 when compared to treating with TGF-beta-2 alone. In our characterization of this molecular event, we have expanded our studies to include evaluation of normal epithelial and mesenchymal cell lines as well as several cancer cell lines. Our studies are aimed at determining the mechanism of how retinoids can potentiate canonical TGF-beta signaling in an acute timeframe independent of transcriptional events. Results suggest important components of TGF-beta signaling affecting Smad2 activation and nuclear accumulation are regulated by retinoids in a manner that has been unappreciated until now. We have evidence supporting that these interactions may be responsible for the congenital heart defects seen in the RXR alpha -/-.
Anthony P. Leonard
Mentor: Dr. Bernie Maria
Hyaluronan-CD44 interactions and regulation of BCRP/ABCG2 in human spinal and cerebral gliomas
Leonard, AP; Gilg, AG; Toole, BP; Maria, BL.
Departments of Pediatrics and Cell Biology and Anatomy, Charles P. Darby Children's Research Institute, Medical University of South Carolina.
Malignant human spinal cord gliomas are extremely rare tumors which are associated with a dismal prognosis for survival and high spinal neurological morbidity. Hyaluronan oligomers (o-HA) that inhibit HA interactions with cell surface receptors (e.g. CD44, RHAMM) inhibit tumor growth and invasiveness in a highly malignant C6 rodent spinal cord glioma model. In vitro, o-HA reverse malignant properties of C6 cells including the expression of breast cancer resistance protein (BCRP/ABCG2), an ATP-drug effluxer abundant in highly tumorigenic CD133+ glioma progenitors. Interestingly, o-HA treatment in vivo also abrogates recruitment of non-C6 (host-derived) BCRP+ progenitors which primarily express neural (vs. bone marrow) markers. Because little is known of the biology of human spinal cord gliomas and there is limited evidence whether highly chemoresistant BCRP+ cells are relevant in human cerebral or spinal gliomas, we undertook immunohistochemical analyses of rare human spinal cord gliomas to determine the presence of BCRP, CD133 (glioma progenitor marker), matrix metalloproteinase inducer EMMPRIN (which regulates HA synthesis), and multi-lineage stem/progenitor marker nestin. Twelve paraffin-embedded human spinal cord gliomas ranging from WHO grade 1 to 4 obtained from the MUSC Tumor Bank were used in this analysis. Most tumors exhibited an increased expression of the above antigens, including both grade IV spinal cord glioblastomas. It is not known if abundant BCRP+ cells in high grade human spinal cord gliomas are tumor cells, tumor progenitor cells, or recruited non-glioma progenitors (as observed in our rat spinal model). High levels of expression of CD133 and BCRP in subcutaneous xenografts of human cerebral glioblastomas in nude mice suggest BCRP could well be expressed at high levels in tumor cells and not just in recruited progenitors. Taken together, these data suggest that BCRP+ cells present in our animal model are present in both cerebral and spinal cord human gliomas, which is indeed confirmed via confocal microscopy of human brain GBM staining for BCRP, CD133, and CD44. Imaging confirms tumor-wide presence of a subpopulation of cells with BCRP+/CD133+ phenotype both adjacent and distal to BCRP+ vessels. Further studies are needed to determine the origin(s) and function of BCRP+ cells in gliomas now that their presence has been confirmed in the human disease.
Michael P. O'Quinn
Mentor: Dr. Rob Gourdie
A Peptide Incorporating a Carboxy-Terminal Domain of Cx43 Improves Recovery of Cardiac Function Following Injury
Michael P. O'Quinn (1), Brett S. Harris (1), Tim C. McQuinn (2,1), Richard P. Visconti (1), Kyu-Ho Lee (2,1), Carol A. Eisenberg (1), Leonard M. Eisenberg (1), and Robert G. Gourdie (1)
(1) Department of Cell Biology and Anatomy, (2) Department of Pediatrics, Medical University of South Carolina, Charles P. Darby Children's Research Institute, Charleston, South Carolina, USA
Disease-associated remodeling of Cx43 gap junctions (GJ) has been linked to increased incidence of cardiac arrhythmias. We have previously shown that dynamic changes in interaction between Cx43 and the actin-binding protein ZO-1 are an important determinant of GJ size and distribution in cardiomyocytes (Hunter et al., 2006). As part of this work we developed a membrane-permeant peptide incorporating a carboxy-terminal domain of Cx43, designed to competitively inhibit the interaction between Cx43 and ZO-1. Subsequent investigations demonstrated that treatment of cutaneous wounds with the Cx43 peptide, in a pluronic gel, enhanced wound healing; decreasing inflammation and accelerating closure, as well as improving mechanical properties and decreasing granulation tissue deposition. Based on these results, we initiated studies of the effects of the peptide in cardiac wound healing. Since coronary artery ligation produces injuries of variable size and geometry, we developed a novel epicardial cryoinjury model that enabled production of a discrete wound displaying minimal variation. Additionally, we re-formulated peptide delivery for cardiac application from pluronic gel to an adherent methylcellulose patch to provide timed, localized release. Echocardiographic studies of cryoinjured hearts treated with the Cx43-based peptide exhibited significantly improved ventricular function (as evidenced by reduced dilation) compared to groups exposed to a control peptide. Interestingly, this change was most pronounced at seven days, though it persisted throughout the 8-week time course of the experiment. Quantitative histological analysis of these hearts showed that treated scars have more uniform collagen organization, which may explain decreased dilation occurring in response to the peptide. Cryoinjury of transgenic mice expressing a LacZ reporter under the control of a GATA-6 enhancer indicates upregulation of a fetal gene program in association with injury. In preliminary observations, the level of LacZ expression is increased in cryoinjuries treated with the Cx43 based peptide. In conclusion, our data suggest a Cx43 carboxy-terminal sequence may upregulate an adaptive response to cardiac injury that results in an inhibition of dilation.
Joseph A. Palatinus
Mentor: Dr. Rob Gourdie
The effects of a gap junction derived peptide on diabetic skin wounds
Joseph A. Palatinus, Gautam Ghatnekar, and Robert G. Gourdie
Department of Cell Biology and Anatomy, Children's Research Institute, Medical University of South Carolina, Charleston, SC 29403, USA
Diabetes is the 6th leading cause of death in the United States and over 20
million people currently suffer from this metabolic disease. One of the most
devastating complications of the diabetic condition is the decreased ability to
heal. Chronic wounds are a hallmark of diabetic patients. These wounds
frequently appear in the distal limbs and are refractory to standard wound care
measures. Diabetic wounds frequently become gangrenous and require limb
amputation presumably because of microvascular occlusion resulting in decreased
tissue perfusion and intercellular communication. There is a need for new
interventions to increase the rate of diabetic wound healing. Our lab has
developed a peptide (dubbed ACT1) composed of the carboxyl terminus of the gap
junction protein Connexin 43. This peptide has been shown to increase the rate
of wound repair and decrease scar tissue formation in skin wounds of normal CD1
mice. Currently studies are underway to determine the efficacy of the peptide
in diabetic models of injury.
Juan C. Varela
Mentor: Dr. Steve Tomlinson
Downregulation of Crry on tumor cells leads to the induction of an anti-tumor T-cell response in a metastatic model of bladder cancer
Juan Carlos Varela, Masaki Imai, Carl Atkinson and Stephen Tomlinson
Department of Microbiology and Immunology, Children's Research Institute, Medical University of South Carolina, Charleston, SC 29403, USA
It is believed that many types of tumors protect themselves from the effects of the complement system by upregulating the expression of membrane-bound complement inhibitors on their cell surface. Complement inhibitory proteins expressed on cancer cells can provide protection from anti-tumor antibodies and may modulate the induction of an immune response to tumor-associated antigens. In the current set of studies we investigated: (1) the mechanisms leading to the upregulation of complement inhibitors on tumors from bladder cancer patients, and (2) the consequences of complement inhibitor downregulation on the effector and inductive phases of an immune response to bladder cancer in a syngeneic model of mouse bladder cancer.
Paired samples of tumor and normal tissue from 22 bladder cancer patients were analyzed for expression of MUC1, CD46, CD55 and CD59, and matched serum samples analyzed for anti-MUC1 IgM and IgG levels. MUC1 was upregulated in 86% of tumor samples. CD46 was upregulated in 77%, CD55 in 55% and CD59 in 59% of tumors. Analysis of the relationship between anti-MUC1 antibody levels and complement inhibitor expression revealed a significant correlation between the expression of the complement inhibitors CD46 and CD55 on tumor cells and the presence of IgM and IgG anti-MUC1 antibodies in the serum of bladder cancer patients. Hence, we propose a mechanism where selection of tumor cells with higher expression of complement inhibitors is mediated by antibodies against the tumor antigen MUC1.
In a separate set of studies, stable siRNA-mediated downregulation of the complement inhibitor Crry led to an increase in C3 deposition and complement-mediated lysis of bladder cancer cells in vitro. In vivo studies determined that mice injected i.v. with bladder cancer cells expressing lower levels of Crry had a significant decrease in tumor burden and a significant increase in survival compared to mice inoculated with bladder cancer cells expressing normal levels of Crry. While anti-tumor antibody responses were detected in the mice injected with Crrylow tumor cells, the decreased tumor burden and improved survival was determined to be caused by the enhancement of an anti-tumor T cell response and was dependent, at least in part, on a functional complement system. The current data indicates that the complement system and complement inhibitors play a significant role in the induction and/or enhancement of T cell responses against tumors in a syngeneic model of mouse bladder cancer.
Khaled Moussawi
Mentor: Dr. Peter Kalivas
Rewiring Cocaine Addiction: The Role of N-Acetylcysteine in Reversing Impaired Plasticity after Chronic Cocaine
Department of Neurosciences, Children's Research Institute, Medical University of South Carolina, Charleston, SC 29403, USA
A major feature of cocaine addiction is the vulnerability to relapse. This vulnerability is thought to be rooted in the long term neuroadaptations in the neurocircuitry comprising the cognitive-motor interface. While some electrophysiological studies have tried to assess neuroplasticity in the ventral striatum after chronic withdrawal in brain slices, we looked into the prefrontal-accumbal changes after prolonged withdrawal from cocaine self administration in vivo. To address this question, we measured the ability of this pathway to undergo long term potentiation (LTP) and long term depression (LTD) in vivo. Rats were trained to self-administer cocaine for 10 days, followed by 21 days withdrawal (2 weeks extinction and 1 week abstinence). Field potentials in the dorsomedial nucleus accumbens were recorded after ventral prelimbic prefrontal cortex stimulation. LTP induction (2 trains of50 Hz, 2seconds each) resulted in around 50% potentiation in the yoked saline animals, and was severely impaired in the cocaine group (<10% potentiation). LTD (3 trains of 5 Hz, 3minutes each) was also blunted (~20% depression) after chronic withdrawal as compared to the control (~55% depression). Interestingly, systemic injection of N-acetylcysteine (100mg/kg), an antioxidant related to glutamate homeostasis, restored both LTP and LTD in the chronic cocaine animals. Together, these results indicate that previous hypotheses suggesting that addiction is associated with an enduring LTD state at the excitatory synapses in the accumbens are incomplete. Rather, a loss of both LTD and LTP indicates that cocaine addiction shifts the accumbens into a pathological state that is deficient in physiological adaptation to the new, important changes in afferent stimulation embodied in LTP and LTD. It is possible that this state may underlie decreased reinforcement of natural rewards and increased vulnerability to relapse observed in cocaine addiction.
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