2017 Rhode Island NIH IDeA Symposium
Poster Session #1 8:00 – 11:45 a.m.
Eposterboard #1
IL-1beta-driven Atherosclerotic Calcification is mediated by Macrophage Rac-signaling
Coronary artery disease caused by atherosclerosis is the leading cause of morbidity and mortality in the world. Calcification of atherosclerotic plaque has predictive value in terms of cardiovascular event risk and mortality. Plaque calcium composition appears critical to determining cardiovascular risk. Inflammation can influence calcification of plaque, but immune modulators of plaque calcification are minimally defined. Rac1 and Rac2 are small GTPases that influence cytokine expression in plaque macrophages. We have defined a Rac-based signaling mechanism that regulates macrophage IL-1β expression. Using an atherosclerotic-prone mouse model, we identified that progressive calcification depends on altered Rac expression and activation (GTP-binding) through consequent effects on macrophage IL-1β production and IL-1R signaling.IL-1β production and its downstream signaling were critical determinants of atherosclerotic plaque calcification.Enhanced plaque expression of the osteogenic transcription factors, RUNX2, SOX9, OSX and MSX2 was associated with increased atherosclerotic calcification.IL-1β production was driven by NF-ĸB and reactive oxygen species (ROS) signaling through effects on mRNA expression and caspase1 activation. Bone marrow transplantation confirmed the calcification attributable to Racgene deletions was dependent on the hematopoietic compartment. In short, Rac2 expression determined the degree of Rac1 GTP-binding, acting as a brake on Rac1-dependent IL-1β production. Whereas, macrophage IL-1β production and atherosclerotic calcification depended on myeloid Rac1. Furthermore, primary macrophages treated with HMG-CoA reductase inhibitors (statins), expressed higher IL-1β mRNA and secreted more IL-1β protein in response to inflammasome activation by TLR-coupled cholesterol crystal phagocytosis. Therapeutic inhibition of macrophage Rac-mediated IL-1β expression has potential to be a treatment strategy for progressive, inflammatory atherosclerotic calcification.
Eposterboard #2
Activation of Anoctamin-1 Causes Apoptosis of Pulmonary Endothelial Cells Mediated via p38
A Allawzi1,2, A Vang1; N Kue1; G Choudhary1,3
1Vascular Research Laboratory, Providence VA Medical Center
2Department of Molecular Pharmacology, Physiology, and Biotechnology, Brown University
3The Warren Alpert Medical School of Brown University
Rationale: Aberrant endothelial cell (EC) proliferation plays an important role in the vascular remodeling observed in Pulmonary Arterial Hypertension (PAH). Anoctamin-1 (Ano1), a calcium activated chloride channel, has been implicated in the regulation of proliferation and cell cycle in tumors. Our objective was to determine the role of Ano1 in PAH and elucidatethe underlying mechanism.
Methods: Ano1 expression was assessed by immunoblot, qPCR, and immunoflouresnce in microvascular endothelial cells and rat lungs. PAH was induced by injecting Sprague-Dawley rats with 20mg/kg SU5416 subcutaneously and placed into hypoxia (10% FiO2) for 3 weeks and then 4 weeks of normoxia (SUH). Ano1 activity was evaluated using whole-cell patch clamp and DiBAC4(3) fluorescence. Cell death/apoptosis was assessed by cell count, flow cytometry, caspase-3 activity assay and immunoblot.
Results: Ano1 is expressed in RLMVECs, conducts current, and its activation results in EC hyperpolarization that is attenuated with DIDS and low chloride physiological saline solution. Activation of Ano1 decreases cell number that is attenuated by DIDS, SB203580 (p38 inhibitor), and NAC (ROS scavenger). Ano1 activation causes apoptosis as demonstrated by an increase in Annexin V/PI staining and in Caspase-3 activity and cleavage. Furthermore, activation of Ano1 increases p38 phosphorylation. Ano1 expression was significantly elevated in hyperproliferative ECs in SUH animals and plexiform-like lesions compared to normoxic animals.
Conclusions: Ano1 expression in increased in hyperproliferative ECs in PAH and activation of Ano1 causes p38-mediated apoptosis of pulmonary ECs. Hence, Ano1 may serve as a therapeutic target to attenuate EC proliferation in PAH.
Contact Information Name: Gaurav Choudhary Institution: PVAMC Department: Medicine Phone #: 401-273-7100 x2029 Email:
Eposterboard #3
Mechanism of RV Dysfunction Associated with Cigarette Smoke Exposure
AVang1; RT Clements1,4,5; HChichger1,3; N Kue1,Ayed Allawzi1,2; KO’Connell1,3; EM Jeong3,5, S Dudley, Jr3,5; PSakhatskyy1,3; Q Lu1,3; P Zhang3,5; SRounds1,3;GChoudhary1,3
1Vascular Research Laboratory, Providence VA Medical Center
2Department of Molecular Pharmacology, Physiology, and Biotechnology, Brown University
3The Warren Alpert Medical School of Brown University
4Department of Surgery, Rhode Island Hospital
5Cardiovascular Research Center, Rhode Island Hospital
BACKGROUND: Cigarette smoke (CS) is one of the major risk factors for chronic obstructive pulmonary disease (COPD). COPD in smokers is associated with RV dysfunction. RV dysfunction has been noted in COPD in absence of significant pulmonary hypertension. However, the mechanism of RV dysfunction in smoking-related COPD remains unclear. The objective of this study was to evaluate the effect of CS exposure on RV function and elucidate the underlying mechanism.
METHODS: AKR mice were exposed to room air (RA) or CS for 6 weeks followed by transthoracic echocardiography. Adult rat cardiac fibroblasts (CF) were isolated from Sprague-Dawley rats for in vitro studies. Cigarette smoke extract (CSE) was prepared by smoking Kentucky Research cigarettes (3R4F) through PBS for 5 min each (100% CSE). Cell proliferation was assessed using hemocytometer after 24 hr serum deprivation. Collagen content was performed using Sircol Assay.
RESULTS: AKR mice exposed to CS had TAPSE, suggesting decreased RV function compared to RA mice without any evidence of pulmonary hypertension. RV dysfunction in CS mice was associated with increased RV collagen content (11.5±0.6 vs 8.9±0.7, p<0.05). In vitro, CFs exposed to 10% CSE demonstrated increased proliferation as assessed by relative cell counts that was attenuated by inhibition of nicotinic acetylcholine receptors (nAChR). Similarly to CSE, nicotine exposure resulted in CF proliferation that was blocked by nAChR antagonists and was associated with significantly decreased p38 phosphorylation.
CONCLUSION: CS causes RV dysfunction and collagen deposition mediated via activation of nAChR by nicotine and associated with reduced p38 phosphorylation.
Contact Information Name: Gaurav Choudhary Institution: PVAMC Department: Medicine Phone #: 401-273-7100 x2029 Email:
Eposterboard #4
Nicotinic Acetylcholine Receptor Signaling is Associated with RV Dysfunction in PAH
AVang1; RClements1,4; A Allawzi1,2; NKue1; T Mancini1, G Choudhary1,3
1Vascular Research Laboratory, Providence VA Medical Center
2Department of Molecular Pharmacology, Physiology, and Biotechnology, Brown University
3The Warren Alpert Medical School of Brown University
4Department of Surgery and Cardiovascular Research Center, Rhode Island Hospital
Introduction: Limited therapeutic options are available for RV failure-associated with Pulmonary Arterial Hypertension (PAH). We investigated the role of cholinergic pathway and nAChR activation in a rat model of PAH as well as isolated cardiac fibroblasts.
Methods: Rats were exposed to Sugen/Hypoxia for 3 wks (PAH), followed by 2 and 4 wks of normoxia. Vehicle/normoxia animals served as controls (Con). Echocardiography was performed and tissue harvested. Rat cardiac fibroblasts (CF) were isolated for in vitro studies. Quiesced CF were exposed to either acetylcholine or nicotine with or without α-bungarotoxin (α-BTX) or mecamylamine (nAChR blockers) followed by proliferation or collagen production measurement. In additional experiments, cells were exposed to TGF-β or Ang II with or without α-BTX or α7 nAChR siRNA.
Results: PAH caused RV dysfunction (increased RV and RA pressures, decreased TAPSE, PAT as determined by echocardiography), increased RV collagen content and RV hypertrophy. RV dysfunction was associated with increased expression of acetylcholine synthetic/signaling proteins in the RV but not LV. nAChR agonists applied to isolated CF significantly increased proliferation and collagen content that was blocked by the nAChRantagonists α-BTX and mecamylamine. nAChR blockade had no effect on TGF-β-induced fibrosis but blocked Ang II-induced proliferation and collagen production.
Conclusions: RV hypertrophy and dysfunction in PAH-associated with increased expression of ACh synthetic enzymes and nAChR. In addition, nAChR activation can directly increase proliferation and collagen production of CF and is required for Ang II-induced CF proliferation. Therefore, CF nAChR signaling may be an important therapeutic target to limit RV dysfunction.
Contact Information Name: Gaurav Choudhary Institution: PVAMC Department: Medicine Phone #: 401-273-7100 x2029 Email:
Eposterboard #5
Stress management: Resilience to polymicrobial pulmonary infections
Kayla M. Lee, Yun Xu, Christina Lee, Lillian Dominguez and Amanda M. Jamieson
Brown University
Bacterial pneumonia is a common complication of respiratory virus infection that leads to increased morbidity and mortality. It is becoming increasingly clear that anti-microbial drugs are not universally effective in treating polymicrobial infections of the lung, therefore novel treatment strategies will be necessary to prepare ourselves for the next IAV pandemic. Both pathogen clearance and host resilience are important processes for surviving a given infection. Resilience is the ability of a host to tolerate and survive the effects of given pathogen burden. Numerous studies have shown that IAV infection decreases bacterial clearance. We propose that host resilience mechanisms are also altered by IAV/bacterial coinfection, and by focusing on this aspect of coinfection we can develop novel treatment strategies for lung infections. Studying host resilience to coinfection is hampered by the fact that the massive increase in bacterial burden after IAV infection masks potential changes in host resilience responses. In order to focus specifically on host resilience mechanisms, we developed IAV/bacterial coinfection modelsin whichpathogen clearance in unchanged but survival is decreased. We hypothesize that IAV/bacterial coinfection leads to decreased hostresilience compared to either infection alone because of a skewing towards inflammatory innate immune responsewithout adequate compensatory anti-inflammatory and tissue repair responses.We found several lines of evidence that support this evidence, by using both a bioinformatics and a cellular immunology approach. We found that regulation of tissue repair and inflammation were both crucial in host resilience to coinfection. We found that there was a decrease in some putative innate immune resilience responses, while there was an increase damage causing aspects of the innate immune response. In order to investigate host resilience to IAV/bacterial coinfection we have combined our in vivo mouse models with in vitro models to to continue to investigate resilience mechanisms on a whole host, cellular, and molecular level.
Eposterboard #6
MOLECULAR MECHANISMS UNDERLYING THE INTERACTION OF Salmonella enterica WITH LEAFY GREENS.
K. Beaton, N. Donahue, H. Kennedy, B. LoCascio, A. Reid.Department of Biology and Biomedical Sciences, Salve Regina University, Newport, RI.
Salmonella entericacauses foodborne gastroenteritis, which is typically linked to the consumption of undercooked eggs and poultry. Recent years have seen an increase in the number of outbreaks linked to the consumption of fresh fruits and vegetables. An understanding of the molecular mechanisms that contribute to S. enterica’s fitness in/on this commodity could lead to strategies to improve food safety and reduce the burden of salmonellosis. Attachment and colonization of fresh produce is likely to be mediated by molecules on the surface of the bacterium, such as polysaccharides, outer membrane proteins and flagella. The aim of this research is to determine whether expression of these surface structures varies from one S. entericaserovar to another, and whether these differences correlate with a particular serovar’s ability to attach to and colonize leafy greens. We also seek to understand whether conditions used to prepare inocula for in vitro attachment and colonization studies influence the expression of these surface structures, and consequently influence the outcome of these studies. The expression of O antigens, flagella and cellulose was studied under a range of growth conditions (media type and physical state, incubation temperature). Where possible, the expression of these structures was correlated to a phenotype (biofilm formation, motility). Serovar-specific differences in flagellin expression, motility and biofilm formation were detected. The expression of these structures also varied with the inoculum preparation conditions used. Future studies will seek to determine whether enhanced expression of these structures correlates with improved attachment and adherence to leafy greens.
Contact Information
Name: Anne Reid, PhD Institution: Salve Regina University, Newport, RI
Department: Biology and Biomedical Sciences
Phone #: (401) 341-7464 Email:
Eposterboard #7
Streptococcus pneumoniaeLytB inhibition by diamides.
J. Gravier*, S. Nayyab*, M. Saladino**, BA. Haubrich*, A. Basu**, CW. Reid*; * Dept. of Science and Technology, Bryant University, Smithfield, RI; ** Dept. of Chemistry, Brown University, Providence, RI
Problem: Streptococcus pneumoniae is a Gram-positive bacterium which causes a wide variety of human disease. The threat of antibiotic resistance demonstrates a need for alternative drug targets. Reports from the CDC indicate up to 30 % of invasive S. pneumoniae infections demonstrated resistance to at least one antibiotic. The objective of this study was to develop diamide inhibitors for LytB, an endo-acting N-acetylglucosaminidase, and study the interactions between inhibitor and enzyme. Methods: Diamide inhibitors were synthesized by 4-component Ugi reaction. MICs were determined by the rezasurin method against S. pneumoniae grown in 96-well plates at varying concentrations of diamide inhibitor. Docking experiments and virtual screens were performed with AutodockVina with known partial substrates of LytB and diamides. Results: Two compounds, fgbband fgkc, were found to be potent inhibitors (MIC 3.6 µg/mL and 5.2 µg/mL, respectively). Molecular docking revealed favored binding in Zone 3 of the active site of LytB. Conclusions: The autolysin from S. pneumoniae is a promising target, and diamide inhibitors fgbband fgkc show antibacterial activity. Future directions include enzymatic screening of the diamide inhibitors against recombinant LytB and co-crystallization with fgbbandfgkc.
Contact Information Name: Brad A Haubrich Institution: Bryant University Department: Science and Technology Phone #: 401-232-0232 Email:
Eposterboard #8
H.J. Axen, J. Gambardello, F. Talone, JD Swanson Department of Biology and Biomedical Sciences, Salve Regina University, Newport, RI 02840
Gastrointestinal cancers are especially lethal, with a 5-year survival rate less than 30%. Standard treatments are non-specific and expensive, with adverse side effects. Nutraceuticals, naturally occurring plant compounds, such as the phenolic gallic acid (GA) offer potential for treatment and prevention. GA specifically targets cancer cells, halting cellular proliferation. We investigated high (100uM) and low doses (20uM) of GA on gene expression compared to control 0uM treated cells using microarrays in the immortal gastric cancer cell lines MKN-28 and AGS treated for durations of 0, 3, 6, 12, 18, 24, 36, and 48 hours.
Extracted RNA was quantified and hybridized to Affymetrix'sGeneChip® Human Transcriptome Arrays 2.0 at the Brown University genomics center. In AGS cells the 6-hour-100uM GA-treated cells displayed the greatest number of differentially expressed genes compared to untreated cells; 70 genes were up regulated and 147 genes were down regulated. In MKN28 the 100uM GA treatment for duration of 6 and 36 hours showed the greatest differentially expressed transcripts compared to untreated cells, with 438 genes up-regulated, and 332 genes down-regulated in the 6hr sample, and 237 up-regulated and 241 genes down-regulated in the 36hr sample. The functional role of differentially expressed transcripts was then determined to have roles potentially involved in anti-proliferation included genes known to be cell cycle regulators. This suggests that gallic acid treatment affecting genes key to cell cycle regulation that is allowing for the anti-proliferation of AGS and MKN-28 gastric cancer cells.
Contact information: Heather Axen: Salve Regina University: Biology and Biomedical Sciences: (401) 341-7470:
Eposterboard #9
HYPEROXIA INCREASES MITOCHONDRIAL UTILIZATION OF FATTY ACIDS IN NEONATAL LUNG ENDOTHELIAL CELLS: IMPLICATIONS FOR INJURY AND REPAIR
H. Yao1, J.F. Carr1, A.L. Peterson1, P.A. Dennery1,2; 1Dept. of Molecular Biology, Cell Biology & Biochemistry; 2Dept.of Pediatrics, Warren Alpert Medical School of Brown University
Problem: In neonates, hyperoxic exposure causes lung injury characterized by arrested vascularization and simplified alveolarization. These are the hallmarks of bronchopulmonary dysplasia. Although endothelial cells (ECs) mainly rely on glycolysis for bioenergetics during vascularization, they have metabolic flexibility to maintain cell function under stress. We hypothesized that hyperoxia alters EC metabolism leading to cell dysfunction and lung injury. Methods: Mouse fetal lung EC line (MFLM-91U) and primary lung ECs isolated from neonatal mice were exposed to hyperoxia (95% O2/5% CO2) for the measurement of glycolysis, mitochondrial respiration and fuel utilization by the Seahorse XF Analyzer. Fatty acid (FA) metabolism gene was analyzed using the RT² PCR Arrays. Cell proliferation was measured by the Click-iT™ EdUFlowCytometry Assay. Results: Hyperoxic exposure for 24 h increased glycolytic flux but reduced mitochondrial respiration in MFLM-91U cells. Interestingly, mitochondrial FA oxidation was significantly increased in MFLM-91U and mouse primary lung ECs exposed to hyperoxia, which was not observed in mouse embryonic fibroblasts or alveolar epithelial cells. Enhanced FAoxidation was associated with augmented expression of Fabp2, Slc27a5, Cpt1c, and Ehhadh genes. No effects of hyperoxia were observed on glutamine or glucose oxidation. Similar to hyperoxia, inhibition of long-chain FA entry into mitochondria for β-oxidation through carnitine palmitoyltransferase 1 inhibition by etomoxir (100 μM, 24 h) reduced proliferation in MFLM-91U cells. Conclusions: Hyperoxiareduces mitochondrial respiration but augments glycolysis andFA oxidation uniquely in lung ECs. This may serve to preserve endothelial cell proliferation and alveolarizationin order to mitigate neonatal hyperoxic lung injury.
Supported by the Institutional Development Award (IDeA) from the NIGMS of NIH under grant # P20GM103652.
Contact Information Name: Hongwei Yao; Institution: Warren Alpert Medical School of Brown University; Department: Molecular Biology, Cell Biology & Biochemistry; Phone #: 401-863-6754; Email:
Eposterboard #10
Screening for Small Molecule Blockers for the Bax Inhibitor (BI-1) Calcium Channel Linked to Human Cancers
James Mullin, Nicholas Mello, and NicanorAustriaco, OP
Providence College, Providence, RI
Yeast Bax inhibitor-1 (BXI1/YBH3) encodes a protein that belongs to the Bax Inhibitor (TMBIM) family of proteins, which has been linked to different tumor types in human patients. The crystal structure of a prokaryotic member of the family, BsYetJ, has revealed that the Bax inhibitor proteins are pH sensitive calcium leaks.Our laboratory has shown that Bxi1p is localized to the yeast ERand vacuole and our genetic studies suggest that the protein is a channel that controls the efflux of calcium from the ER.Wehave also over expressed Bxi1p inE.coliand have used a fura-2 based calcium assay to show that the protein facilitates the influx of extracellular calcium into the cell.Further studies have suggested that the influx of calcium can be altered by the pH of the extracellular environment. We have initiated a screen to identify small molecule blockers for the channel in the hopes of identifying drugs that would kill cancer cells that are addicted to Bax inhibitor.[Our laboratory is supported by grant NIGMS R15 GM110578, awarded to N. Austriaco.]
Eposterboard #11
Transcriptional Profiling of Circulating Exosomes Defines Molecular Phenotypes of Preeclampsia
Jessica Schuster, AlperUzun, Mackenzie Brigham*, Joan Stabila, James Padbury, Dept. of Pediatrics, Women and Infants Hospital, Providence, RI; *Warren Alpert Medical School, Providence, RI