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BACKGROUND Interleukin 13 (IL-13) is a pleiotropic cytokine that has been shown to be important in the pathogenesis of chronic rhinosinusitis with nasal polyps (CRSwNP) and other type 2 inflammation-related diseases. Increased IL-13 expression can elicit several pro-inflammatory effects,including eosinophilia,and pathology such as increased mucus secretion. Polypogenesis in chronic rhinosinusitis (CRS) can be caused by hypoxia,which can also lead to hyperpermeability of airway epithelium and epithelium-to-mesenchymal translation through the upregulation of hypoxia-associated genes,such as HIF1. Whether T-helper 2 (Th2) inflammatory cytokines,such as IL-13,can also induce sinonasal epithelial hypoxia-associated genes is currently unknown. METHODS Human air-liquid interface (ALI) sinonasal epithelial cell cultures treated with recombinant IL-13 were analyzed by real-time polymerase chain reaction (PCR) and flow cytometry to determine the effect on epithelial cells. RESULTS Whole tissue from CRSwNP subjects showed increased HIF1A gene expression. Treatment of fully differentiated human ALI cultures with IL-13 resulted in a concurrent increase in HIF1A and ARNT messenger RNA (mRNA) expression. However,the level of EPAS1 expression was significantly reduced. IL-13 also had a dose-dependent response on the expression of HIF genes and the time course experiment showed peak expression of HIF1A and ARNT at 5 to 7 days poststimulation. Remarkably,CD73 surface expression also peaked at day 5 poststimulation. CONCLUSION Our data suggests that IL-13 can induce hypoxia signaling pathway genes leading to surface expression of CD73,which has an anti-inflammatory effect. View Publication

Perhexiline is a potent prophylactic anti-anginal agent that has been shown to inhibit myocardial utilization of long-chain fatty acids and to inhibit the mitochondrial enzyme carnitine palmitoyltransferase (CPT)-1. We compared the hemodynamic and biochemical effects of perhexiline (0.5 and 2.0 microM) and of another CPT-1 inhibitor,oxfenicine (0.5 mM),in Langendorff-perfused rat hearts subjected to 60 min of low-flow ischemia (95{\%} flow reduction) followed by 30 min of reperfusion. Both perhexiline (2 microM only) and oxfenicine attenuated (p {\textless} 0.003,p {\textless} 0.0002,respectively) increases in diastolic tension during ischemia,without significant effects on developed tension,or on cardiac function during reperfusion. Myocardial concentrations of long-chain acylcarnitines (LCAC),products of CPT-1 action,were decreased (p {\textless} 0.05) by oxfenicine,unaffected by 2 microM perhexiline,and increased slightly by 0.5 microM perhexiline. Perhexiline,but not the active metabolite of oxfenicine,also inhibited cardiac CPT-2 with similar IC50 and Emax,although lower Hill slope,compared with CPT-1. Oxfenicine,but not perhexiline,reduced concentrations of the endogenous CPT-1 inhibitor,malonyl-CoA. Perhexiline,but not oxfenicine,inhibited myocardial release of lactate during normal flow. We conclude that (a) perhexiline protects against diastolic dysfunction during ischemia in this model,independent of major changes in LCAC accumulation and (b) this may result from simultaneous effects of perhexiline on myocardial CPT-1 and CPT-2. View Publication

The mechanism of the anti-anginal effect of perhexiline is unclear but appears to involve a shift in cardiac metabolism from utilization of fatty acid to that of carbohydrate. We tested the hypothesis that perhexiline inhibits the enzyme carnitine palmitoyltransferase-1 (CPT-1),which controls access of long chain fatty acids to the mitochondrial site of beta-oxidation. Perhexiline produced a concentration-dependent inhibition of CPT-1 in rat cardiac and hepatic mitochondria in vitro,with half-maximal inhibition (IC50) at 77 and 148 mumol/L,respectively. Amiodarone,another drug with anti-anginal properties,also inhibited cardiac CPT-1 (IC50 = 228 mumol/L). The rank order of potency for inhibition was malonyl-CoA {\textgreater} 4-hydroxyphenylglyoxylate (HPG) = perhexiline {\textgreater} amiodarone = monohydroxy-perhexiline. Kinetic analysis revealed competitive inhibition of cardiac and hepatic CPT-1 by perhexiline with respect to palmitoyl-CoA but non-competitive inhibition with respect to carnitine. Curvilinear Dixon plots generated apparent inhibitory constant (Ki)" values for perhexiline which indicated a greater sensitivity of the cardiac than the hepatic enzyme to inhibition by perhexiline. Perhexiline inhibition of CPT-1 unlike that of malonyl-CoA and HPG was unaffected by pretreatment with the protease nagarse. These data establish for the first time that two agents with proven anti-anginal effects inhibit cardiac CPT-1. This action is likely to contribute to the anti-ischaemic effects of both perhexiline and amiodarone." View Publication

Airway inflammation is a critical feature of lower respiratory tract infections caused by viruses such as respiratory syncytial virus (RSV). A growing body of literature has demonstrated the importance of extracellular matrix (ECM) changes such as the accumulation of hyaluronan (HA) and versican in the subepithelial space in promoting airway inflammation; however,whether these factors contribute to airway inflammation during RSV infection remains unknown. To test the hypothesis that RSV infection promotes inflammation via altered HA and versican production,we studied an ex vivo human bronchial epithelial cell (BEC)/human lung fibroblast (HLF) co-culture model. RSV infection of BEC/HLF co-cultures led to decreased hyaluronidase expression by HLFs,increased accumulation of HA,and enhanced adhesion of U937 cells as would be expected with increased HA. HLF production of versican was not altered following RSV infection; however,BEC production of versican was significantly downregulated following RSV infection. In vivo studies with epithelial-specific versican-deficient mice [SPC-Cre(+) Vcan-/-] demonstrated that RSV infection led to increased HA accumulation compared to control mice which also coincided with decreased hyaluronidase expression in the lung. SPC-Cre(+) Vcan-/- mice demonstrated enhanced recruitment of monocytes and neutrophils in bronchoalveolar lavage fluid and increased neutrophils in the lung compared to SPC-Cre(-) RSV-infected littermates. Taken together,these data demonstrate that altered ECM accumulation of HA occurs following RSV infection and may contribute to airway inflammation. Additionally,loss of epithelial expression of versican promotes airway inflammation during RSV infection further demonstrating that versican's role in inflammatory regulation is complex and dependent on the microenvironment. View Publication

Impaired protein stability or trafficking underlies diverse ion channelopathies and represents an unexploited unifying principle for developing common treatments for otherwise dissimilar diseases. Ubiquitination limits ion channel surface density,but targeting this pathway for the purposes of basic study or therapy is challenging because of its prevalent role in proteostasis. We developed engineered deubiquitinases (enDUBs) that enable selective ubiquitin chain removal from target proteins to rescue the functional expression of disparate mutant ion channels that underlie long QT syndrome (LQT) and cystic fibrosis (CF). In an LQT type 1 (LQT1) cardiomyocyte model,enDUB treatment restored delayed rectifier potassium currents and normalized action potential duration. CF-targeted enDUBs synergistically rescued common ($\Delta$F508) and pharmacotherapy-resistant (N1303K) CF mutations when combined with the US Food and Drug Administation (FDA)-approved drugs Orkambi (lumacaftor/ivacaftor) and Trikafta (elexacaftor/tezacaftor/ivacaftor and ivacaftor). Altogether,targeted deubiquitination via enDUBs provides a powerful protein stabilization method that not only corrects diverse diseases caused by impaired ion channel trafficking,but also introduces a new tool for deconstructing the ubiquitin code in situ. View Publication

BACKGROUND The conducting airway epithelium is repaired by tissue specific stem cells (TSC). In response to mild/moderate injury,each TSC repairs a discrete area of the epithelium. In contrast,severe epithelial injury stimulates TSC migration and expands the stem cell's reparative domain. Lung transplantation (LTx) can cause a moderate/severe airway injury and the remodeled airway contains a chimeric mixture of donor and recipient cells. These studies supported the hypothesis,LTx stimulates TSC migration resulting in epithelial chimerism. We tested this hypothesis in cystic fibrosis (CF) LTx patients. METHODS Airway mucosal injury was quantified using bronchoscopic imaging and a novel grading system. Bronchial brushing was used to recover TSC from 10 sites in the recipient and allograft airways. TSC chimerism was quantified by short tandem repeat analysis. TSC self-renewal and differentiation potential were assayed using the clone forming cell frequency and air-liquid-interface methods. Electrophysiology was used to determine if TSC chimerism altered epithelial ion channel activity. RESULTS LTx caused a mild to moderate airway mucosal injury. Donor and recipient TSC were identified in 91{\%} of anastomotic sites and 93{\%} of bronchial airways. TSC chimerism did not alter stem cell self-renewal or differentiation potential. The frequency of recipient TSC was proportional to CF Transmembrane Conductance Regulator (CFTR)-dependent ion channel activity and 33{\%} of allograft regions were at risk for abnormal CFTR activity. CONCLUSIONS LTx in CF patients stimulates bidirectional TSC migration across the anastomoses. TSC chimerism may alter ion homeostasis and compromise the host defense capability of the allograft airway epithelium. View Publication

The endoplasmic reticular Ca(2+)-ATPase inhibitor,thapsigargin,was used to study the role of an increase in cytosolic free calcium concentration ([Ca2+]i) as a signal for the activation of thymocyte apoptosis. Treatment of rat thymocytes with thapsigargin resulted in an early sustained increase in [Ca2+]i followed by extensive DNA fragmentation. Agarose gel electrophoresis revealed that the pattern of DNA fragments was typical of endonuclease-mediated internucleosomal cleavage. In addition,confocal microscopy studies showed the formation of apoptotic nuclei in thapsigargin-treated thymocytes. The concentrations of thapsigargin required to induce DNA fragmentation and [Ca2+]i increase in thymocytes were identical and so were the kinetics of thapsigargin-induced DNA fragmentation and formation of apoptotic nuclei. The lowest concentration of thapsigargin needed to activate apoptosis was 1 nM. Thapsigargin-induced [Ca2+]i increase and thymocyte apoptosis were inhibited in cells incubated in nominally Ca(2+)-free medium or pretreated with the intracellular Ca2+ chelator,bis-(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid/acetoxymethyl ester. Removal of extracellular free Ca2+ with 5 mM EGTA at different time points after thapsigargin addition revealed a time dependency of about 2 h for the sustained increase in [Ca2+]i to trigger apoptosis in thymocytes. Thus,we conclude that the signal provided by the thapsigargin-induced [Ca2+]i increase is sufficient to activate thymocyte apoptosis. View Publication

Perhexiline maleate,originally classified as a calcium antagonist,is in use as an antianginal agent. The mechanism of its protective effect is unknown,but there is speculation that it involves a modification of myocardial substrate utilization,in which glycolytic sources are used rather than fatty acids. This hypothesis was tested by employing [13C]NMR isotopomer analysis to measure substrate selection in the working rat heart. Substrate utilization was measured from a mixture of substrates present at their physiological concentration,as follows: acetoacetate,glucose,lactate and long-chain fatty acids. Control perfusions were compared with those perfused with perhexiline. It was found that perhexiline increased lactate utilization,which reduced the extent of fatty acid and endogenous substrate oxidation. There was also a significant increase in cardiac output for a small and insignificant increase in oxygen consumption,which suggested an improvement in myocardial efficiency. Thus,it was confirmed by direct measurement that this drug does modify substrate oxidation,which suggests that further investigations of the role that this agent can play in the management of ischemic heart disease would be beneficial. View Publication

Retinal pigment epithelium (RPE) performs numerous functions critical to retinal health and visual function. RPE senescence is a hallmark of aging and degenerative retinal disease development. Here,we evaluated the temporal expression of key nicotinamide adenine dinucleotide (NAD+)-biosynthetic genes and associated levels of NAD+,a principal regulator of energy metabolism and cellular fate,in mouse RPE. NAD+ levels declined with age and correlated directly with decreased nicotinamide phosphoribosyltransferase (NAMPT) expression,increased expression of senescence markers (p16INK4a,p21Waf/Cip1,ApoJ,CTGF and $\beta$-galactosidase) and significant reductions in SIRT1 expression and activity. We simulated in vitro the age-dependent decline in NAD+ and the related increase in RPE senescence in human (ARPE-19) and mouse primary RPE using the NAMPT inhibitor FK866 and demonstrated the positive impact of NAD+-enhancing therapies on RPE cell viability. This,we confirmed in vivo in the RPE of mice injected sub-retinally with FK866 in the presence or absence of nicotinamide mononucleotide. Our data confirm the importance of NAD+ to RPE cell biology normally and in aging and demonstrate the potential utility of therapies targeting NAMPT and NAD+ biosynthesis to prevent or alleviate consequences of RPE senescence in aging and/or degenerative retinal diseases in which RPE dysfunction is a crucial element. View Publication

Older organs represent an untapped potential to close the gap between demand and supply in organ transplantation but are associated with age-specific responses to injury and increased immunogenicity,thereby aggravating transplant outcomes. Here we show that cell-free mitochondrial DNA (cf-mt-DNA) released by senescent cells accumulates with aging and augments immunogenicity. Ischemia reperfusion injury induces a systemic increase of cf-mt-DNA that promotes dendritic cell-mediated,age-specific inflammatory responses. Comparable events are observed clinically,with the levels of cf-mt-DNA elevated in older deceased organ donors,and with the isolated cf-mt-DNA capable of activating human dendritic cells. In experimental models,treatment of old donor animals with senolytics clear senescent cells and diminish cf-mt-DNA release,thereby dampening age-specific immune responses and prolonging the survival of old cardiac allografts comparable to young donor organs. Collectively,we identify accumulating cf-mt-DNA as a key factor in inflamm-aging and present senolytics as a potential approach to improve transplant outcomes and availability. View Publication

Interleukin-4 (IL-4) suppresses the development of multiple sclerosis in a murine model of experimental autoimmune encephalomyelitis (EAE). Here,we show that,in mice with EAE,the accumulation and persistence in the lymph nodes and spleen of a systemically administered serum albumin (SA)-IL-4 fusion protein leads to higher efficacy in preventing disease development than the administration of wild-type IL-4 or of the clinically approved drug fingolimod. We also show that the SA-IL-4 fusion protein prevents immune-cell infiltration in the spinal cord,decreases integrin expression in antigen-specific CD4+ T cells,increases the number of granulocyte-like myeloid-derived suppressor cells (and their expression of programmed-death-ligand-1) in spinal cord-draining lymph nodes and decreases the number of T helper 17 cells,a pathogenic cell population in EAE. In mice with chronic EAE,SA-IL-4 inhibits immune-cell infiltration into the spinal cord and completely abrogates immune responses to myelin antigen in the spleen. The SA-IL-4 fusion protein may be prophylactically and therapeutically advantageous in the treatment of multiple sclerosis. View Publication

Accumulating evidence suggests important roles for the receptor tyrosine kinase Axl in cancer progression,invasion,metastasis,drug resistance,and patient mortality,highlighting Axl as an attractive target for therapeutic development. We have generated and characterized a potent and selective small-molecule inhibitor,R428,that blocks the catalytic and procancerous activities of Axl. R428 inhibits Axl with low nanomolar activity and blocked Axl-dependent events,including Akt phosphorylation,breast cancer cell invasion,and proinflammatory cytokine production. Pharmacologic investigations revealed favorable exposure after oral administration such that R428-treated tumors displayed a dose-dependent reduction in expression of the cytokine granulocyte macrophage colony-stimulating factor and the epithelial-mesenchymal transition transcriptional regulator Snail. In support of an earlier study,R428 inhibited angiogenesis in corneal micropocket and tumor models. R428 administration reduced metastatic burden and extended survival in MDA-MB-231 intracardiac and 4T1 orthotopic (median survival,{\textgreater}80 days compared with 52 days; P {\textless} 0.05) mouse models of breast cancer metastasis. Additionally,R428 synergized with cisplatin to enhance suppression of liver micrometastasis. Our results show that Axl signaling regulates breast cancer metastasis at multiple levels in tumor cells and tumor stromal cells and that selective Axl blockade confers therapeutic value in prolonging survival of animals bearing metastatic tumors. View Publication