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Wang Q.,University of Toronto | Wang Y.,University of Toronto | Fritz D.,University of Toronto | Rajshankar D.,University of Toronto | And 3 more authors.
Journal of Biological Chemistry | Year: 2014

Interleukin-1 (IL-1) signaling in fibroblasts is mediated through focal adhesions, organelles that are enriched with adaptor and cytoskeletal proteins that regulate signal transduction. We examined interactions of the focal adhesion kinase (FAK) with protein-tyrosine phosphatase-α(PTP-α) in IL-1 signaling. In wild type and FAK knock-out mouse embryonic fibroblasts, we found by immunoblotting, immunoprecipitation, immunostaining, and gene silencing that FAK is required for IL-1-mediated sequestration of PTPαto focal adhesions. Immunoprecipitation and pulldown assays of purified proteins demonstrated a direct interaction between FAK and PTPα, which was dependent on the FAT domain of FAK and by an intact membraneproximal phosphatase domain of PTPα. Recruitment of PTPα to focal adhesions, IL-1-induced Ca2+ release from the endoplasmic reticulum, ERK activation, and IL-6, MMP-3, and MMP-9 expression were all blocked in FAK knock-out fibroblasts. These processes were restored in FAK knock-out cells transfected with wild type FAK, FAT domain, and FRNK. Our data indicate that IL-1-induced signaling through focal adhesions involves interactions between the FAT domain of FAK and PTPα. © 2014 by The American Society for Biochemistry and Molecular Biology, Inc.

Cumbo-Nacheli G.,Cleveland Clinic | Samavati L.,Sleep and Critical Care Medicine | Guzman J.A.,Cleveland Clinic
Journal of Critical Care | Year: 2011

Introduction: Venous thromboembolism is a common problem in the intensive care unit (ICU). To decrease its incidence, prophylactic pharmacologic interventions are part of the ICU routine. However, common ICU conditions may impair the bioavailability of subcutaneously administered agents. The present study evaluates the bioavailability of prophylactic subcutaneous fondaparinux to critically ill patients. Purpose: The purpose of the study was to evaluate vasopressor effect on the bioavailability of subcutaneously administered fondaparinux. Materials and Methods: A 2-center, prospective, observational study was performed. Forty patients were enrolled and divided into 2 groups depending on their vasopressor requirements. All subjects were critically ill patients admitted to a medical ICU for an anticipated stay of more than 72 hours. Interventions: All patients received subcutaneous fondaparinux 2.5 mg/d, and serum anti-Xa factor was serially assessed during the first 100 hours of medical ICU stay. Results: Therapeutic anti-factor Xa levels among patients receiving vasopressors were observed. In hemodynamically normal patients, subtherapeutic concentrations were detected during the first 48 hours of fondaparinux administration. Conclusions: Vasopressor therapy does not appear to affect fondaparinux bioavailability or to reduce anti-factor Xa levels. Subtherapeutic concentrations were detected during the first 48 hours of fondaparinux administration in hemodynamically stable patients. The clinical significance of reduced levels during the first 2 days of fondaparinux administration remains unknown. © 2011 Elsevier Inc.

Klinger J.R.,Vascular Research Laboratory | Klinger J.R.,Brown University | Tsai S.-W.,Vascular Research Laboratory | Tsai S.-W.,Brown University | And 7 more authors.
Journal of Applied Physiology | Year: 2013

Atrial natriuretic peptide (ANP) inhibits agonist-induced pulmonary edema formation, but the signaling pathway responsible is not well defined. To investigate the role of the particulate guanylate cyclase-linked receptor, natriuretic peptide receptor-A (NPR-A), we measured acute lung injury responses in intact mice and pulmonary microvascular endothelial cells (PMVEC) with normal and disrupted expression of NPR-A. NPR-A wild-type (NPR-A+/+), heterozygous (NPR-A+/-), and knockout (NPR-A-/-) mice were anesthetized and treated with thrombin receptor agonist peptide (TRAP) or lipopolysaccharide (LPS). Lung injury was assessed by lung wet-to-dry (W/D) weight and by protein and cell concentration of bronchoalveolar lavage (BAL) fluid. No difference in pulmonary edema formation was seen between NPR-A genotypes under baseline conditions. TRAP and LPS increased lung W/D weight and BAL fluid cell counts more in NPR-A-/- mice than in NPR-A+/- or NPR-A+/+ mice, but no genotype-related differences were seen in TRAP-induced increases in bloodless lung W/D weight or LPS-induced increases in BAL protein concentration. Pretreatment with ANP infusion completely blocked TRAP-induced increases in lung W/D weight and blunted LPSinduced increases in BAL cell counts and protein concentration in both NPR-A-/- and NPR-A+/+ mice. Thrombin decreased transmembrane electrical resistance in monolayers of PMVECs in vitro, and this effect was attenuated by ANP in PMVECs isolated from both genotypes. Administration of the NPR-C-specific ligand, cANF, also blocked TRAP-induced increases in lung W/D weight and LPSinduced increases in BAL cell count and protein concentration in NPR-A+/+ and NPR-A-/- mice. We conclude that ANP is capable of attenuating agonist-induced lung edema in the absence of NPR-A. The protective effect of ANP on agonist-induced lung injury and pulmonary barrier function may be mediated by NPR-C. Copyright © 2013 the American Physiological Society.

Levinson A.T.,University of Rhode Island | Klinger J.R.,Sleep and Critical Care Medicine
Therapeutic Advances in Respiratory Disease | Year: 2011

Multiple medical therapies have been developed for the treatment of pulmonary arterial hypertension (PAH) over the last decade and a half. Unfortunately, none of these medications is curative and the majority of patients develop disease progression despite treatment. Presently available medications target one of three known pathways that have been implicated in disease pathogenesis. The multiplicity of pulmonary vascular abnormalities identified in PAH provides the rationale for a therapeutic strategy that targets more than one mechanism at a time. Although a handful of studies have demonstrated clinical improvement in PAH patients who have a second medication added to stable background therapy in a randomized, placebo-controlled fashion, it is unclear whether the derived benefit is due to the combination of two therapies or merely the response to the new agent. This review discusses the rationale for combination therapy, critically reviews the findings of presently completed combination studies and outlines the need for new studies that are better designed to determine whether combination therapy is more efficacious than single agent therapies for the treatment of PAH. © SAGE Publications 2011.

Casserly B.,Memorial Hospital of Rhode Island | Casserly B.,Brown University | Pietras L.,Sleep and Critical Care Medicine | Schuyler J.,Rhode Island Hospital | And 4 more authors.
Life Sciences | Year: 2010

Aims: Atrial natriuretic peptide (ANP) is released from the heart in response to hypoxia and helps mitigate the development of pulmonary hypertension. However, the mechanism of hypoxia-induced ANP release is not clear. The cardiac atria are the primary source of ANP secretion under normal conditions, but right ventricular ANP expression is markedly up-regulated during adaptation to hypoxia. We sought to better understand mechanisms of cardiac ANP release during adaptation to hypoxia. Main methods: We measured hypoxia-induced ANP release from isolated perfused rat hearts obtained from normoxia and hypoxia-adapted rats before and after removal of the atria. Key findings: In both normoxia- and hypoxia-adapted hearts, ANP levels in the perfusate increased within 15 min of hypoxia. Hypoxia-induced ANP release was greater from hypoxia-adapted than normoxia-adapted hearts. Baseline and hypoxia-induced ANP release were considerably greater with the atria intact (213 ± 29 to 454 ± 62 and 281 ± 26 to 618 ± 87 pg/ml for normoxia- and hypoxia-adapted hearts respectively, P< 0.001 for both) than with atria removed (94 ± 17 to 131 ± 32 and 103 ± 26 to 201 ± 55 pg/ml, respectively, P< 0.002 for both). Hypoxia-induced ANP release was reduced over 80% by removing the atria in both normoxia- and in hypoxia-adapted hearts. Acute hypoxia caused a transient increase in lactate release and reductions in pH and left ventricular generated force, but no differences in pH or left ventricular generated force were seen between normoxia- and hypoxia-adapted rats. Significance: We conclude that the right ventricle is not a major source of cardiac ANP release in normoxia- or hypoxia-adapted rats. © 2010 Elsevier Inc.

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