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MSTP Student Research Day
Mohammad Dahrouj (back to top)
Using SD-OCT to Study the Effect of VEGF on Fluid Transport Across the RPEin vivo
Mohammad Dahrouj, Jake Chambliss McMillin, Craig E. Crosson,
Purpose:Macular edema is a common symptom in several eye conditions and a major reason for the loss of visual acuity. Anti-VEGF therapy is thought to be effective in treating edema by targeting the leakiness of retinal vessels. However, it is the retinal pigment epithelium (RPE) that maintains the outer retina in a dehydrated state. Our laboratory has recently shown that VEGF can regulate the barrier function of the RPE in vitro. Utilizing spectral domain optical coherence tomography (OCT), we have developed an in vivo model system to study the effects of VEGF on RPE barrier function and fluid transport.
Methods:Rabbits were anaesthetized, intraocular pressure (IOP) was maintained at 10, 15 or 20 mmHg. Subretinal blebs (1-5mL) containing PBS,(100ng/mL) of BSA (100ng/mL), VEGF (100ng/mL),or PLGF (100ng/mL)were created. Bleb reabsorption was followed for 90 minutes with infrared fundus images and high resolution OCT scans across the entire bleb-filled area. In selected experiments 5 mL of ZM323881 (10mM), a relatively selective VEGF-R2 receptor inhibitor, was injected into the vitreous one hour prior to the creation of VEGF-filled blebs. Fluorescein angiograms (100 mg/kg, intravenous injection) were used to visualize RPE barrier leakage.
Results:In blebs containing PBS and at IOP of 10 mmHg fluid transport across the RPE was 6±1.2 mL/cm2hr. Raising IOP to 15 and 20 mmHg increased transport rates to 15±1.2 and 23±1.8 mL/cm2hr respectively. Blebs containing BSA exhibited a fluid transport rate of 5.3±1.2 mL/cm2hr. VEGF containing blebs did not show any significant reabsorption over the time blebs were monitored. Pretreatment with ZM323881 inhibited the VEGF induced effect on fluid transport across the RPE.PLGF did not cause any statistically significant alteration in the rate of reabsorption compared to BSA. VEGF-filled blebs showed significant early-phase fluorescein leakage while PBS-filled bleb did not show any early phase leakage.
Conclusions: In these experiments, OCT technology was successfully applied to increase our understanding of basic physiological properties of the RPE barrier. The result that VEGF induces a significant reduction in the reabsorption is consistent with a localized breakdown of the RPE barrier function. These data establish the RPE as an important factor in the pathophysiology of edema and VEGF as a major cytokine that can modulate
RPE function in vivo.
Thomas Beckham (back to top)
Sphingosine 1-Phosphate And Acid Ceramidase Modulate The Subcellular Localization Of The PTEN Tumor Suppressor
Thomas H. Beckham, Joseph C. Cheng, Ping Lu, Tucker Marrison, Xiang Liu, James S. Norris
Department of Microbiology and Immunology, Medical University of South Carolina, Charleston, South Carolina
PTEN is a critically important tumor suppressor that canonically functions as a PIP3,4,5 phosphatase, antagonizing PI3K and the activation of Akt. More recently PTEN has been found to have tumor suppressive roles away from the cytoplasmic membrane including in the nucleus where it regulates cell cycle progression and chromosome stability, however the disease-relevant mechanisms that influence PTEN cytoplasmic/nuclear trafficking remain unidentified or poorly characterized. The pleiotropic signaling lipid sphingosine 1-phosphate (S1P) signals through 5 GPCRs to affect diverse cell signaling pathways, often promoting oncogenic events. Investigating the impact of S1P on PTEN in prostate cancer cells, we determined that S1P, through PI3K and Akt activation, promotes a reduction in nuclear PTEN and an increase in cytoplasmic PTEN. Previous work on PTEN nucleocytoplasmic localization implicate conformational changes associated with phosphorylation of the c-terminus in dictating trafficking between the nucleus and the cytoplasm. We find that phosphorylated PTEN seems to preferentially be involved with S1P induced alterations in PTEN localization, leading us to propose a model by which S1P, through Akt activation, promotes nuclear egress of phoshoporylated PTEN. As the tumor suppressive functions of PTEN have been shown to be dose-dependent, we believe that S1P signaling may promote nuclear insufficiency of PTEN tumor suppression, possibly promoting oncogenic changes.
David Perry (back to top)
Differential Regulation of IL-6 Production by Sphingolipid Catabolism: Role of Acid Sphingomyelinase in p38 Activation
David M. Perry, Benjamin Newcomb, Bill Wu, Kazuyuki Kitatani,
Yusuf A. Hannun
Department of Biochemistry, MUSC
Abstract: Interleukin-6 (IL-6) is a pleiotropic cytokine that is involved in many aspects of cancer biology including tumorigenesis, invasion, and metastasis. Previous work from our laboratory has shown that catabolism of glycosphingolipids by glucocerebrosidase (acid beta-glucosidase, GBA) is a negative regulator of IL-6 production in MCF-7 breast cancer cells. Upon further investigation, it was found that loss of Acid Sphingomyelinase (ASM) resulted in the inhibition of IL-6 production in response to the phorbol ester, 4 beta-phorbol 12-myristate 13-acetate (PMA), suggesting that ASM is a positive regulator of IL-6 production in contrast to GBA. Inhibition of ASM in HeLa cells also reduced IL-6 production in response to TNF-alpha. Furthermore, loss of ASM in MCF-7 lead to a decrease in p38 phosphorylation in response to PMA, demonstrating a role for ASM upstream of p38. Further investigation is needed to elucidate the mechanisms by which ASM and GBA regulate p38 and to define the pathophysiological consequence of ASM functions in cancer and inflammatory disorders.
Sarah Brice (back to top)
The Role of Sphingolipids and (Dihydro) Ceramide Synthase 5 in Diabetic Cardiomyopathy in Mice
Sarah E. Brice, Catalin F. Baicu, An Van Laer, Tuoyu Geng, Harinath Kasiganesan, Michael R. Zile, L. Ashley Cowart
Diabetic cardiomyopathy (DbCM), which consists of cardiac hypertrophy and failure in the absence of traditional risk factors, is a major contributor to the increase in heart failure risk experienced by patients with Type 2 diabetes. In transgenic rodent models of DbCM, cardiac hypertrophy and dysfunction have been shown to depend upon fatty acid oversupply and de novo sphingolipid synthesis in cardiomyocytes. However, it is not known whether these effects are mediated by bulk saturated fatty acids (SFA) and sphingolipids or by individual lipid species. In this study, we reveal that a diet high in SFA induced cardiac hypertrophy, left ventricular systolic and diastolic dysfunction, and autophagy in wild type mice. Furthermore, treatment with the SFA myristate (C14:0), but not palmitate (C16:0), induced hypertrophy and autophagy in adult primary cardiomyocytes. Strikingly, de novo sphingolipid synthesis was required for induction of all of the pathological features observed both in vitro and in vivo, and induction of hypertrophy was dependent on sphingolipid-dependent autophagy in vitro. Finally, we implicated a specific ceramide N-acyl chain length in this process and demonstrated a requirement for (dihydro)ceramide synthase 5 (CerS5) in cardiomyocyte autophagy and myristate-mediated hypertrophy. Thus, this report revealed, for the first time, a requirement for a specific sphingolipid metabolic route and a in individual dietary SFA in the molecular pathogenesis of lipotoxic cardiomyopathy and hypertrophy.
Alton Sutter (back to top)
TLR4 Knockout Protects Obese Mice from Steatosis Associated Live
Inflammation and Injury
Sutter, AG; Chavin, KD
Studies in animal models and humans indicate a link between Toll-like receptor 4 (TLR4), metabolism, and non-alcoholic steatohepatitis. We were curious what the long-term effects of TLR4 knockout would be upon the development of steatosis and inflammation within the liver. TLR4/Leptin double KO mice were generated through cross breeding of C57BL/6 mice carrying the TLR4 KO allele, Tlr4lps-del, with those carrying the leptin KO, or Lepob, mutation. Mice were then allowed access to normal chow, ad libitum. At 5 and 9 months mice were sacrificed and serum ALT and AST were measured. Real-time PCR was used to examine mRNA expression of cytokines, intracellular adhesion molecules, and other genes associated with progression of NAFLD and NASH. Liver tissue was fixed and stained for histology. ob/ob mice showed elevated serum ALT and AST levels, markers of hepatocellular injury. In addition, histology revealed increased inflammatory cell infiltrate and hepatocellular ballooning, as compared to their lean counterparts. TLR4 KO ameliorated these changes. Furthermore, ob/ob mice displayed elevated expression of IL-1b, ICAM-1, TNFα, and TGF-β. These changes were also not present in the TLR4 KO ob/ob animals. These data indicate: 1) leptin is not necessary for the development of inflammation in the steatotic liver, 2) hyperphagia and subsequent steatosis alone are sufficient for the development hepatic inflammation and a NASH-like phenotype, 3) TLR4 is integral to the development inflammation in the setting of steatosis without a second insult.
Obaidullah Aseem (back to top)
The Epigenetic Regulation of Multi-Ligand Receptor Cubilin
Obaidullah Aseem, Brian T. Smith, Marion A. Cooley and
W. Scott Argraves
Department of Regenerative Medicine and Cell Biology, Medical University of South Carolina, Charleston, SC 29425 USA
Cubilin is a peripheral membrane, glycoprotein receptor capable of mediating the endocytosis of multiple ligands including High Density Lipoprotein apolipoprotein A-I and albumin. Cubilin is highly expressed on the epithelium of intestinal villi and renal proximal tubules. In the kidney, endocytosis mediated by cubilin and its co-receptor megalin is the only established mechanism for the proximal tubule reabsorption of proteins filtered through the glomerulus. As such, cubilin is implicated in proteinuria associated with nephropathy, including diabetic nephropathy, acute kidney injury and chronic kidney disease. Despite its functional importance in health and disease, little is known about the regulation of cubilin expression. In this study, we have discovered evidence of epigenetic regulation of cubilin and co-receptor megalin. Through study of mice heterozygous for targeted cubilin gene deletion and EGFP knockin, we show that cubilin in the proximal tubule epithelial cells is expressed in a monoallelic fashion. Monoallelic gene expression is often regulated by epigenetic processes such as DNA and histone methylation and histone acetylation/deacetylation. Thus, we treated NRK-52E and Caco2 cells with the DNA methyltransferase inhibitor 5-Azacytidine (5Aza) and Histone Deacetylase (HDAC) inhibitor Trichostatin A (TSA). Treatment with either 5Aza or TSA led to a dose dependent increase in the expression of both cubilin and megalin. These findings have implications for the therapeutic regulation of cubilin and megalin expression in kidney disease. Interestingly, previous studies demonstrated that HDAC inhibitor treatment in mice resulted in attenuation of chronic renal injury and reduced proteinuria. We are currently focused on studying the effects of 5Aza and TSA treatment on cubilin and megalin expression in a mouse model of kidney injury.
Brandon Bentzley (back to top)
Microinjection Of Muscimol Into The Subthalamic Nucleus Attenuates Cocaine Consumption At High Price
Brandon Bentzley and Gary Aston-Jones
The current study determined the role of the subthalamic nucleus (STN) in regulating cocaine demand (consumption) as a function of cocaine price. Male Sprague-Dawley rats were given 0.3 μL bilateral microinjections into the STN of either artificial cerebrospinal fluid (aCSF) or the GABAA receptor agonist muscimol (0.4 mM) prior to threshold procedure testing. The threshold procedure exposes animals to a series of decreasing doses of intravenous cocaine under a fixed ratio-1 schedule of reinforcement in a single 110-min operant session. The dose is decreased every 10 min, effectively raising the unit price of cocaine; i.e., the number of responses required to obtain 1 mg of cocaine. We found that muscimol pretreatment significantly attenuated the demand of cocaine at high price but did not affect demand at the lowest price point. Hence, function of the STN is required to drive cocaine demand when price is high.
Fahmin Basher (back to top)
Effects of Fli1 on T Cell Function in Systemic Lupus Erythematosus
Fahmin Basher, Zainab Amani, Marlene Bunni, and Tamara Nowling
Dept of Microbiology & Immunology, Dept of Medicine/Rheumatology, Ralph H. Johnson VA Medical Center, Charleston, SC
Lupus is an autoimmune disease characterized by abnormal activation of T and B cells and consequent production of autoantibodies, inflammation, and deposition of immune complexes in peripheral organs. Overexpression of the transcription factor Fli-1 in non-autoimmune mice leads to a lupus-like disease state, while reduction of Fli-1 expression results in decreased T cell infiltration in the kidney as well as decreased inflammation and increased survival. In this study we investigated effects of Fli-1 on T cell function in both non-autoimmune (C57BL/6) and autoimmune (MRL/lpr) mouse models with respect to proliferation and apoptosis after stimulation with PMA/ionomycin and anti-CD3/anti-CD28. While a decrease in proliferation after stimulation with PMA/ionomycin was seen in wildtype B6 mice compared to Fli-1+/-, no differences were observed in pre-disease (10-12 wk) MRL mice or diseased (>17 wk) mice, although proliferation of CD8+ T cells was significantly increased in diseased Fli1+/- mice. Fli-1’s known anti-apoptotic effects were observed in the B6 mice, with a decrease in early apoptosis in wildtype mice compared to Fli-1+/-, and a similar trend was seen in the MRL model with pre-disease but not diseased mice. Preliminary studies using anti-CD3/anti-CD28 stimulation in T cells isolated from diseased mice demonstrate no significant differences in overall proliferation but a trend towards decreased CD8+ proliferation in contrast to stimulation with PMA/ionomycin. In addition, analysis of cytokine production by diseased MRL/lpr mice show trends toward reduction of IL-4, IL-6, and IL-13, increases in IL-10 and IL-17a, and no changes in IFNy, TNF, and IL-2 in Fli1+/- mice vs. wildtype. The presence of surface receptors in both pre-disease and diseased mice, specifically follicular T cell markers CXCR4 and CXCR5 and regulatory T cell markers CD25 and CTLA-4, were also decreased in unstimulated and stimulated T cells from Fli1+/- mice vs. wildtype. We adoptively transferred T cells isolated from young wildtype MRL/lpr mice into Fli-1+/- mice, and vice versa and monitored serum antibodies and proteinuria over 12 weeks. IgG levels in Fli-1+/- mice receiving wildtype cells did not increase significantly above levels of Fli-1+/- controls, although wildtype mice receiving Fli-1+/- cells did not exhibit a decrease in IgG levels. However, the transfer of T cells with lower levels of Fli-1 reduced proteinuria levels in wildtype recipients, while the transfer of wildtype T cells to Fli-1 +/- recipients only modestly increased proteinuria levels. These results indicate that presence of lower levels of Fli-1 in endogenous or transferred T cells may have a protective effect on the levels of circulating autoantibodies but perhaps more profoundly on progression of renal disease.
Haley Buff Lindner (back to top)
pGlcNAc Nanofibers Stimulate A Scarless Wound Healing Program
Haley Buff Lindner1, Aiguo Zhang4, Juanita Eldridge1,
Marina Demcheva3, Arun Seth4, Philip Tsichlis5, Rick Visconti1
Amy Bradshaw2, John Vournakis3 and Robin C. Muise-Helmericks1
1Department Regenerative Medicine and Cell Biology and 2Department of Medicine, Medical University of South Carolina, Charleston, SC 29425, USA, 3Marine Polymer Technologies, Inc., Danvers, MA 01923 , 4Sunnybrook Research Institute, University of Toronto, Ontario, Canada, M5G 1G6 and 5Tufts University Medical Center, Boston, MA
Our published findings show that treatment of cutaneous wounds with poly-N-acetyl-glucosamine nanofibers (pGlcNAc), a novel polysaccharide material derived from a marine diatom, results in increased kinetics of wound healing and in innate immune responses resulting in antibacterial activities in infected wound models. We have also shown that the serine threonine kinase, Akt1 plays a central role in the regulation of pGlcNAc-induced activities. Here, we show that pGlcNAc treatment also results in decreased scar sizes as compared to untreated wounds. In correlation with decreased scaring, treatment of cutaneous wounds with pGlcNAc results in increased tensile strengths and elasticity as compared to untreated wounds; equal to if not exceeding tensile strengths obtained using unwounded tissue. Visualization of collagen content using Masson trichrome staining suggests that pGlcNAc treated wounds display a reduction in collagen content and an organized collagen phenotype where collagen fibrils are aligned similarly to unwounded tissue, suggesting a “scarless” wound healing program. Indeed, pGlcNAc treatment reduces smooth muscle actin staining within the wound, suggesting a reduction in myofibroblast content. Using whole transcriptome analyses we show that a series of interferon responsive genes are up-regulated by pGlcNAc treatment. We have confirmed that treatment with pGlcNAc nanofibers results in an increased expression of Epithelial Stromal Interaction Protein 1 (EPSTI1), a novel protein involved in epithelial/stromal interactions. pGlcNAc stimulation of EPSTI1 expression is dependent on Akt1, both in vitro and in vivo. Interestingly, co-staining of EPSTI1 and Hsp47 (a collagen chaperone) shows that this protein is up regulated in properly aligned collagen producing cells. Taken together, our findings suggest that pGlcNAc nanofibers stimulate an Akt1 dependent pathway that results in the proper alignment of fibroblasts by a mechanism that is dependent EPSTI1 leading to decreased scaring and increased tensile strength.
Bryce Johnson (back to top)
Total body irradiation enhances the anti-tumor efficacy of adoptively transferred CD8+ T cells stimulated ex vivo with IL-12
C. Bryce Johnson1,2, Colleen A. Cloud1, Mark P. Rubinstein1,2 and David J. Cole1
Departments of Surgery1 and Microbiology/Immunology2 at MUSC
Stimulation of T cells with IL-12 improves their cytolytic capabilities and ability to persist in vivo. Despite these enhanced functional characteristics, we have found that these cells can only eliminate established tumors in mice preconditioned with cyclophosphamide (CTX). While this result demonstrates the importance of lymphodepletion in adoptive cell transfer (ACT) regimens, it is unclear if other forms of lymphodepletion would also enhance the efficacy of IL-12 primed cells. Since the most successful lymphodepleting regimen in metastatic melanoma clinical trials uses total body irradiation (TBI), we sought to determine if TBI preconditioning combined with ACT of IL-12 stimulated pmel-1 CD8+ T cells might be similarly synergistic. When we adoptively transferred pmel-1 CD8+ T cells primed with antigen and IL-12 (pmelIL-12) into mice preconditioned with TBI, we saw significantly elevated numbers of donor T cells compared with mice adoptively transferred with pmel-1 CD8+ T cells primed with antigen alone (pmelsham).
Importantly, the increase in donor T cell numbers seen with the combination of TBI + pmelIL-12 translated to significant delays in melanoma outgrowth. Strikingly, the addition of pmelsham to TBI preconditioning resulted in no improvement in anti-tumor immunity compared to TBI alone. Thus both CTX and TBI host preconditioning greatly enhance the anti-tumor efficacy of transferred T-cells stimulated with IL-12.
Anthony Leonard (back to top)
High Content Imaging-based Automated Classification of Mitochondrial Morphology
Anthony Leonard1,2, Yuri Peterson2, Nathan Perron2, Craig Beeson2, Baerbel Rohrer1,3
1 – Dept. of Ophthalmology, 2 – Dept. of Pharmaceutical Sciences, 3 – Dept. of Neurosciences
Mitochondrial morphology is one indicator of mitochondrial function that has been used to assess neurodegenerative processes. Mitochondrial dysfunction is implicated in the inherited retinal degeneration Retinitis Pigmentosa (RP). Based on the hypothesis that preservation of mitochondrial function would aid in photoreceptor survival, our lab used an in vitro model of RP (661W photoreceptor cell line) to screen for small molecules (termed the CB’s) that rescue cells from Ca2+-mediated mitochondrial toxicity. We verified via Seahorse respirometry that these molecules assist in preventing decreases in mitochondrial respiration. Subsequently, we have demonstrated efficacy of a series of these compounds in protecting photoreceptor viability in organ culture models and in an in vivo model of oxidative stress. In 661W cells, mitochondrial fission protein Drp1 levels are decreased ~20%; meanwhile, the fusion-promoting protein mitofusin 2 levels increase ~75% with CB12 treatment. Interestingly, preliminary results using confocal microscopy show a significant induction of mitochondrial fusion with CB12 treatment in these cells. However, current measurement of mitochondrial morphology depends on subjective visual classification of mitochondrial subtypes; a technique prone to sampling and observer bias. In order to avoid these biases in our morphology analyses, we developed a computational method for classifying mitochondria from high content imaging. For the first time, we report the morphological quantification of every single mitochondrion in hundreds of thousands of individual cells. Morphologies are binned into fragmented, swollen, rod-like, and networked subtypes. Small variants in these morphological subpopulations defined by mitochondrial complexity, size, and shape were reliably quantified. The image acquisition, analysis pipeline, and results will be discussed in the context of our pharmacological model of RP.
Keely Morris (back to top)
Pathogenic Natural IgM Antibodies Initiate The Inflammatory Response Important For Both Hepatic Ischemia/Reperfusion Injury And Liver Regeneration
After Partial Hepatectomy
Keely Morris, Fei Qiao, Song He, Carl Atkinson, L Kulik,
Michael Holers, Stephen Tomlinson
Complement activation and inflammation are linked to both hepatic ischemia/reperfusion injury (IRI) and to liver regeneration. The complement activation products C3a and/or C5a are known to be involved in the priming phase of liver regeneration. Here, we report on the complement activation event and initiation of a post-ischemic and post-resection inflammatory response. Previous studies using models of intestine, heart, brain and hindlimb IRI have shown that natural IgM Abs trigger complement activation upon reperfusion. These natural Abs bind to neoepitopes exposed on post-ischemic endothelium and activate the lectin and/or classical pathway to induce injury that is dependent upon alternative pathway amplification. Utilizing separate models of hepatic IRI and 70% partial hepatectomy (Phx), we characterized injury and regeneration in Ab-deficient (Rag1-/-) mice, with and without reconstitution with different IgM mAbs. Rag1-/- mice were protected from hepatic IRI, but had significantly increased injury and an impaired regenerative response after Phx. Injury in the IRI model (ALT, necrotic index) and the regenerative response in the Phx model (mitotic index, BrdU staining) were restored to wild type levels in Rag1-/- mice injected of B4 or C2 IgM mAbs, specific for annexin IV and a subset of phospholipids, respectively. Analysis of liver sections after either IRI or PHx showed IgM deposition co-localized with C3 deposition. There was no detectable IgM or C3 in sections from untreated Rag1-/- mice. These studies show that different pathophysiologically important epitopes recognized by IgM natural Abs are similarly expressed after hepatic IR and Phx, and that these Abs play a critical role in initiating the inflammatory response that is important for injury after IR and for regeneration after PHx.
Laura Briggs (back to top)
The Role of Bone Morphogenetic Protein 4 in the Developing Inflow and Outflow Regions of the Heart
Laura Briggs, Jayant Kakarla, Aimee Phelps, Arno Wessels
Although it will eventually assume a four-chambered conformation, the heart initially develops as a tube consisting of little more than an outer ring of myocardium and an inner layer of endocardium. Blood flows through this linear tube in one direction only, entering at a region termed the inflow (arterial pole) and exiting through the venous pole. While the initial tube is derived from a precursor population of cells termed the primary heart field, additional components are added at inlet and outlet extremities by cells of the second heart field. Eventually, a four-chambered organ results, with cells of the primary heart field contributing primarily to the left ventricle and second heart field cells forming the right ventricle and the outflow tract. Prior studies have indicated that the growth factor Bone Morphogenetic Protein 4 (BMP4) is essential for proper heart development, possibly by playing a role in neural crest migration and epithelial-to-mesenchymal transformation. In order to determine the role BMP4 plays in the formation of both the arterial and venous poles of the heart, we have utilized a cre-lox strategy to conditionally delete the BMP4 receptor Alk3 from the second heart field. Further illustrating the importance of this growth factor in the development of the heart, we have found both inflow and outflow abnormalities in mutant mice. Here, we will histologically and immunohistochemically describe these mutants and further characterize the role of BMP4 in the developing heart.
David Perry (back to top)
REGULATION OF p38ð BY Gba: MECHANISTIC INSIGHTS AND IMPLICATIONS FOR DISEASE
David M. Perry, Kazuyuki Kitatani, Patrick Roddy, and Yusuf Hannun
Department of Biochemistry and Molecular Biology, MUSC
Sphingolipids have long been implicated in stress responses while molecular mechanisms have been more elusive to delineate, particularly for ceramide. Complexity of sphingolipid/ceramide biology arises from several factors: structural diversity within a given class, subcellular restriction of the sphingolipid-metabolizing enzymes, and intricate metabolic pathways involving the enzymes and sphingolipids. Therefore, studying ceramide in the context of a specific enzyme is important as this may dictate its function and downstream target. One such enzyme is Gba (glucosylceramidase, EC-18.104.22.168), which is proposed to have an anti-inflammatory role, distinguishing it from other ceramide-producing enzymes. Previous work from our lab has shown that loss of Gba results in decreased ceramide formation leading to hyperphosphorylation of p38 and overproduction of IL-6 in breast cancer cells, with indirect evidence that the p38ð isoform is preferentially hyperphosphorylated by loss of Gba. In an effort to identify novel targets of Gba-derived ceramide, we discovered PP2Cα (PPM1A), a stress-related phosphatase, to be activated by ceramide. Interestingly, both IL-6 and p38 are proposed to have pro-migratory and pro-metastatic roles in breast cancer, and conversely, PP2Cα has been reported to be a negative regulator of migration. Taken together, our overall hypothesis is that Gba-derived ceramide activates PP2Cα to dephosphorylate p38ð as part of a negative feedback mechanism in stress signaling, which plays a role in suppressing invasiveness of breast cancer cells.
Joseph Cheng (back to top
Sphingolipid Metabolism In The Radiation Resistance And Relapse Of Prostate Cancer
Joseph C. Cheng,1 Xiang Liu,1 Thomas H. Beckham,1 Thomas E. Keane,2 Yusuf A. Hannun,3 James S. Norris.1 Departments of 1Microbiology & Immunology, 2Urology, and 3Biochemistry, Medical University of South Carolina, Charleston, SC, USA
The escape of a subset of prostate tumor cells from ionizing radiation (IR)-induced killing may lead to disease progression and relapse. Bioactive sphingolipids such as ceramide and sphingosine 1-phosphate (S1P) influence signal transduction pathways regulating stress response. In particular, enhanced ceramide metabolism into sphingosine and S1P constitutes an important cell survival adaptation of irradiated cells, and places ceramidase activity at the crux of this process. Enzyme activity assays, and mRNA and protein analyses demonstrated selective upregulation of the lysosomal ceramide-metabolizing enzyme acid ceramidase (AC) in cancer cells exposed to IR. In vitro modeling of fractionated radiotherapy (RT) applied to prostate cancer cells demonstrated logarithmic kill, but a majority of surviving clonogens constitutively over-expressed AC. Moreover, levels of AC expression correlated with radioresistance and proliferation rate, suggesting AC plays a role in response to and relapse after RT. Immunohistochemical analyses of prostate cancer tissues from failed RT patients revealed higher levels of AC than adenocarcinoma tissues at diagnosis, PIN, or normal tissues. Promoter-reporter and ChIP-qPCR analyses demonstrated IR transactivated AC through AP-1 activity that is sensitive to inhibition by myriocin or fumonisin, elaborating a critical feedback trigger of IR-induced ceramide generation via de novo biosynthesis. Radiosensitization of prostate cancer cells in vivo and in vitro by a dominant negative mutant of JUN was rescued, in part, by forced overexpression of AC. This work was sponsored by NCI P01 CA097132 and NCRR UL1 RR029882.
David Sentelle (back to top
Ceramide Mediates LC3B Dependent Mitochondrial Dysfunction
and Tumor Suppression
R. David Sentelle1,2 and Besim Ogretmen1,2
1Department of Biochemistry and Molecular Biology, 2Hollings Cancer Center
Ceramide is a signaling sphingolipid recognized for limiting tumor growth. Specifically, the product of Ceramide Synthase 1 (CerS1), C18-ceramide, regulates an unknown mechanism which limits head and neck squamous cell carcinoma growth. During the investigation of this mechanism we reconstituted C18-ceramide by a tetracycline inducible system and treatment with the analog C18-pyridinium ceramide (C18PC). Exogenous treatment of 22A cells with 10mM C18PC induced LC3B-II protein levels in the presence and absence of ZVAD-fmk, increased mature autophagosomes, and significantly increased trypan blue positive cells at 18 hr. Knockdown of both Atg3 and Atg7 significantly decreased the number of trypan blue positive cells. Similarly, Atg5 -/- mouse embryonic fibroblasts showed significantly less trypan blue positive cells as compared to the wild type. Conversely, Bax-/-Bak-/- and caspase 3-/-7-/- showed no protection from C18PC treatment. C18PC treatment at 2hr showed a significant increase in mitochondria (MTR) merged with autophagosomes (LTR) that correlated with a decrease in respiration.
Reconstitution of wt-CerS1 increased protein levels of LC3B-II, LC3B-GFP punctate, and decreased respiration after 24 hr induction when compared with reconstitution of a catalytically inactive mutant CerS1 (mut-CerS1). Furthermore, a significant decrease in ATP production by wt-CerS1 induced cells was rescued when the wt-CerS1 cells constitutively expressed LC3B shRNA. Next, the necessity of CerS1 was examined. Cells were treated with a common mitophagy inducer, Sodium Selenite (SS). Transient knockdown of CerS1 with RNAi prior to treatment with with 10mM of SS rescued the cells from increased autophagosome formation and engulfment of mitochondria resulting in decreased respiration. Confocal imaging following wt-CerS1 induction showed that ceramide-specific antibody, cfp-LC3B, and the mitochondria (MTR) colocalized. Generation of LC3B shRNA and non-targeting shRNA, wt-CerS1 cell lines showed a decrease in respiration rate that was rescued in the LC3B shRNA cell line. Xenografts with LC3B shRNA and non-targeting shRNA in combination with wt-CerS1 were established on the flanks of SCID mice. In vivo results showed that knocking down LC3B with shRNA enhanced tumor growth in the presence of wt-CerS1 while induction of wt-CerS1 with non-targeting shRNA reduced tumor growth. Overall, these findings suggest that C18-ceramide mediates an LC3B-dependent mitochondrial dysfunction pathway culminating in tumor suppression.
Support: This work was supported by the National Institutes of Health Award Number F30DE021295 from the National Institute Of Dental & Craniofacial Research and Award DE-016572 to BO. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institute Of Dental & Craniofacial Research or National Institutes of Health.
Joshua Smith (back to top)
Identifying Telomerase as a Target for Estrogen Neuroprotection Following Spinal Cord Injury
Joshua A. Smith, Sookyoung Park, Arabinda Das, Kenkichi Nozaki, James S. Krause, and Naren L. Banik
Spinal cord injury (SCI) is a devastating and debilitating disorder for which no cure exists. Multiple studies have demonstrated that estrogen (17b-estradiol; E2) has neuroprotective effects in the setting of CNS injury and neurodegenerative disorders. Previous work from our laboratory indicated that E2 attenuates cellular damage and restores locomotor function following SCI in rats even at low, physiologic doses (10 mg/kg). However, the mechanisms by which E2 exerts its neuroprotective effects remain largely unknown. Mounting evidence suggests that E2 may be potent modulator of both expression and activity of telomerase, a ribonucleoprotein complex involved in maintenance of the chromosome end and DNA repair. Recent studies have also demonstrated important roles for telomerase reverse transcriptase (TERT), the catalytic subunit of telomerase, in neuroprotection and normal CNS function. Thus, we hypothesized that E2 administration increases expression and/or activity of the telomerase enzyme in neurons and/or glial cells to promote neuroprotection. In order to determine the effects of E2 treatment on telomerase expression in vivo, male Sprague Dawley rats were subjected to moderately severe (40 gcm force) SCI and treated with 100 mg/kg Premarin, an FDA approved formulation containing 10% 17b-estradiol for 7 days post-injury. Western blot analysis indicated that Premarin treatment significantly increased expression of TERT in spinal cord injured animals when compared to vehicle alone. To further understand how estrogen may modulate expression/activity of telomerase in neural cells, primary cortical neurons cultured from the neonatal rat brain were also subjected to estradiol treatment in vitro for 24 h. Changes in expression of TERT and the telomerase RNA component (TERC) were determined at the mRNA level by reverse transcriptase-polymerase chain reaction (RT-PCR) and telomerase activity was assessed by the telomeric repeat amplification protocol (TRAP). Our preliminary findings indicate that E2 administration in vitro at doses up to 1 mM does not alter mRNA expression of either the telomerase reverse transcriptase (TERT) or its RNA template (TERC). Interestingly, E2 significantly enhanced telomerase activity as measured by the TRAP assay in a dose dependent manner. Taken together, these findings indicate that the telomerase enzyme may be an important mediator of the neuroprotective effects of E2. However, further work is necessary to determine whether TERT is essential for E2 neuroprotection and the mechanisms by which E2 modulates telomerase activity/expression.
This work was supported in part by funding from the National Institutes of Health National Institute of Neurological Disorder and Stroke (NS031622) and the State of South Carolina Spinal Cord Injury Research Fund.
Danielle Clark (back to top)
NAC and Vit D Treatment Improving Hypoxic Ischemic Injury in the Neonatal Rat Model
Danielle Clark1, Jessica Perkel1, Jamie Fraser2, Laura Grace Rollins3, Xingju Nie1, Inderjit Singh1, and Dorothea Jenkins1
1 Department of Pediatrics, MUSC, Charleston, SC 2 Pediatrics, Children's National Medical Center, Washington, DC 3 Clinical Psychology, University of Massachusetts Boston, MA
Neonatal hypoxic ischemic (HI) injury can be a devastating ending to an apparently normal pregnancy. Current standard of treatment is hypothermia, a neuroprotective therapy that decreases oxygen consumption and preserves energy levels in the brain. In this study, we compared hypothermia treatment alone, combination therapy with N-acetylcysteine (NAC), and triple therapy with NAC and Vitamin D in a neonatal HI rat model. NAC has antioxidant and anti-inflammatory properties and been shown to improve behavioral testing and infarct volume. Vitamin D (Vit D), an immunomodulator, is depleted during inflammatory processes. In one report, Vit D treatment was neuroprotective in neonatal hypoxic ischemic encephalopathy. We hypothesized triple therapy of hypothermia, NAC and Vit D will result in better outcomes than hypothermia treatment alone or with NAC in the rat HI model. Using a standard HI model on PND seven rats, we randomized ten rats to each treatment, giving IP injections of saline, NAC, or NAC and Vit D daily for seven days followed by PO for four days. Behavioral testing was done daily for negative geotaxis and every three days for other measures until sacrifice at eleven days post HI insult. Infarct volume, as measured by gross brain pathology scoring, showed a trend of improvement in triple therapy rats compared with other treatments. A trend in behavioral improvement with triple therapy was also observed in first day of successful negative geotaxis, measure of neurological reflexes and locomotion. Poor brain pathology scores showed a significant association with post injury poor weight gain or weight loss. Spleen weight was significantly adversely affected by eleven days post injury and correlated with both worse brain pathology scores and post injury weight loss. In conclusion, our data may provide insight into future possible treatment combinations as well as predicting outcome severity using a non-invasive measure.
Anna-Maria De Costa (back t top)
Administration Of A Vaccine Composed Of Dendritic Cells Pulsed With Premalignant Oral Lesion Lysate To Mice Bearing Carcinogen-Induced Premalignant Oral Lesions Stimulates A Protective Immune Response
Anna-Maria De Costa, Danielle Justis, Corinne Schuyler, Rita Young
The use of dendritic cell (DC) vaccines as treatment for malignancy is complicated by immune evasion tactics often employed by carcinomas such as head and neck squamous cell carcinoma (HNSCC). The present study aims to determine if an immune response can be elicited by administering a DC vaccine during the premalignant stages of HNSCC, prior to development of immune escape. Mice treated with the carcinogen 4NQO in drinking water develop premalignant oral lesions that progress to HNSCC. As previous studies demonstrated that premalignant lesions and HNSCC overexpress common tumor antigens, bone marrow-derived DCs were pulsed with premalignant lesion lysate (DCpm) and administered to 4NQO-treated mice exhibiting premalignant lesions. Lesion progression was tracked through endoscopy, which revealed that DCpm vaccination and control vaccination with dendritic cells pulsed with normal tongue epithelium lysate (DCnt) significantly decreased lesion burden at 8 weeks. Analysis of lymph node cells revealed that while DCnt vaccination resulted in a rapid increase in total lymphocyte count, levels of activated conventional CD4+ T cells and Th1, Tc1, Th17, Tc17, and Th2 cells, DCpm vaccination results in a delayed, yet substantial, increase in these immune effector mechanisms. This suggests that dendritic cell vaccination may have a beneficial effect on clinical outcome regardless of type of antigenic stimulation Also, pulsing DCs with premalignant lysate rather than normal tongue epithelium lysate affects the dendritic cells in a way that delays the immune effector response upon vaccination of premalignant lesion-bearing mice.
Michael DeFee (back to top)
Membrane-Associated Hsp90 Regulates NF-kB Activation
In Herpesvirus Oncogenesis
DeFee M, Qin Z, Dai L, Toole B, Isaacs J, and Parsons C
Numerous studies suggest that the transcription factor known as nuclear factor kappa B (NF-kB) plays a critical role in several processes integral to Kaposi's sarcoma-associated herpesvirus (KSHV) pathogenesis, including viral gene expression and the secretion of soluble mediators of angiogenesis. Our lab has previously demonstrated that Heat Shock Protein 90 associated with the cell surface (csHsp90) serves as a co-factor for KSHV gene expression during de novo infection. Therefore, using a specific inhibitor of csHsp90 (dimethylaminoethylamino-17-demethoxygeldanamycin-N-oxide or DNo) and monoclonal antibodies targeting csHsp90, we sought to determine whether csHsp90 regulates KSHV-mediated NF-kB activation and related downstream pathogenic events, including secretion of pro-angiogenic factors and cell migration. We found that targeting csHsp90 significantly reduced KSHV-mediated activation of NF-kB, secretion of VEGF and IL-8, and KSHV-initiated cell invasion. Furthermore, vector mediated NF-kB overexpression restored IL-8 and VEGF secretion in the presence of Dno treatment. These results suggest an important role for csHsp90 in KSHV-mediated NF-kB activation and the potential utility of targeting csHsp90 for the reduction of cytokine-mediated angiogenesis and KS tumor progression.
Russell Glenn (back to top)
Retrospective Noise Correction in Diffusional Kurtosis Imaging
Russell Glenn, Ali Tabesh
Radiology and Radiological Sciences, MUSC
Diffusional kurtosis imaging (DKI) is a novel, non-invasive, magnetic resonance imaging (MRI) technique that can be used to measure the properties of water diffusion in vivo to assess tissue microstructure and pathophysiology. DKI is an extension of conventional diffusion tensor imaging (DTI), which, unlike DTI, does not assume that water diffusion is Gaussian. Since movement of water in biological tissues is often non-Gaussian, DKI provides a more accurate representation of water diffusion patterns than DTI. The additional information contained in DKI has been shown to be useful in assessing disease-related tissue changes. To access this complementary information, MRI data should be obtained at higher diffusion weightings than required by DTI. This requirement poses a challenge since increasing the diffusion weighting results in a considerably lower signal-to-noise ratio than with DTI. A confounding factor is that the noise follows a non-negative, non-Gaussian distribution. The combination of having a relatively high positive expected value for the noise and a relatively low diffusion signal can significantly bias the measured signal and therefore result in inaccurate estimates of the diffusion parameters. This effect can be particularly dramatic in images acquired with small voxel size, thereby limiting the resolution of DKI, and in iron-rich regions of the brain, such as the globus pallidus, it can result in significantly biased estimates of diffusion parameters. Consequently, by removing the noise bias, it may be possible to improve the efficacy of DKI by increasing its accuracy and/or resolution. We present a retrospective method for noise bias removal from DKI images. A phantom study was performed to assess the efficacy of the method. The retrospective method was also tested on human DKI datasets. This method has been incorporated into Diffusional Kurtosis Estimator, the standard software platform for DKI post-processing and will be provided to researchers worldwide.
Daniel Grass (back to top)
Emmprin (CD147) Is A Novel Regulator Of Invadopodia Formation And Activity
G. Daniel Grass, Momka Bratoeva, Bryan P. Toole
A defining feature of malignant tumor progression is cellular penetration through the basement membrane and interstitial matrices that separate various cellular compartments. Accumulating evidence supports the notion that invasive cells employ specialized structures termed invadopodia, which are actin-based, lipid raft-enriched membrane protrusions abundant in membrane-type-1 matrix metalloproteinase (MT1-MMP) and several signaling proteins, to breach these structural barriers. CD147 (emmprin; basigin), an immunoglobulin superfamily protein that is associated with tumor invasion and metastasis, induces the synthesis of various matrix metalloproteinases in multiple systems. In this study we show that up-regulation of CD147 is sufficient to induce MT1-MMP expression, invasiveness, and formation of invadopodia-like structures in non-transformed, non-invasive, breast epithelial cells. We also demonstrate that CD147 and MT1-MMP are in close proximity within these invadopodia-like structures and in membrane sub-fractions with the properties of lipid rafts. Moreover, manipulation of CD147 levels in invasive breast carcinoma cells causes corresponding changes in MT1-MMP expression, invasiveness, and invadopodia formation and activity. These findings indicate that CD147 regulates invadopodia formation and activity, most likely via assembly of MT1-MMP-containing complexes within lipid raft domains of the invadopodia.
Craig Kutz (back to top)
Noncanonical, Rapid Downstream Crosstalk Between TGFβ and Retinoic Acid Signaling Pathways As Revealed By the Proximity Ligation Assay
Craig Kutz1, James Atkison2, Jayne Bernanke3, Steven Kubalak3; 1
Medicine, MUSC, 2Graduate Studies, MUSC, 3Regenerative Medicine and Cell Biology, MUSC
The importance of transforming growth factor beta (TGFβ) and retinoic acid (RA) signaling in a variety of physiological and pathological processes has been well documented. Yet, despite the traditional canonical roles of these signaling pathways independently, there are few reports exploring unconventional crosstalk between them. Our lab has previously found novel interactions of downstream Smad cofactors in TGFβ signaling directly binding to intracellular receptors for retinoid acid, retinoid X receptor alpha (RXRα). Importantly, this interaction takes place faster and at traditionally suboptimal concentrations of RA (low nM range), suggesting a role for unliganded retinoid receptors independent of transcriptional regulation. Using a novel form of in situ immunohistochemistry, the proximity ligation assay, direct intracellular protein-protein interactions between RXRα and TGFβ signaling cofactors were explored. We found that in addition to canonical interactions between Smad2 and Smad4, RXRα demonstrates direct interactions for both these TGFβ cofactors. In particular, we are the first to show that RXRα binds with Smad4. The RXRα/Sma4 interaction increased with TGFβ treatment, but was blunted with administration of RA.
These results are critical because they suggest that unliganded retinoid receptor modulation may be more extensive than originally perceived and may regulate heterologous signal pathways independent of retinoid receptor-driven transcription.
Whether these effects are a result of direct heterodimerization or a novel multimeric complex is the focus of future studies.
Tucker Marrison (back to top)
Sphingosine 1-Phosphate Receptor Signaling In Mesenchymal Stem Cells
S Tucker Marrison, Joseph C Cheng, Thomas H Beckham, Ping Lu, Xiang Liu, James S Norris
Mesenchymal Stem Cells (MSCs) are a pluripotent cell population acquired most prominently from bone marrow with the capacity to differentiate into osteoblasts, chondrocytes, adipocytes, cardiomyoctes, fibroblasts and other cell types. The immunoprivileged nature of these cells combined with their ability to home to sites of injury enhances therapeutic interest in this stem cell population. MSCs have been proposed and evaluated with some success in Phase I/II clinical trials as a therapeutic option after acute myocardial infarction (MI), graft vs. host diseases, spinal cord injury, and a number of other conditions. The signaling pathways that direct the maintenance, migration, and engraftment of these cells are therefore subject to extensive, ongoing research. One facet of MSC signaling that remains largely unexplored is that of sphingosine 1-phosphate (S1P) and its associated receptors. S1P is a biologically active sphingolipid with many known functions including proliferation, inhibition of apoptosis, inflammation, and angiogenesis. These activities are largely mediated by interactions with the 5 G-protein coupled S1P receptors (S1PR1-5). Serum S1P levels (100-600 nM) are significantly higher than both bone marrow and tissue levels and as such MSCs, in the process of homing to a site of injury, experience a dramatic increase in S1P stimulation followed by a subsequent decrease. S1P2, through its emphasis of G12/13 Rho dependent signaling, has been shown to promote and inhibitory role in migration and proliferation and to promote the enhancement of vascular barrier integrity. We have observed that S1P2 signaling is important for MSC maintenance. Inhibition of S1PR2 with competitive antagonist JTE013 in human hTert immortalized MSC results in increases phosphorylation of Akt and Erk, key mediators of MSC self-renewal, in the presence of S1P. Furthermore, this change is accompanied by increased proliferation and migration of the same cells with JTE treatment. Primary murine bone marrow derived MSCs show enhanced proliferation as assessed by clonogenic assay with JTE treatment in the presence of S1P. We therefore propose the role of S1P signaling as important for MSC maintenance and function.
Jamie Mills (back to top)
MicroRNA 510 as a Predictive Marker for Response to Platinum-Based Chemotherapy in Triple Negative and HER2+ Breast Cancer Patients
Jamie N Mills, Savannah G Bandurraga, Rita Kramer, Tihana Rumboldt, Carola Neumann, David P Turner and Victoria J Findlay
BC is becoming increasingly recognized as a heterogenous disease with multiple subtypes based on the expression of the estrogen receptor (ER), progesterone receptor (PR) and human epidermal growth factor receptor 2 (HER2). Systemic treatment to decrease the risk of metastasis is stratified according to receptor status. Unfortunately no targeted therapy is of proven efficacy in triple negative breast cancer (TNBC) defined as ER-/PR-/HER2-. Systemic chemotherapy remains the mainstay of treatment for women with this aggressive subtype of disease. Clinical data regarding the use of platinum agents in TNBC are still limited. A recent study has shown single agent cisplatin induced a response in a subset of patients with TNBC and suggests identification of biomarkers to predict response. Platinum agents are also widely used in the Her 2 amplified subset of breast cancer. The development of markers of sensitivity to platinum agents will allow identification of patients most likely to benefit from this agent and avoidance of toxicity from treatment in those most likely to be resistant.
Recent evidence has suggested a role for microRNAs as promising predictive markers of drug response in cancer. We have published studies examining the role of miR-510 in breast cancer and have performed in situ hybridization of human breast tissue microarrays (TMAs) and observed that miR-510 is expressed in a subset of malignant breast tumors (~40%), however, miR-510 expression was not observed in normal and benign breast tissue, suggesting miR-510 has biomarker potential. We identified, amongst others, the antioxidant peroxiredoxin 1 (Prdx1) as a novel direct target of miR-510. Prdx1 plays a protective role against cisplatin cytotoxicity, therefore we performed drug cytotoxicity assays and identified increased sensitivity to both cisplatin and carboplatin when miR-510 is over-expressed in vitro. Of interest, miR-510 was able to restore sensitivity to resistant mesenchymal-like breast cancer cell lines. Based on these data we propose the specific hypothesis that miR-510 is a prognostic indicator of response to platinum-based chemotherapy in triple negative and Her2+ breast cancer patients, and that identifying those patients with elevated miR-510 levels will result in an increase in response rate to therapy.
Lee Wheless (back to top)
A Community-Based Study Of Nucleotide Excision Repair PolymorphismsIn Relation To Risk Of Non-Melanoma Skin Cancer
Lee Wheless1, Emily Kistner-Griffin1,2,Timothy J. Jorgensen3, Ingo Ruczinski4, Yvette Berthier-Schaad5,8, Bailey Kessing5, Judith Hoffman-Bolton6, Lesley Woodward6, Yin Yao7, Paul T. Strickland8,9, W.H. Linda Kao8, Rhoda M. Alani10, Michael W. Smith11, Anthony J. Alberg1,2,8
1Division of Epidemiology and Biostatistics, Department of Medicine, and 2Hollings Cancer Center, Medical University of South Carolina, Charleston, SC; 3Department of Radiation Medicine, Georgetown University School of Medicine, Washington, DC; 4Department of Biostatistics, The Johns Hopkins University Bloomberg School of Public Health, Baltimore, MD; 5Laboratory of Genomic Diversity, NCI-Frederick; 6George W. Comstock Center for Public Health Research and Prevention, Washington County, MD; 7Division of Intramural Research Programs, National Institute of Mental Health; 8Department of Epidemiology, The Johns Hopkins University Bloomberg School of Public Health, Baltimore, MD; 9Department of Environmental Health Sciences, The Johns Hopkins University Bloomberg School of Public Health, Baltimore, MD; 10Department Dermatology, Boston University, Boston, MA; 11Genetics and Genomics Group, Advanced Technology Program, SAIC-Frederick, Inc., NCI-Frederick, Frederick, MD
Nucleotide excision repair (NER) is responsible for protecting DNA in skin cells against ultraviolet radiation-induced damage. Using a candidate pathway approach, a matched case-control study nested within a prospective, community-based cohort was carried out to test the hypothesis that single nucleotide polymorphisms (SNPs) in NER genes are associated with susceptibility to non-melanoma skin cancer (NMSC). Histologically-confirmed cases of NMSC (n=900) were matched to controls (n=900) on age, gender, and skin type. Associations were measured between NMSC and 221 SNPs in 26 NER genes. Using the additive model, two tightly linked functional SNPs in ERCC6 were significantly associated with increased risk of NMSC: rs2228527 (odds ratio (OR) 1.57, 95% confidence interval (CI) 1.20 – 2.05), and rs2228529 (OR 1.57, 95% CI 1.20 – 2.05). These associations were confined to basal cell carcinoma of the skin (BCC) (rs2228529, OR 1.78, 95% CI 1.30 – 2.44; rs2228527 OR 1.78, 95% CI 1.31 – 2.43). These novel, hypothesis-generating findings suggest functional variants in ERCC6 may be associated with an increased risk of NMSC that may be specific to BCC.
Peter Tang (back to top)
RPE65 Is Present Within Human Cone Photoreceptors And Is Required For Efficient Cone Visual Pigment Regeneration
PlH. Tang1, L. Wheless2, J.X. Ma3, R.K. Crouch1.4
Departments of Neuroscience1, Medicine – Division of Biostatistics and Epidemiology2, and Ophthalmology4, Medical University of South Carolina, Charleston, SC; Department of Physiology3, University of Oklahoma, Oklahoma City, OK
Purpose: Previous studies have suggested that RPE65, the retinoid isomerase of the classic visual cycle, is present within cones of various animal species. In the present study, we evaluate the presence of RPE65 within cones of the rod-dominant retina, including human and mice, and investigate its possible role within cones.
Methods: Antibodies targeting residues 150-164 (PETLET) or 473-486 (DALEED) of bovine RPE65 were developed. Donor human retinas were obtained from two patients (11 & 21 years). To generate mice with varying levels of RPE65 in the RPE, we crossed C57BL6 mice with BALB/c or Rpe65-/- mice. Immunoblots were performed to verify antibody specificity and to quantify RPE65. Immunohistochemistry techniques were utilized to localize and quantify the amount RPE65 within cones. Mice were treated with either 9-cis retinal or saline and then dark-adapted for 12 hours before electroretinography recordings were performed.
Results: Both PETLET and DALEED antibodies showed similar RPE65 staining within the outer segments of human cones, and this pattern was reflected in mouse cones. Across the various mouse strains/lines, an inverse relationship in the amount of RPE65 was observed between cones and the RPE. Chromophore treatment enhanced UV-cone responses in mice with low or undetectable cone RPE65, but had no effect in mice with high cone RPE65. The rod responses were unchanged.
Conclusions: Antibodies with high affinity and specificity for RPE65 are essential to detect the protein within cones. Our data show RPE65 is present within human cones and mouse is an appropriate model. Cones with low or undetectable RPE65 appear to be partially chromophore deprived, suggesting that RPE65 within cones is essential to maintain fully regenerated visual pigments.
Jennifer Scott (back to top)
Absence of Estrogen Receptor Alpha Reduces the Number and Function of Plasmacytoid Dendritic Cells in Lupus Prone Mice
Jennifer Scott1, Melissa Cunningham2,3, Osama Naga2,
1College of Graduate Studies, Department of Microbiology & Immunology. Med. Univ. of South Carolina, Charleston, SC 2Division of Rheumatology & immunology, Department of Medicine. Med. Univ. of South Carolina, Charleston, SC 3Ralph H. Johnson VA Medical Center, Charleston, SC
Systemic Lupus Erythematosus (SLE) is a chronic autoimmune disease that affects women at a 9:1 ratio compared to men. As a result of this female predominance, the role of estrogen and its receptors are under investigation. Previous work in our laboratory shows that estrogen receptor alpha (ERa) deficient lupus-prone mice have significantly increased survival and less renal disease compared to wild type lupus prone mice. The improvement occurs despite similar levels of renal immune complex deposition and autoantibody production in ERa deficient and wild type lupus prone mice. Since autoantibody production and immune complex deposition remain intact in ERa deficient lupus prone mice, we hypothesize that ERa deficiency improves SLE by reducing the innate immune system’s ability to respond to this immune activation. To study the innate immune response this research focuses the role of the ERa in the plasmacytoid dendritic cell (pDC). pDCs are the body’s major producers of type I interferon, a key cytokine in the innate defense against viruses. In addition to type I interferon’s role in host defense, its serum levels are elevated in SLE and it can cause the dissolution of tolerance when given to healthy individuals. Our findings show a decrease in pDC levels in ERa deficient lupus prone mice compared to wild type lupus prone mice. pDCs account for 60% of total bone marrow derived dendritic cells in wild type lupus prone mice, but only 35% in the ERa deficient lupus prone mice. Additionally, the pDCs from ERα deficient lupus prone mice produce decreased levels of type I IFN compared to wild type lupus prone mice. Given these findings, the next step will be determining the mechanism by which ERα deficiency reduces plasmacytoid dendritic cell number and decreases type I interferon production.
Thomas Mullen (back to top)
Complex Regulation Of Sphingoid Base Metabolism By
Multiple Sphingolipid Enzymes
Thomas D. Mullen1,3, Christopher J. Clarke2, Christopher R. Gault1,3, Ashley J. Snider1,3, Bill X. Wu2, Linda A. Heffernan1,3,
Yusuf A. Hannun2, and Lina M. Obeid1,2,3
In mammals, several enzymes use sphingoid bases as substrates, and these enzymes include ceramide synthases (CerS), sphingosine kinases (SK), and reverse ceramidase (CDase) activities. Many researchers have used radioactive or odd-chain sphingoid bases as in situ metabolic labels to study sphingolipid metabolism, yet the contribution of each of the sphingoid base-metabolizing enzymes to the acute uptake and incorporation of exogenous sphingosine (Sph) has not been well established. In this study, we investigated the uptake and incorporation of the odd-chain sphingoid base, 17-carbon Sph (17C-Sph) into ceramides using high performance liquid chromatography/mass spectrometry (LC/MS). Results show that after incubating cells with 17C-Sph for 30 minutes, C16:0-ceramide was the predominant ceramide species produced. Conversely, very long-chain ceramides were produced in low levels despite the high expression of CerS2 in MCF-7 cells and predominance of very long-chain CerS activity in vitro. The 17C16:0-CerS activity was further characterized as being sensitive to the CerS inhibitor fumonisin B1 and to knockdown of CerS5 and/or CerS6. We next investigated the role of reverse CDase activities (i.e., aCDase and nCDase) and discovered that, although neither aCDase or nCDase knockout fibroblasts had altered 17C16:0-ceramide formation, deficiency of either of these CDase reduced 17C-Sph-1-phosphate (17C-S1P) production. The effects of SK1 and SK2 deficiency on 17C-Sph metabolism were also examined, and the results showed that deficiency in either SK1 or SK2 reduced 17C-S1P formation; loss of each of these enzymes also affected ceramide generation in distinct ways. Overall, this study provides valuable insight into the metabolism of exogenous Sph and the use of Sph as an in situ metabolic label.
Xiaoyi (Tina) Zhang (back to top)
MKP-1 Deficiency Enhances Murine Oral Neoplasia
Xiaoyi Zhang1, Hong Yu1, Nisha J. D'Silva2, and Keith L. Kirkwood1
1Department of Craniofacial Biology and Center for Oral Health Research, Medical University of South Carolina College of Dental Medicine, Charleston, SC, USA; 2Departments of Periodontics and Oral Medicine and Pathology, University of Michigan, Ann Arbor, MI, USA
Head and neck cancer accounts for approximately 6% of diagnosed malignancies in the United States, the most common form being head and neck squamous cell carcinoma (HNSCC). Despite research efforts, survival rates remain lower than more common malignancies. Cytokines and pro-inflammatory factors have been shown to play a critical role in various steps of malignant transformation, many of which are regulated by mitogen-activated protein kinases (MAPK). These kinases are negatively regulated by MAPK phosphatases (MKPs), the founding member of this class being mitogen-activated protein kinase phosphatase-1 (MKP-1). Initial studies revealed significant over-expression of MKP-1 in the early stages of disease with levels of MKP-1 expression falling progressively in tumors of higher histological grade and in metastases. Previous work has demonstrated MKP-1 is a crucial negative regulator of innate immunity. Therefore, we hypothesize MKP-1 inhibits tumor-promoting inflammation in the HNSCC microenvironment. To address how MKP-1 signaling and downstream MAPK-regulated inflammatory cytokine expression may affect HNSCC development and progression, MKP-1 deficient and wild-type mice were treated with 4-nitroquinoline 1-oxide, a surrogate for tobacco exposure, to induce oral squamous cell carcinomas. MKP-1 deficient animals show a significant increase in tumor development, characterized by onset of disease, tumor size, histological grade, and inflammation. In addition, isolated tissues demonstrate increased levels of inflammatory cytokines and receptors by qPCR and altered MAPK activity by immunoblot. Ongoing work will be conducted to assess how MKP-1 regulates expression of newly identified inflammatory targets CXCL1 and IL-1b and their impact on tumor development and progression. Elucidating the regulation of inflammatory mediators in the tumor microenvironment may reveal critical tumor suppressive interactions mediated by MKP-1 that are lost during cancer initiation and progression.
Supported by 1R01DE018290 (KK), 1R01DE021423 (KK), 2P20 RR017696 (KK), R01DE018512 (NJD), R21DE017977 (NJD), K02DE0219513 (NJD)
Ryan Whitaker (back to top)
Stimulation of Mitochondrial Biogenesis with PDE3 Inhibitors
Ryan Whitaker, Lauren Wills, PhD, and Rick Schnellmann, PhD
Department of Pharmaceutical and Biomedical Sciences, Medical University of South Carolina, Charleston, SC
Acute kidney injury (AKI) is a severe pathological state characterized by rapid loss of renal function with high levels of morbidity and mortality. AKI is a consequence of a variety of conditions including renal ischemia-reperfusion (I/R) and drug or toxin exposure. With no effective therapeutic options and an array of increasing risk factors, AKI is a growing problem that needs to be addressed to both improve patient care and reduce healthcare costs. The development of new therapies for AKI has been largely unsuccessful, and treatment outcomes from AKI have remained unchanged for several decades. Therefore, the development of new, effective therapies requires the identification of novel molecular targets. Following I/R injury, mitochondrial dysfunction contributes to oxidative stress, persistent energy depletion and impairment of energy dependent repair mechanisms, ultimately leading to end organ damage and failure in a variety of tissues. Our laboratory has demonstrated persistent mitochondrial dysfunction and depletion of mitochondrial proteins following renal I/R injury that correlates with sustained renal tubular dysfunction in these animals. These results indicate that restoration of mitochondrial function may be an effective therapeutic strategy for accelerating recovery from AKI. Mitochondrial biogenesis is the process by which new mitochondria are formed within the cell and serves as the primary mechanism of increasing energy output during pathological times of need. Currently, there are few pharmacological agents that are capable of inducing mitochondrial biogenesis. We tested PDE inhibitors due to the potential role of cAMP in the regulation of peroxisome proliferator-activated receptor gamma coactivator-1α (PGC-1α), the “master regulator” of mitochondrial biogenesis. We demonstrated that the PDE3 inhibitors angrelide, cilostamide, enoximone and trequinsin stimulate mitochondrial biogenesis as assessed by increases in FCCP-uncoupled respiration in vitro in primary renal proximal tubule cells (RPTC) following 24 hour treatment. Interestingly, a panel of PDE4 inhibitors failed to produce an increase in uncoupled respiration. Additionally, mRNA expression of PGC-1α, as well as complex I proteins ND6 (mitochondrial-encoded) and NDUFB8 (nuclear-encoded) was increased. No changes were observed in mitochondrial DNA copy number. Cilostamide and trequinsin, plus the clinically approved compound cilostazol were selected for in vivo testing in naïve C57BL/6 mice. Mice were given an intraperitoneal injection of cilostamide (0.3 mg/kg or 3 mg/kg), trequinsin (0.3 mg/kg or 3 mg/kg), cilostazol (0.3 mg/kg), or vehicle control. Mice were sacrificed at 24 hours post-injection and kidneys were collected for RT-PCR and immunoblot analysis. Cilostamide, trequinsin and cilostazol all caused an increase in mRNA expression of PGC-1α, NDUFB8 and ND1 in the kidney cortex. Protein expression of mitochondrial complex proteins NDUFB8, ATP synthase β, and COX1 were all increased compared to controls. Mitochondrial DNA copy number was also significantly increased in kidney cortex from these animals. This data demonstrates that PDE3 inhibitors are capable of stimulating mitochondrial biogenesis both in vitro in RPTCand in vivo in kidney, and suggests that these compounds may serve as viable pharmacological agents for the treatment of AKI. As mitochondrial biogenesis is a recovery process occurring throughout the body, these agents may also prove effective in acute injury in other organs including heart, liver, lung and brain.
Dan Neitzke (back to top)
Repetitive Transcranial Magnetic Stimulation of the Left Dorsolateral Prefrontal Cortex Promotes Resilience, Prevents Learned Helplessness
Daniel Neitzke, Joseph Taylor, George Khouri, Jeffrey Borckardt,
Department of Psychiatry and Behavioral Sciences, Medical University of South Carolina
Application of an aversive stimulus over which the subject has no control has been shown to produce a measurable decline in cognitive performance, relative to subjects who can terminate the stimulus, in humans and other mammals. This “Learned Helplessness” paradigm has been used as a model for Depression and Post Traumatic Disorder (PTSD), allowing the efficacy of potential therapies for these disorders to be assessed in healthy subjects. It has been further demonstrated in mouse models by Maier and colleagues that the feeling of control that is missing in the helpless subjects is actually a signal from the Prefrontal Cortex to the dorsal Raphe Nucleus and that blocking this signal pharmacologically can produce helpless behavior in non helpless animals. The present study sought to translate this work to human subjects and to demonstrate the beneficial effect of knocking in the pathway rather than the detrimental effect of knocking it out. Repetitive Transcranial Magnetic Stimulation (rTMS) is a non-invasive form of brain stimulation in which a localized pulsed magnetic field applied externally to the subject’s head is able to induce depolarization of superficial cortical neurons. Although left dorsolateral prefrontal rTMS has been approved for the treatment of refractory depression, little is known about the mechanism by which it alleviates this condition. We utilized a 2x2 study design in which subjects received either real or sham rTMS and either did or did not have the ability to terminate a thermal pain stimulus that was delivered simultaneously with the rTMS. Helpless subjects were yoked to subjects with control of the stimulus to eliminate total time of aversive stimulus exposure as a potential variable. Subjects were subsequently asked to solve a series of anagrams and the average time to correct solution, number of trials in which the subject gave up, and average time it took the subject to give up were determined. Our data show that subjects who do not have control of the aversive stimulus perform worse on the cognitive task than subjects who have control. Furthermore, the application of rTMS to the left dorsolateral prefrontal cortex caused the performance of helpless subjects to recover to the level of that of subjects with control. It appears that activation of this “control circuit” during the experience of stimuli over which the subject does not in fact have control protects the subject from the negative cognitive effects of the stimulus. This indicates a possible mechanism for the efficacy of rTMS in the treatment of depression and suggests a new approach in the treatment of PTSD.
Joseph Alge (back to top)
Urinary Angiotensinogen Predicts Acute Kidney Injury After Cardiac Surgery
J.L. Alge1, M.G. Janech1,2, A.D. Shaw3, L.S. Chawla4, J.A. Tumlin5 and J.M. Arthur1,2
1Department of Medicine, Division of Nephrology, Medical University of South Carolina; 2Ralph H. Johnson VA Medical Center, Charleston, SC; 3Department of Anesthesiology, Duke University Medical Center; 4Department of Anesthesiology and Critical Care Medicine and Division of Nephrology George Washington University; 5Department of Medicine, Division of Nephrology, University of Tennessee at Memphis
Acute kidney injury (AKI) is a common and serious post-operative complication of cardiac surgery that is associated with an increased risk of several adverse outcomes. The poor outcomes of these patients are partially attributable to the limitations of the current method of diagnosis, which is based upon an increase in serum creatinine (sCr). The discovery of novel biomarkers of AKI could improve patient outcomes by allowing for earlier and more accurate diagnosis and prognosis. Using a proteomic approach, we have identified novel candidate biomarkers of AKI. In this study, we validated one of these biomarkers, urinary angiotensinogen (uAng), in a set of samples that included pre-operative samples, post-operative samples from patients who did not develop AKI, and patients who developed AKI of increasing severity (stages I, II, and III). Ten patients developed severe AKI requiring renal replacement therapy (RRT). Patients with severe AKI had higher levels of uAng compared to the pre-operative group, and uAng was predictive of the progression of sCr elevation after urine collection (AUC= 0.66, Sensitivity= 74.4%, Specificity= 52.1%). However, it was not a good predictor of AKI, severe AKI, or the need for RRT. Importantly, however, we found that it was an excellent predictor of severe AKI (AUC= 0.86, Sensitivity=100%, Specificity= 70.1%), the need for RRT (AUC= 0.89, Sensitivity= 100%, Specificity= 81.8%), and the future increase of sCr (AUC= 0.74, Sensitivity=100%, Specificity= 64.3%) in the subset of patients who did not undergo intraoperative cardiopulmonary bypass (CPB). Analysis of paired pre-op and post-op samples suggested that CPB increased uAng. These results are the first to demonstrate the potential diagnostic and prognostic utility of uAng in patients at risk of renal injury.