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 Rac gene 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 elucidate the 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: Gaurav_Choudhary@Brown.Edu

Eposterboard #3 Mechanism of RV Dysfunction Associated with Cigarette Smoke Exposure

A Vang1; RT Clements1,4,5; H Chichger1,3; N Kue1, Ayed Allawzi1,2; K O’Connell1,3; EM Jeong3,5, S Dudley, Jr3,5; P Sakhatskyy1,3; Q Lu1,3; P Zhang3,5; S Rounds1,3; 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, 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: Gaurav_Choudhary@Brown.Edu

Eposterboard #4 Nicotinic Acetylcholine Receptor Signaling is Associated with RV Dysfunction in PAH

A Vang1; R Clements1,4; A Allawzi1,2; N Kue1; 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 nAChR antagonists 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: Gaurav_Choudhary@Brown.Edu 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 models in which pathogen clearance in unchanged but survival is decreased. We hypothesize that IAV/bacterial coinfection leads to decreased host resilience compared to either infection alone because of a skewing towards inflammatory innate immune response without 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 enterica causes 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. enterica serovar 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: anne.reid@salve.edu

Eposterboard #7

Streptococcus pneumoniae LytB 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 Autodock Vina with known partial substrates of LytB and diamides. Results: Two compounds, fgbb and 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 fgbb and fgkc show antibacterial activity. Future directions include enzymatic screening of the diamide inhibitors against recombinant LytB and co-crystallization with fgbb and fgkc.

Contact Information Name: Brad A Haubrich Institution: Bryant University Department: Science and Technology Phone #: 401-232-0232 Email: bhaubrich@bryant.edu

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's GeneChip® 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: Heather.Axen@Salve.edu

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™ EdU Flow Cytometry 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 FA oxidation 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: Hyperoxia reduces mitochondrial respiration but

augments glycolysis and FA oxidation uniquely in lung ECs. This may serve to preserve endothelial cell proliferation and alveolarization in 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: Hongwei_yao@brown.edu Eposterboard #10 Screening for Small Molecule Blockers for the Bax Inhibitor (BI-1) Calcium Channel Linked to Human Cancers James Mullin, Nicholas Mello, and Nicanor Austriaco, 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 ER and vacuole and our genetic studies suggest that the protein is a channel that controls the efflux of calcium from the ER. We have also over expressed Bxi1p in E.coli and 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, Alper Uzun, Mackenzie Brigham*, Joan Stabila, James Padbury, Dept. of Pediatrics, Women and Infants Hospital, Providence, RI; *Warren Alpert Medical School, Providence, RI

Problem: Preeclampsia is a multi-system hypertensive disorder of pregnancy affecting 2-8 % of deliveries in the US. It is characterized by elevated blood pressure, proteinuria and fetal growth retardation. There is currently no effective intervention short of delivery of the fetus. We know that nearly all tissues and cell types release vesicles from their plasma membrane. The contents of these vesicles, including mRNA and miRNA, are tissue specific, can be translated into proteins by target cells, and can lead to alterations in downstream signaling. We hypothesize that the mechanism for severe preeclampsia can be revealed by the transcript profile of the

exosomes from the whole blood of affected women. Methods: We enrolled patients with early onset severe pre-eclampsia and gestational age matched controls. We used differential centrifugation of whole blood to isolate exosomes, extracted total RNA and preformed paired end sequencing using HighSeq 2500. We will analyze differential expression and deconstruct the transcription profiles of the exosomal cargo into the contribution from their tissues of origin. Results: We isolated exosomes ranging in size from 88-200nm. The RNA yield ranged from 5ng-30ng with a mean of 11.7ng. We aligned 60-70% of the paired end reads to the reference genome. Between 1-5% of the bases aligned to coding regions. Conclusion: With this preliminary data we have demonstrated the feasibility of our research design for isolating exosomes from plasma and deep sequencing the resulting RNA cargo. Identification of the transcript profiles in these exosomes will provide insights into the mechanisms and pathogenesis of severe preeclampsia.

Jessica Schuster, PhD Women and Infants Hospital Department of Pediatrics (401) 274-1100 ex 48015 jschuster@wihri.org

Eposterboard #12

CELL ISOLATION AND ORGAN FUNCTION CORE ENHANCES CARDIOPULMONARY VASCULAR BIOLOGY RESEARCH IN RHODE ISLAND Elizabeth O. Harrington, Julie Newton, Julie Braza, Sharon Rounds. CardioPulmonary Vascular Biology COBRE, Ocean State Research Institute, Providence VAMC, Brown University, Providence, RI The Cell Isolation/Organ Function Core has been established to provide essential services in: i) isolation, characterization, and propagation of primary pulmonary and cardiac endothelial cells (EC), ventricular fibroblasts, and myocyte cells, stem cell derived macrophages and ii) provide measurements of heart and lung function in vivo and ex vivo. Procedures include isolating ECs using immuno-affinity procedures, cardiac ventricular fibroblasts and myocytes using Langendorff perfusion protocols. Lung and heart function protocols determine a multitude of organ function endpoints using the FlexiVent™ system and echocardiography, pressure/volume loops, and lung edema formation. Additional services are ELISA, histological slide scanning and analysis, intravascular injections of liposomes, flow cytometry and protocol/methodology development. Thus, the centralized core assists Investigators in data acquisition and analysis, improves reproducibility, relieves efforts by project personnel, and increases the efficiency and productivity of projects. Contact Information: Julie Braza and Julie Newton Brown University Julie_braza@brown.edu Phone Number: 401-273-7100 ext:3645

Eposterboard #13 FINE DISSECTION OF THE MICROBIOME IN A SPATIAL CONTEXT

K. Duncan, S. Vaishnava Problem: A dysregulated gut microbiome has been shown to have large contributions to gastrointestinal pathologies, but mechanistically is poorly understood. Traditional methods for studying the gut microbiome have hinted which organisms are present, but loses any sort of spatial context in its local environment. Only by understanding the gut flora in its ecological framework can we begin to look into the host-microbe interactions in this ecosystem. Methods: We have developed a robust methodology that allows us to isolate distinct transverse regions to elucidate the biogeography of the microbiome. Specific fixation and sectioning techniques are employed that maintain the structural integrity of the gut flora, and by coupling laser-capture microdissection and 16s rRNA sequencing, the community composition of each layer is revealed. Results: Employing this methodology, we are able to explore organization of the colon mucosa. A PCoA plot comparing distinct regions of the mucosa shows that the populations that reside in similar regions across mice are more similar than they are to sequential regions within the same mouse. We are also able to show differences in the abundances of specific groups of bacteria between the two regions, showing finer spatial organization within the mucosal layers than what was previously known. Conclusions: Our approach of spatial microdissection of microbial communities in situ will be applied towards exploring immune mechanisms that maintain biogeography of intestinal microbial communities. We anticipate this line of investigation will provide tremendous insight into the metabolic networks that govern assembly of complex bacterial communities during health and disease.

Contact Information: Kellyanne Duncan, Brown University, Department of Molecular Microbiology and Immunology. Kellyanne_duncan@brown.edu

Eposterboard #14 Purification and Characterization of a Ni(II) and Co(II) Responsive Transcriptional Regulator from Mycobacterium tuberculosis K. Higgins, G. Swanson, A. Miller, V. Surette, K. Gonzalez, M. McGowan, Department of Chemistry, Salve Regina University, Newport, RI Mycobacterium tuberculosis (M. tuberculosis), the causative agent of tuberculosis (TB), infects nearly one-third of the world’s population and is responsible for the death of almost two million people annually. With an increase in the number of multiresistant, extensively resistant, and totally drug-resistant strains the development of new therapeutic strategies that target other essential pathways in the bacteria is critical. The bacteria requires Ni(II) for two Ni(II) containing proteins, urease and glyoxalase I. The former, urease catalyzes the conversion of urea to ammonia and carbamate, which further decomposes to give an additional molecule of ammonia and bicarbonate. The ammonia produced contributes to the nitrogen availability in the bacteria and is

thought to be important for the survival of the bacteria in the human host, as it neutralizes the pH of the phagosome, resulting in the inhibition of lysosome-phagosome fusion. The bacteria also requires Co(II) for vitamin B12 biosynthesis, which may also play a role in the survival of the bacteria in the host. The fact that there are two regulators that are associated with the expression of two different exporters for Ni(II) and Co(II) in M. tuberculosis suggests that maintaining the intracellular Ni(II) and Co(II) concentrations is critical to the bacteria. We have purified KmtR using series of ammonium sulfate cuts, affinity/anion and size exclusion chromatography. To gain insights into the mechanism of metal recognition in KmtR, binding affinities and structural details regarding the complexes formed with cognate and non-cognate metal ions are being determined. Salve Regina University: Department of Chemistry: 401-341-3215: khadine.higgins@salve.edu Eposterboard #15 INSIGHT INTO THE CONTRIBUTION OF BIGLYCAN AND DECORIN TO PRETERM PREMATURE RUPTURE OF FETAL MEMBRANES

*L.A. Underhill, **G. Lambert-Messerlian, *N. Avalos, *C. Barbaros and *B.E. Lechner

*Dept. of Pediatrics, Women and Infants Hospital and The Warren Alpert Medical School at Brown University, Providence, RI. ** Dept. of Pathology, Laboratory Medicine and Obstetrics and Gynecology, Women and Infants Hospital and The Warren Alpert Medical School at Brown University, Providence, RI.

Problem: Preterm birth is a leading cause of neonatal mortality and morbidity. Connective tissue abnormalities such as Ehlers-Danlos Syndrome (EDS) are associated with an increase in preterm premature rupture of membranes (PPROM). One subtype of EDS is caused by abnormal secretion of the proteoglycans biglycan and decorin. The objective of this study was to investigate the gestational phenotype of a large cohort of women with EDS, as well as examine the expression of biglycan and decorin in maternal serum during pregnancy to identify biomarkers of PPROM. Methods: A web-based survey was developed to evaluate the contribution of EDS to birth complications. Quantitative data sets including EDS population and pregnancy descriptors were collected for analysis. For the biomarker study, 20 cases of patients diagnosed with PPROM were chosen by stringent criteria and assigned 5 controls matched for gestational age and maternal race. Results: A total of 1682 people responded to the survey, representing 22 countries globally. Interestingly, an increased incidence in both preterm birth and PPROM was determined in EDS women. During the second trimester of pregnancy, serum biglycan and decorin expression were significantly (p=0.006 and p=0.003 respectively) altered in patients who later delivered prematurely due to PPROM compared to matched controls. Conclusions: Understanding the pathophysiology leading to PPROM is important for identifying those patients who may be at risk, as well as developing interventions. The e-survey data will be used as an epidemiologic study to elucidate genetic components leading to PPROM. The altered pattern of serum expression of biglycan and decorin in patients whose preterm delivery was due to PPROM, were seen during the second trimester of pregnancy, prior to clinical symptoms. Therefore; these proteoglycans may have the potential to be used as part of a protein serum biomarker panel to identify patients at risk of PPROM.

Contact Information: Beatrice Lechner, MD; Dept of Pediatrics, Women and Infants Hospital and The Warren Alpert Medical School at Brown University; Blechner@wihri.org. Eposterboard #16

Community-Engaged Tribal Research to Assess Dietary Exposures to Mercury and Polychlorinated Biphenyls (PCBs)

Marcella R Thompson1, Elizabeth M. Hoover2, Dinalyn Spears3 Mentors: Alison Field4, Gregory Wellenius4

1College of Nursing, University of Rhode Island; 2American and Ethnic Studies, Brown University; 3Narragansett Tribe, 4School of Public Health, Brown University

Aim. To describe Narragansett tribal members’ consumption of fish through a culturally appropriate household based survey. Specific Aim 1. To create a culturally appropriate household based survey to estimate Narragansett tribal members’ current and seasonal fish consumption rates. Specific Aim 2. To establish an infrastructure (interviewers, recruitment, data collection, data management and analyses) that will serve as a basis for future studies. Background. Cultural and economic factors are important determinants of health; in particular, fishing and fish consumption among indigenous populations. While seeking to protect the public from harmful health impacts, fish advisories do not take into account the impact of the absence of fish on indigenous culture. Conversely, continuing tribal fishing traditions in communities where fishing is critically linked to cultural identity has the potential to place tribal members at increased risk for health impacts from environmental contaminants. However, fish is an important source of protein, selenium (Se), and omega-3 polyunsaturated fatty acids (Ω-3 PUFAs). It is known that selenium can inhibit the absorption of methylmercury. There is evidence that consuming fish has beneficial effects for preventing cardiovascular disease and mortality. Recent testing of fish tissues have revealed elevated levels of two persistent and pervasive environmental neurotoxicants, mercury and polychlorinated biphenyls (PCBs). The next step in translating this wildlife research is to estimate its effects on tribal members’ health. Together with results of fish tissue and sediment analyses as well as information gleaned from Talking Circles (group discussions with tribal elders, leaders and others), these fish consumption rates will inform tribal leaders’ decisions about fish advisories and their implementation. Methods. A semi-quantitative cross-sectional household based survey will be structured to capture fishing-related activities, preparation and consumption patterns. For those Narragansett that fish, this survey will concentrate on fish species commonly found in local freshwaters, the Bay and offshore with specific questions regarding the fish found in two tribal ponds (Schoolhouse and Deep Ponds). Qualitative inquiries will ask about consumption suppression. Natives will be hired and trained as interviewers through the Indigenous Empowerment Network. Native interviewers will be paired with graduate students from URI College of Nursing and Brown University’s School of Public Health and American Studies for culturally based experiential learning. Conclusions and Implications. The infrastructure developed will serve as a basis for conducting a larger fish consumption survey aimed at determining the frequency of recreational and subsistence fishing among the Narragansett and estimating their annual dose of mercury and polychlorinated biphenyls (PCBs) given contaminant levels in fish living in two tribal ponds. Findings from this study will inform The Namaus (All Things Fish) Project leading to a health impact assessment, improving environmental health literacy, and conducting an environmental

risk assessment in advance of remediating and restoring these natural resources of significant cultural importance. ACKNOWLEDGMENTS. This work was supported by the National Institute of General Medical Sciences (NIGMS) under Grant U54GM115677 through Advance Center for Clinical Translational Science; National Institutes of Health (NIH) National Institute of Environmental Health Sciences (NIEHS) under Grant 2P42ES013660-11 through Brown University’s Superfund Research Program. The content is solely the responsibility of the authors and does not necessarily represent the official views of NIH, NIEHS, NIGMS or other funding agency. The authors do not have any financial conflicts of interest to declare. Contact: marcy_thompson@uri.edu +1.401.569.7548

Eposterboard #17 PATHOLOGIC ROLE OF MICRORNA-365 IN THE HEART

Nedyalka Valkov 1, 2, Tae Yun Kim1, Man Liu1, Jacob Moeller1, Michelle King1, Jin O-Uchi1, Qian Chen3, Bum-Rak Choi1, Peng Zhang1 1Cardiovascular Research Center, Rhode Island Hospital & Alpert Medical School of Brown University, Providence, RI; 2Dept of Molecular Pharmacology, Physiology and Biotechnology, Brown University, Providence, RI; 3Dept of Orthopaedics, Rhode Island Hospital & Alpert Medical School of Brown University, Providence, RI

Cardiac remodeling occurs in many cardiovascular diseases and can severely impair ventricular function. A better understanding of cardiac signaling mechanisms is critical for development of effective therapies. MicroRNAs (miRNAs) are important regulators in the heart. MiRNA-365 is upregulated upon cardiac stress in vitro (phenylephrine stimulation) and in vivo (after myocardial infarction). Our in vitro data suggests pro-hypertrophic potential of miRNA-365 in neonatal cardiac myocytes, evidenced by increase in cell size and atrial natriuretic factor (ANF) expression. The goal of this study is to delineate the functional importance and regulatory mechanisms of miRNA-365 in the heart. We generated a transgenic mouse model with cardiac specific, α myosin heavy chain-driven overexpression of miRNA-365. Significant increase of ANF expression in the ventricles of miRNA-365 transgenic mice suggests the activation of hypertrophy signaling. Ex vivo optical mapping data indicates highly inducible ventricular tachycardia (VT) in the heart of miRNA-365 transgenic mice and discordant calcium alternans is the major driven force for the VT events. Importantly, this discordant calcium alternans can be eliminated by Dantrolene, suggesting uncontrolled SR calcium release may contribute to the VT induction. Our data further show that spatially discordant calcium alternans can be reversed to concordant alternans by mitoTEMPO, suggesting that increased mitochondrial reactive oxygen species could be the upstream of the uncontrolled SR calcium release. These findings are supported by single cell patch clamps, in which the isolated myocytes from miRNA-365 overexpressing hearts show increased early afterdepolarizations (EAD), which are majorly due to elevated cytosolic calcium concentrations. Taken together, our results suggest miRNA-365 has a critical pathologic role in the heart and may regulate both structural and electrical cardiac remodeling, which warrants further investigation.

Contact Information Name: Nedyalka Valkov Institution: Cardiovascular Research Center Department: Medicine Phone #: 401-444-9856 Email: nedyalka_valkov@brown.edu

Eposterboard #18 CELL-FREE DNA AS A BIOMARKER FOR HEALTHY AGING Yee Voan Teo1, Miriam Capri3, Claudio Franceschi3, Nicola Neretti1,2 1Department of Molecular Biology, Cell Biology & Biochemistry, 2Center for Computational Molecular Biology, Brown University, 3University of Bologna Cell-free DNA (cfDNA) is present in the circulating plasma and other body fluids, and is thought to originate from apoptotic cells. Although, most of these cells belong to the hematopoietic lineage, apoptotic cells from other tissues can also contribute to the cfDNA pool. Recent studies have demonstrated that the fragmentation patterns of cfDNA can reveal the nucleosome landscape of the cells of origin. Here, we provide the first in vivo evidence of global and local chromatin changes in human aging by analyzing the cfDNA from the blood of individuals of different ages. Several studies have previously demonstrated changes to the chromatin structure as cells age, but so far, genome-wide chromatin reorganization has been primarily studied in model organisms and human cell cultures. Our results show that nucleosome signals inferred from cfDNA are consistent with the redistribution of heterochromatin observed in cellular senescence and aging in other model systems. It also revealed age and deteriorating health status correlate with the enrichment of signals from cells in the small intestine. We also detected an overall nucleosome loss at several genomic locations, such as transcription start and termination sites, and at the 5’UTR of L1Hs retrotransposons. Our results show that circulating cfDNA from human blood plasma can be used as a non-invasive way to study age-associated changes to the epigenome in vivo. Contact Information Name: Yee Voan Teo Institution: Brown University Department: Department of Molecular Biology, Cell Biology & Biochemistry Phone #: 401-863-3445 Email: yee_voan_teo@brown.edu

Eposterboard #19 Chronic Endurance Exercise Attenuates Impairments in VO2 max in Rats With Pulmonary Arterial Hypertension

N Kue1; D McCullough5; R Clements1,4; A Vang1; 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 5Edward Via College of Osteopathic Medicine, Auburn Campus – Auburn, AL

Rationale: While there are therapies, pulmonary arterial hypertension (PAH) is a progressive disease and patients continue to suffer from physical deconditioning. The mechanisms associated with exercise-induced improvements remain largely unidentified. This study was to evaluate the effect of chronic endurance exercise on cardiac function and exercise capacity in a rat model of PAH.

Methods: Male Sprague-Dawley rats were placed into normoxia or 3 wks of hypoxia (10% FiO2) after SU5416 injection followed by 5 wks of normoxia. Animals were then assigned to sedentary (SED) or exercise training (ExT; 1 hr/d @ 55-60% VO2max, 5 d/wk, 5 wks). Exercise capacity (VO2max) was determined via a metabolic treadmill and a graded exercise protocol, before (Pre) and after (Post) 5 weeks. To confirm PAH, transthoracic echocardiography was performed.

Results: PAH was confirmed by cardiac function measurements. Pre-VO2max was significantly lower in the PAH vs. Norm. However, there was significant improvement in Post VO2max in ExT-PAH vs. SED-PAH. There was no change in cardiac function between SED-PAH and ExT-PAH. Capillary density in soleus muscle was not affected by exercise. There was also no change pAMPKα or Opa-1. There was an increase in PGC1α expression in ExT-PAH. Relative mtDNA content did not change suggesting mitochondrial-independent pathways.

Conclusions: Exercise training significantly attenuated PAH-associated exercise intolerance, despite no change to cardiac function. This suggests exercise-induced improvements may be attributed to events occurring in the skeletal muscles and not cardiac or peripheral circulation effects. Differences in improved VO2max may involve modulation of mitochondrial and metabolism signaling involving PGC1α.

Contact Information Name: Gaurav Choudhary Institution: PVAMC Department: Medicine Phone #: 401-273-7100 x2029 Email: Gaurav_Choudhary@Brown.Edu Eposterboard #20 DIFFERENTIAL EFFECT OF LUBRICIN/PROTEOGLYCAN 4 (PRG4) IN MONOSODIUM (MSU)-CRYSTAL INDUCED INFLAMMATION ON Mφ, M1 AND M2 MACROPHAGES

Rashid Ahmed1, BS; Changqi Sun1, PhD; Nicole Yang1, MD; Keith Q. Wu2, MD; Tannin A. Schmidt3, PhD; Khalid A. Elsaid4, PhD; Olin Liang2, PhD; Gregory D. Jay5, MD, PhD; and Anthony M. Reginato1, PhD, MD.1Division of Rheumatology, Rhode Island Hospital, The Warren Alpert School of Medicine of Brown University, Providence, RI. USA; 2Department of Orthopedic Surgery, The Warren Alpert Medical School of Brown University, Providence, RI. USA; 3Deparment of Kinesiology and Schulich of Engineering, University of Calgary, AB, Calgary, Canada; 4Department of Pharmaceutical Science and School of Pharmacy, MCHS University, Boston, MA. USA; and 5Division of Emergency Medicine, Rhode Island Hospital, The Warren Alpert Medical School of Brown University, Providence, RI. USA.

Problem: Gouty arthritis arises when monosodium urate (MSU) crystals deposits in joints. Macrophage polarization influences the inflammatory cascade in rheumatic diseases. However, its role in gout has not been clearly defined. Recent studies have suggested that Lubricin/proteoglycan-4 (PRG4) has anti-inflammatory properties. The objective of this study was to investigate the anti-inflammatory effects of recombinant human PRG4 (rhPRG4) in MSU-induced polarized macrophages using cytokine array, immunohistochemistry, and Western blot analysis. Methods: The human monocytic cell line THP-1 was differentiated into macrophages (Mφ) using PMA. The macrophages were polarized by LPS and IFN-γ for classical macrophage activation (M1) or by IL-4 and IL-13 for alternative activation (M2). Mφ, M1, and M2 macrophages were exposed for MSU-induced inflammation in the presence or absence of rhPRG4 or colchicine. Results: Mφ showed a 2-fold increase in IL-1β, IL-8, TNF-α, IL-6, MCP-1, GRO, and GRO-α after MSU-induced inflammation. M1 showed a 1.5-fold increase in TNF-α and IL-6. In M2, RANTES showed a 5-fold decrease. Both colchicine and PRG4 had anti-inflammatory effects on RANTES, IL-1β, IL-8, TNF-α, IL-6, and MCP-1. In Mφ, osteopontin showed a 1.8-fold increase when treated with colchicine. rhPRG4 showed a mild decrease in IL-1β, TNF-α, and IL-6 cytokines in M1. In M2, rhPRG4 significantly decreased RANTES, MCP-1, MCP-2, IP-10, and IL-10 compared to colchicine treated M2. Conclusions: Cytokine profiling shows differential activation of the macrophage subgroups Mφ, M1, and M2 by MSU crystals. These results also confirm a differential cytokine inhibitory function on the various macrophage subgroups by rhPRG4 or colchicine.

Contact Information

Name: Rashid H. Ahmed

Institution: The Warren Alpert School of Medicine of Brown University

Department: Division of Rheumatology, Rhode Island Hospital

Phone #: (401) 444-1676

Email: rashidahmed0188@gmail.com

Eposterboard #21 ANTI-INFLAMMATORY ROLE OF LUBRICIN/PROTEOGLYCAN-4 (PRG4) IN MONOSODIUM URATE (MSU)-CRYSTAL INDUCED ARTHRITIS

Anthony M. Reginato1, PhD, MD; Rashid Ahmed1, BS; Marwa Qadri2, BA; Changqi Sun1, Nicole Yang3, MD, Khalid A. Elsaid2, PhD and Gregory D Jay4, PhD, MD.1Division of Rheumatology, Rhode Island Hospital, 2Department of Pharmaceutical Science, School of Pharmacy, MCHS University, Boston, 3Department of Medicine, Miriam Hospital, and 4Division of Emergency Medicine, Rhode Island Hospital, The Warren Alpert School of Medicine of Brown University, Providence, RI. USA

Problem: Lubricin/proteoglycan-4 (PRG4) is a mucinous glycoprotein secreted by synovial fibroblasts and superficial zone chondrocytes involved in joint boundary lubrication, lowering friction of apposed cartilage surfaces, and may have an endogenous anti-inflammatory role. The objective of this study was to evaluate the anti-inflammatory properties of PRG4 in monosodium urate (MSU) – acute gout inflammation. Methods: Synovial fluid (SF) aspirates from normal and patients with acute gout flares were used to evaluate the activation of TLR2 and TLR4 on HEK cells. Activation of TLR2 and TLR4 on HEK cells was assessed by immunoprecipitation of PRG4 from gout SF. We evaluated the impact of human recombinant PRG4 (hRPRG4) on MSU-induced release of IL-1β, TNF-α, and IL-8 by human monocytic cell line, THP-1 using ELISA, immunohistochemistry, and western-blot analysis. We also evaluated the role of hrPRG4 in inhibiting the NLRP3 inflammasome in THP-1 cells. Results: Using TLR-HEK cells systems, we found that synovial fluid from acute gout activated primarily through TLR2 rather than TLR4. Removal of PRG4 from gout SF by immunoprecipitation with a monoclonal anti-PRG4 antibody resulted in higher TLR2-HEK activation compared to gout SF and untreated controls. In a dose dependent manner, hrPRG4 significantly inhibited IL-1β, TNF-α, and IL-8 production by human monocytic cell line, THP-1 in response to MSU using ELISA, western blot analysis and immunohistochemistry. Immunohistochemistry of NLRP3 showed a dose dependent inhibition by hrPRG4 comparable to its inhibition by colchicine. Conclusions: PRG4 retards progression of MSU-crystal induced arthritis by binding to TLR2, mediating a novel anti-inflammatory role for PRG4 in joint homeostasis.

Contact Information

Name: Anthony M. Reginato

Institution: The Warren Alpert School of Medicine of Brown University

Department: Division of Rheumatology, Rhode Island Hospital

Phone #: (401) 649-4040

Email: areginato@lifespan.org

Eposterboard #22 BKCa-dependent cardioprotection is associated with increased mitochondrial supercomplex formation. Sodha, Neel R., Cordeiro, Brenda, Terntyeva, Radmila, Potz, Brittany A., Abid, Ruhul M., Kolodziejczak, Martin, Sellke, Frank W., Terentyev, Dmitry., and Clements, Richard T.

Vascular Research Lab, Providence VAMC and Cardiothoracic Surgery Research, Department of Surgery, Rhode Island Hospital

Introduction:Large conductance Ca++-acitvated K+ channel(BKCa) activation in cardiomyocyte mitochondria are thought to enhance myocardial protection through beneficial reduction in mitochondrial ROS and improved respiration. Individual electron transport chain (ETC) complexes can physically associate to create mitochondrial supercomplexes (SC) which can potentially provide more efficient respiration and reduced ROS. The current study evaluated

the hypothesis that BKCa-dependent cardioprotection is mediated through enhanced SC formation in cardiomyocyte mitochondria.

Methods:Cardiomyoblast H9c2 cells were subjected to simulated in vitro hypoxic cardioplegia (CP) and 30 min reoxygenation with or without the BKCa channel activator rottlerin followed by cell respiration (seahorse), ROS, supercomplex assembly (BN-PAGE), and mitochondrial membrane potential measurement. For large animal studies 50 kg Yorkshire pigs of either sex were subjected to cardiopulmonary bypass and 1 hour hypothermic cardioplegic arrest with (n=4) and without (n=4) rottlerin followed by 1 hour reperfusion and tissue harvest.

Results:Hypoxic CP/R increased ROS, reduced mitochondrial membrane potential (ΔΨm), and reduced respiration which were normalized with BKCa activation. BKCa activation increased mitochondrial SCs which consisted of components of complex I,III, and IV as assessed by BN-PAGE/immunoblot. In pigs subjected to CP/CPB, inclusion of 500 nM rottlerin in the CP solution significantly improved recovery of LVDevP, +/- dP/dt, and cardiac contractility (end systolic Elastance) up to 60 minutes post-reperfusion. BKCa activation in pigs associated also with increased ETC supercomplex formation.

Conclusion:BKCa activation during hypothermic cardioplegic arrest improves cardiac functional recovery upon reperfusion. BKCa-mediated improved cardiac function is associated with enhanced mitochondrial supercomplex formation and likely more efficient respiration and reduced ROS.

Contact Info: Richard T. Clements, Ocean State Research Institute, Providence VAMC, Ph 518-330-1553, richard_clements@brown.edu

Eposterboard #23

NEOCORTICAL CONTROL OF THE THALAMUS S.J. Cruikshank, S.R. Crandall, C.A. Deister, C.I. Moore & B.W. Connors; Department of Neuroscience, Brown University, Providence, RI

Problem: Most sensory information ascends to the perceptually critical neocortex via thalamus. Curiously, the neocortex also sends massive descending “corticothalamic” (CT) input to thalamus—CT axons outnumber ascending “thalamocortical” axons tenfold. This implies that the cortex strongly influences thalamus and, thus, its own input. Understanding CT function has been elusive. The scale (even the sign!) of thalamic modulation by neocortex has varied widely across studies. Circuit complexity has impeded progress, but powerful optogenetic tools can now be brought online. We have pioneered use of such tools for CT study (Cruikshank et al., Neuron, 2010) and the present experiments extend this. We are testing the hypothesis, based partly on our own in vitro data (Crandall, et al., Neuron, 2015), that CT circuits bidirectionally control thalamic sensory processing. We predict that the sign of control is dynamically determined by CT spike rates and short-term synaptic plasticity. Methods and Results: In whole-cell and juxtacellullar recordings from mouse thalamic neurons in vivo, we observed classic voltage-dependent bursting/tonic spiking, and photoactivated CT responses. CT input modulated thalamic excitability as predicted. Low frequency CT stimulation markedly suppressed sensory responses, consistent with feedforward inhibition. Conclusions. We found that most CT properties in vivo were closely consistent with those observed in vitro, supporting our hypothesis. We are now examining high frequency CT effects, and interactions between CT

influence and arousal/behavioral states (assayed via pupillometry, ECoG, locomotion, sensory detection). This project is providing insight into how the neocortex controls thalamus, which is essential for understanding disorders involving CT communication.

Contact Information: Scott J. Cruikshank, Brown University, Department of Neuroscience, 401-338-8094, Scott_Cruikshank@Brown.edu

Eposterboard #24 THE ROLE OF SHP-2 ON NK CELL DEVELOPMENT AND FUNCTIONS

S. M. Shahjahan Miah1, Veronica Sexl2, Wentian Yang1 and Laurent Brossay1. 2University of

Veterinary Medicine Vienna, Austria1. Brown University, Providence, RI

Abstract: The SH2 domain-containing tyrosine phosphatase SHP-2 (PTPN11) has been shown to play essential roles in immune cell signaling. Several inhibitory receptors expressed on natural killer (NK) cells associate with SHP-2 to exert downstream functions. Here we examined the influence of SHP-2 on NK cell development and effector activity. Because SHP-2 is required for embryonic development in mice, we generated mice conditionally deficient for SHP-2 in NK cells. SHP-2fl/fl mice were crossed to NKp46-Cre mice in order to inactivate Ptpn11 in NKp46+ cells. These mice were also crossed to R26R-EYFP reporter mice to track cells with active Cre recombinase. We found an increase in NK cell frequency in these mice compared to littermate controls. The frequency of mature NK cells (CD11b+CD27-) was also increased in the periphery while decreased in the bone marrow. In addition, homeostatic proliferation of SHP-2-deficient NK cells was impaired. However, effector functions were not affected significantly. These included IFN-γ production at the peak of the NK cell response during MCMV infection, rejection of β2-microglobulin deficient splenocytes, and degranulation via Ly49H using M157-transfected cells as targets. Taken together, the data suggest that SHP-2 is dispensable for NK cell effector functions and education, but is important for NK cell maturation.

Funding: COBRE Pilot Grant 4P20GM104317

Contact Information:

Shah Md. Shahjahan Miah

Investigator

Molecular Microbiology and Immunology

Brown University, Providence, RI

Phone: 401-863-9643

Email: shahjahan_miah@brown.edu

Eposterboard #25 PULMONARY ARTERY STIFFNESS IS ASSOCIATED WITH RIGHT VENTRICULAR FIBROSIS IN PULMONARY HYPERTENSION

Siddique Abbasi, MD*, Ryan Hebel, BA*, Wen-Chih Wu, MD, MPH*, Matthew Jankowich, MD*, Gaurav Choudhary, MD*

*Providence VA Medical Center, Providence, RI

Problem: The factors that lead to maladaptive right ventricular (RV) remodeling in pulmonary hypertension (PH) are not well-understood. RV fibrosis is a hallmark of RV failure. Pulmonary artery (PA) stiffness has been associated adverse clinical outcomes in PH; however, the relationship between PA stiffness and RV fibrosis remains unknown. We performed right-heart catheterization and cardiac MRI on fifteen patients with PH. PA stiffness was assessed using pulse wave velocity (PWV). RV fibrosis was assessed using T1 mapping to quantify the RV extracellular volume fraction (ECV). Results: On average, patients were older (mean age = 69.1±7.4 years), obese (mean body mass index = 33.1±10.0 kg/m2), and had preserved biventricular function (mean LVEF = 57.1±7.35%, mean RVEF = 46.8±11.8%). Overall, RV fibrosis (mean ECV = 38.9±0.05%) and PA stiffness were markedly elevated (mean PWV = 3.44±1.0m/s). There was a strong association between RV ECV and PA PWV by Spearman correlation (r = 0.72, p = 0.0051). Using multivariable regression, only PA PWV was significantly associated with RV ECV in a model that included age, gender, and mPAP. Conclusion: Pulmonary artery stiffness is independently associated with RV fibrosis in patients with PH, and may play an important role in the pathogenesis of RV failure.

Siddique Abbasi, MD; Providence VAMC; Department of Medicine/Cardiology;

401-273-7100 x2265; siddique_abbasi@brown.edu

Eposterboard #26

Mechanical Forces Accelerates Fetal Lung Development via Release of Extracellular

Vesicles

Tanbir Najrana1, Laura Goldberg2, Peter J. Quesenberry2, Juan Sanchez-Esteban1.

1Department of Pediatrics, Division of Neonatology, Women & Infants Hospital of Rhode Island

2Department of Medicine, Division of Hematology/Oncology, Rhode Island Hospital.

Background: Lung underdevelopment secondary to extreme prematurity and pulmonary

hypoplasia can cause significant morbidity and mortality to the neonatal population. Currently,

there are no effective interventions to accelerate lung development. Mechanical forces generated

inside the fetal lung are critical for normal lung development. However, the mechanisms by which

mechanical signals stimulate lung development are not fully-characterized. Extracellular vesicles

(EVs), including exosomes and microvesicles, are small, membrane-bound particles, increasingly

recognized as a novel mode of cell-to-cell communication. They play important roles in cancer,

inflammation, immunity, etc. However, the role of EVs in fetal lung development is unexplored.

Hypothesis: Mechanical signals promote fetal lung development via release of EVs.

Methods: EVs were isolated from the E18.5 mouse fetal lungs using differential centrifugation

steps (1000g, 15000 g for 20 mins at 4°C and 110000g for 2h at 4°C). Size and concentration of

EVs were measured by nanoparticle tracking analysis (NTA). Purity of EVs was analyzed by

Western blot and flow cytometry using anti-CD63 and anti-CD9 antibodies. Isolated E18.5 mouse

epithelial cells were cultured on Bioflex plates coated with laminin and exposed to 5% cyclic

stretch (to mimic mechanical forces in lung development) for 24 hours using the Flexercell Strain

Unit. EVs were isolated from cell supernatant following the above mentioned protocol. EVs from

stretched cells were added to the mouse epithelial cell culture to evaluate the effect of cell

differential.

Results: More than 80% of the EVs isolated from the fetal lung fluids have a diameter of around

100nm and tetraspanins surface markers including CD9 and CD63, consistent with exosomes.

Mechanical stretch of fetal epithelial cells increased release of EVs by 2.4 fold when compared to

controls. Moreover, incubation of primary fetal epithelial cells with EVs released from stretched

cells or from EVs isolated from fetal lungs promoted type I epithelial cell differentiation.

Conclusion: EVs are present in the lumen of the fetal lung. Mechanical signals release EVs that

are important for differentiation of fetal type I epithelial cells. Future studies will test this

hypothesis using ex vivo and in vivo models.