Browsing by Author "Natarajan, Viswanathan"

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    Cigarette Smoke and Nicotine-Containing Electronic-Cigarette Vapor Downregulate Lung WWOX Expression, Which Is Associated with Increased Severity of Murine Acute Respiratory Distress Syndrome
    (American Thoracic Society, 2021) Zeng, Zhenguo; Chen, Weiguo; Moshensky, Alexander; Shakir, Zaid; Khan, Raheel; Crotty Alexander, Laura E.; Ware, Lorraine B.; Aldaz, C.M.; Jacobson, Jeffrey R.; Dudek, Steven M.; Natarajan, Viswanathan; Machado, Roberto F.; Singla, Sunit; Medicine, School of Medicine
    A history of chronic cigarette smoking is known to increase risk for acute respiratory distress syndrome (ARDS), but the corresponding risks associated with chronic e-cigarette use are largely unknown. The chromosomal fragile site gene, WWOX, is highly susceptible to genotoxic stress from environmental exposures and thus an interesting candidate gene for the study of exposure-related lung disease. Lungs harvested from current versus former/never-smokers exhibited a 47% decrease in WWOX mRNA levels. Exposure to nicotine-containing e-cigarette vapor resulted in an average 57% decrease in WWOX mRNA levels relative to vehicle-treated controls. In separate studies, endothelial (EC)-specific WWOX knockout (KO) versus WWOX flox control mice were examined under ARDS-producing conditions. EC WWOX KO mice exhibited significantly greater levels of vascular leak and histologic lung injury. ECs were isolated from digested lungs of untreated EC WWOX KO mice using sorting by flow cytometry for CD31+ CD45-cells. These were grown in culture, confirmed to be WWOX deficient by RT-PCR and Western blotting, and analyzed by electric cell impedance sensing as well as an FITC dextran transwell assay for their barrier properties during methicillin-resistant Staphylococcus aureus or LPS exposure. WWOX KO ECs demonstrated significantly greater declines in barrier function relative to cells from WWOX flox controls during either methicillin-resistant S. aureus or LPS treatment as measured by both electric cell impedance sensing and the transwell assay. The increased risk for ARDS observed in chronic smokers may be mechanistically linked, at least in part, to lung WWOX downregulation, and this phenomenon may also manifest in the near future in chronic users of e-cigarettes.
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    Cortactin loss protects against hemin-induced acute lung injury in sickle cell disease
    (American Physiological Society, 2022) Jones, Nicole M.; Sysol, Justin R.; Singla, Sunit; Smith, Patricia; Sandusky, George E.; Wang, Huashan; Natarajan, Viswanathan; Dudek, Steven M.; Machado, Roberto F.; Medicine, School of Medicine
    In patients with sickle cell disease (SCD), acute chest syndrome (ACS) is a common form of acute lung injury and a major cause of morbidity and mortality. The pathophysiology of ACS is complex, and hemin, the prosthetic moiety of hemoglobin, has been implicated in endothelial cell (EC) activation and subsequent acute lung injury (ALI) and ACS in vitro and in animal studies. Here, we examined the role of cortactin (CTTN), a cytoskeletal protein that regulates EC function, in response to hemin-induced ALI and ACS. Cortactin heterozygous (Cttn+/−) mice (n = 8) and their wild-type siblings (n = 8) were irradiated and subsequently received bone marrow cells (BMCs) extruded from the femurs of SCD mice (SS) to generate SS Cttn+/− and SS CttnWT chimeras. Following hemoglobin electrophoretic proof of BMC transplantation, the mice received 35 µmol/kg of hemin. Within 24 h, surviving mice were euthanized, and bronchoalveolar fluid (BAL) and lung samples were analyzed. For in vitro studies, human lung microvascular endothelial cells (HLMVECs) were used to determine hemin-induced changes in gene expression and reactive oxygen species (ROS) generation in cortactin deficiency and control conditions. When compared with wild-type littermates, the mortality for SS Cttn+/− mice trended to be lower after hemin infusion and these mice exhibited less severe lung injury and less necroptotic cell death. In vitro studies confirmed that cortactin deficiency is protective against hemin-induced injury in HMLVECs, by decreasing protein expression of p38/HSP27, improving cell barrier function, and decreasing the production of ROS. Further studies examining the role of CTTN in ACS are warranted and may open a new avenue of potential treatment for this devastating disease.
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    eNAMPT neutralization reduces preclinical ARDS severity via rectified NFkB and Akt/mTORC2 signaling
    (Springer, 2022-01-13) Bermudez, Tadeo; Sammani, Saad; Song, Jin H.; Hernon, Vivian Reyes; Kempf, Carrie L.; Garcia, Alexander N.; Burt, Jessica; Hufford, Matthew; Camp, Sara M.; Cress, Anne E.; Desai, Ankit A.; Natarajan, Viswanathan; Jacobson, Jeffrey R.; Dudek, Steven M.; Cancio, Leopoldo C.; Alvarez, Julie; Rafikov, Ruslan; Li, Yansong; Zhang, Donna D.; Casanova, Nancy G.; Bime, Christian; Garcia, Joe G. N.; Medicine, School of Medicine
    Despite encouraging preclinical data, therapies to reduce ARDS mortality remains a globally unmet need, including during the COVID-19 pandemic. We previously identified extracellular nicotinamide phosphoribosyltransferase (eNAMPT) as a novel damage-associated molecular pattern protein (DAMP) via TLR4 ligation which regulates inflammatory cascade activation. eNAMPT is tightly linked to human ARDS by biomarker and genotyping studies in ARDS subjects. We now hypothesize that an eNAMPT-neutralizing mAb will significantly reduce the severity of ARDS lung inflammatory lung injury in diverse preclinical rat and porcine models. Sprague Dawley rats received eNAMPT mAb intravenously following exposure to intratracheal lipopolysaccharide (LPS) or to a traumatic blast (125 kPa) but prior to initiation of ventilator-induced lung injury (VILI) (4 h). Yucatan minipigs received intravenous eNAMPT mAb 2 h after initiation of septic shock and VILI (12 h). Each rat/porcine ARDS/VILI model was strongly associated with evidence of severe inflammatory lung injury with NFkB pathway activation and marked dysregulation of the Akt/mTORC2 signaling pathway. eNAMPT neutralization dramatically reduced inflammatory indices and the severity of lung injury in each rat/porcine ARDS/VILI model (~ 50% reduction) including reduction in serum lactate, and plasma levels of eNAMPT, IL-6, TNFα and Ang-2. The eNAMPT mAb further rectified NFkB pathway activation and preserved the Akt/mTORC2 signaling pathway. These results strongly support targeting the eNAMPT/TLR4 inflammatory pathway as a potential ARDS strategy to reduce inflammatory lung injury and ARDS mortality.
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    eNAMPT neutralization reduces preclinical ARDS severity via rectified NFkB and Akt/mTORC2 signaling
    (Springer Nature, 2022-01-13) Bermudez, Tadeo; Sammani, Saad; Song, Jin H.; Reyes Hernon, Vivian; Kempf, Carrie L.; Garcia, Alexander N.; Burt, Jessica; Hufford, Matthew; Camp, Sara M.; Cress, Anne E.; Desai, Ankit A.; Natarajan, Viswanathan; Jacobson, Jeffrey R.; Dudek, Steven M.; Cancio, Leopoldo C.; Alvarez, Julie; Rafikov, Ruslan; Li, Yansong; Zhang, Donna D.; Casanova, Nancy G.; Bime, Christian; Garcia, Joe G. N.; Medicine, School of Medicine
    Despite encouraging preclinical data, therapies to reduce ARDS mortality remains a globally unmet need, including during the COVID-19 pandemic. We previously identified extracellular nicotinamide phosphoribosyltransferase (eNAMPT) as a novel damage-associated molecular pattern protein (DAMP) via TLR4 ligation which regulates inflammatory cascade activation. eNAMPT is tightly linked to human ARDS by biomarker and genotyping studies in ARDS subjects. We now hypothesize that an eNAMPT-neutralizing mAb will significantly reduce the severity of ARDS lung inflammatory lung injury in diverse preclinical rat and porcine models. Sprague Dawley rats received eNAMPT mAb intravenously following exposure to intratracheal lipopolysaccharide (LPS) or to a traumatic blast (125 kPa) but prior to initiation of ventilator-induced lung injury (VILI) (4 h). Yucatan minipigs received intravenous eNAMPT mAb 2 h after initiation of septic shock and VILI (12 h). Each rat/porcine ARDS/VILI model was strongly associated with evidence of severe inflammatory lung injury with NFkB pathway activation and marked dysregulation of the Akt/mTORC2 signaling pathway. eNAMPT neutralization dramatically reduced inflammatory indices and the severity of lung injury in each rat/porcine ARDS/VILI model (~ 50% reduction) including reduction in serum lactate, and plasma levels of eNAMPT, IL-6, TNFα and Ang-2. The eNAMPT mAb further rectified NFkB pathway activation and preserved the Akt/mTORC2 signaling pathway. These results strongly support targeting the eNAMPT/TLR4 inflammatory pathway as a potential ARDS strategy to reduce inflammatory lung injury and ARDS mortality.
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    Endothelial eNAMPT amplifies pre-clinical acute lung injury: efficacy of an eNAMPT-neutralising monoclonal antibody
    (European Respiratory Society, 2021-05-06) Quijada, Hector; Bermudez, Tadeo; Kempf, Carrie L.; Valera, Daniel G.; Garcia, Alexander N.; Camp, Sara M.; Song, Jin H.; Franco, Evelyn; Burt, Jessica K.; Sun, Belinda; Mascarenhas, Joseph B.; Burns, Kimberlie; Gaber, Amir; Oita, Radu C.; Reyes Hernon, Vivian; Barber, Christy; Moreno-Vinasco, Liliana; Sun, Xiaoguang; Cress, Anne E.; Martin, Diego; Liu, Zhonglin; Desai, Ankit A.; Natarajan, Viswanathan; Jacobson, Jeffrey R.; Dudek, Steven M.; Bime, Christian; Sammani, Saad; Garcia, Joe G.N.; Medicine, School of Medicine
    Rationale: The severe acute respiratory syndrome coronavirus 2/coronavirus disease 2019 pandemic has highlighted the serious unmet need for effective therapies that reduce acute respiratory distress syndrome (ARDS) mortality. We explored whether extracellular nicotinamide phosphoribosyltransferase (eNAMPT), a ligand for Toll-like receptor (TLR)4 and a master regulator of innate immunity and inflammation, is a potential ARDS therapeutic target. Methods: Wild-type C57BL/6J or endothelial cell (EC)-cNAMPT -/- knockout mice (targeted EC NAMPT deletion) were exposed to either a lipopolysaccharide (LPS)-induced ("one-hit") or a combined LPS/ventilator ("two-hit")-induced acute inflammatory lung injury model. A NAMPT-specific monoclonal antibody (mAb) imaging probe (99mTc-ProNamptor) was used to detect NAMPT expression in lung tissues. Either an eNAMPT-neutralising goat polyclonal antibody (pAb) or a humanised monoclonal antibody (ALT-100 mAb) were used in vitro and in vivo. Results: Immunohistochemical, biochemical and imaging studies validated time-dependent increases in NAMPT lung tissue expression in both pre-clinical ARDS models. Intravenous delivery of either eNAMPT-neutralising pAb or mAb significantly attenuated inflammatory lung injury (haematoxylin and eosin staining, bronchoalveolar lavage (BAL) protein, BAL polymorphonuclear cells, plasma interleukin-6) in both pre-clinical models. In vitro human lung EC studies demonstrated eNAMPT-neutralising antibodies (pAb, mAb) to strongly abrogate eNAMPT-induced TLR4 pathway activation and EC barrier disruption. In vivo studies in wild-type and EC-cNAMPT -/- mice confirmed a highly significant contribution of EC-derived NAMPT to the severity of inflammatory lung injury in both pre-clinical ARDS models. Conclusions: These findings highlight both the role of EC-derived eNAMPT and the potential for biologic targeting of the eNAMPT/TLR4 inflammatory pathway. In combination with predictive eNAMPT biomarker and NAMPT genotyping assays, this offers the opportunity to identify high-risk ARDS subjects for delivery of personalised medicine.
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    Lipid Mediators Regulate Pulmonary Fibrosis: Potential Mechanisms and Signaling Pathways
    (MDPI, 2020-06-15) Suryadevara, Vidyani; Ramchandran, Ramaswamy; Kamp, David W.; Natarajan, Viswanathan; Pathology and Laboratory Medicine, School of Medicine
    Idiopathic pulmonary fibrosis (IPF) is a progressive lung disease of unknown etiology characterized by distorted distal lung architecture, inflammation, and fibrosis. The molecular mechanisms involved in the pathophysiology of IPF are incompletely defined. Several lung cell types including alveolar epithelial cells, fibroblasts, monocyte-derived macrophages, and endothelial cells have been implicated in the development and progression of fibrosis. Regardless of the cell types involved, changes in gene expression, disrupted glycolysis, and mitochondrial oxidation, dysregulated protein folding, and altered phospholipid and sphingolipid metabolism result in activation of myofibroblast, deposition of extracellular matrix proteins, remodeling of lung architecture and fibrosis. Lipid mediators derived from phospholipids, sphingolipids, and polyunsaturated fatty acids play an important role in the pathogenesis of pulmonary fibrosis and have been described to exhibit pro- and anti-fibrotic effects in IPF and in preclinical animal models of lung fibrosis. This review describes the current understanding of the role and signaling pathways of prostanoids, lysophospholipids, and sphingolipids and their metabolizing enzymes in the development of lung fibrosis. Further, several of the lipid mediators and enzymes involved in their metabolism are therapeutic targets for drug development to treat IPF.
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    Micro-RNA-1 is decreased by hypoxia and contributes to the development of pulmonary vascular remodeling via regulation of sphingosine kinase 1
    (American Physiological Society, 2018-03-01) Sysol, Justin R.; Chen, Jiwang; Singla, Sunit; Zhao, Shuangping; Comhair, Suzy; Natarajan, Viswanathan; Machado, Roberto F.; Medicine, School of Medicine
    Sphingosine kinase 1 (SphK1) upregulation is associated with pathologic pulmonary vascular remodeling in pulmonary arterial hypertension (PAH), but the mechanisms controlling its expression are undefined. In this study, we sought to characterize the regulation of SphK1 expression by micro-RNAs (miRs). In silico analysis of the SphK1 3'-untranslated region identified several putative miR binding sites, with miR-1-3p (miR-1) being the most highly predicted target. Therefore we further investigated the role of miR-1 in modulating SphK1 expression and characterized its effects on the phenotype of pulmonary artery smooth muscle cells (PASMCs) and the development of experimental pulmonary hypertension in vivo. Our results demonstrate that miR-1 is downregulated by hypoxia in PASMCs and can directly inhibit SphK1 expression. Overexpression of miR-1 in human PASMCs inhibits basal and hypoxia-induced proliferation and migration. Human PASMCs isolated from PAH patients exhibit reduced miR-1 expression. We also demonstrate that miR-1 is downregulated in mouse lung tissues during experimental hypoxia-mediated pulmonary hypertension (HPH), consistent with upregulation of SphK1. Furthermore, administration of miR-1 mimics in vivo prevented the development of HPH in mice and attenuated induction of SphK1 in PASMCs. These data reveal the importance of miR-1 in regulating SphK1 expression during hypoxia in PASMCs. A pivotal role is played by miR-1 in pulmonary vascular remodeling, including PASMC proliferation and migration, and its overexpression protects from the development of HPH in vivo. These studies improve our understanding of the molecular mechanisms underlying the pathogenesis of pulmonary hypertension.
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    Sphingosine Kinase 1 Deficiency in Smooth Muscle Cells Protects against Hypoxia-Mediated Pulmonary Hypertension via YAP1 Signaling
    (MDPI, 2022-11-22) Chen, Jiwang; Lockett, Angelia; Zhao, Shuangping; Huang, Long Shuang; Wang, Yifan; Wu, Weiwen; Tang, Ming; Haider, Shahzaib; Velez Rendon, Daniela; Khan, Raheel; Liu, Bing; Felesena, Nicholas; Sysol, Justin R.; Valdez-Jasso, Daniela; Tang, Haiyan; Bai, Yang; Natarajan, Viswanathan; Machado, Roberto F.; Medicine, School of Medicine
    Sphingosine kinase 1 (SPHK1) and the sphingosine-1-phosphate (S1P) signaling pathway have been shown to play a role in pulmonary arterial hypertension (PAH). S1P is an important stimulus for pulmonary artery smooth muscle cell (PASMC) proliferation and pulmonary vascular remodeling. We aimed to examine the specific roles of SPHK1 in PASMCs during pulmonary hypertension (PH) progression. We generated smooth muscle cell-specific, Sphk1-deficient (Sphk1f/f TaglnCre+) mice and isolated Sphk1-deficient PASMCs from SPHK1 knockout mice. We demonstrated that Sphk1f/f TaglnCre+ mice are protected from hypoxia or hypoxia/Sugen-mediated PH, and pulmonary vascular remodeling and that Sphk1-deficient PASMCs are less proliferative compared with ones isolated from wild-type (WT) siblings. S1P or hypoxia activated yes-associated protein 1 (YAP1) signaling by enhancing its translocation to the nucleus, which was dependent on SPHK1 enzymatic activity. Further, verteporfin, a pharmacologic YAP1 inhibitor, attenuated the S1P-mediated proliferation of hPASMCs, hypoxia-mediated PH, and pulmonary vascular remodeling in mice and hypoxia/Sugen-mediated severe PH in rats. Smooth muscle cell-specific SPHK1 plays an essential role in PH via YAP1 signaling, and YAP1 inhibition may have therapeutic potential in treating PH.