Publications by authors named "Steven M Dudek"

88 Publications

Development of an Image-Based HCS-Compatible Method for Endothelial Barrier Function Assessment.

SLAS Discov 2021 Jul 16:24725552211030900. Epub 2021 Jul 16.

Department of Advanced Technology R&D, Nexcelom Bioscience LLC, Lawrence, MA, USA.

The recent renascence of phenotypic drug discovery (PDD) is catalyzed by its ability to identify first-in-class drugs and deliver results when the exact molecular mechanism is partially obscure. Acute respiratory distress syndrome (ARDS) is a severe, life-threatening condition with a high mortality rate that has increased in frequency due to the COVID-19 pandemic. Despite decades of laboratory and clinical study, no efficient pharmacological therapy for ARDS has been found. An increase in endothelial permeability is the primary event in ARDS onset, causing the development of pulmonary edema that leads to respiratory failure. Currently, the detailed molecular mechanisms regulating endothelial permeability are poorly understood. Therefore, the use of the PDD approach in the search for efficient ARDS treatment can be more productive than classic target-based drug discovery (TDD), but its use requires a new cell-based assay compatible with high-throughput (HTS) and high-content (HCS) screening. Here we report the development of a new plate-based image cytometry method to measure endothelial barrier function. The incorporation of image cytometry in combination with digital image analysis substantially decreases assay variability and increases the signal window. This new method simultaneously allows for rapid measurement of cell monolayer permeability and cytological analysis. The time-course of permeability increase in human pulmonary artery endothelial cells (HPAECs) in response to the thrombin and tumor necrosis factor α treatment correlates with previously published data obtained by transendothelial resistance (TER) measurements. Furthermore, the proposed image cytometry method can be easily adapted for HTS/HCS applications.
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http://dx.doi.org/10.1177/24725552211030900DOI Listing
July 2021

A Case of Candidemia after Long-term Presence of Urethral Foreign Bodies.

IDCases 2021 8;25:e01176. Epub 2021 Jun 8.

Department of Respirology, Graduate School of Medicine, Chiba University, Chiba, 260-8670, Japan.

A 52-year-old man presented to our hospital complaining of general malaise, cough, and fever. Total body computed tomography revealed scattered pneumonia and urethral foreign bodies that had been inserted during adolescence. was detected in blood and urine cultures. Based on these findings, the patient was diagnosed with candidemia that developed due to urinary tract infection, complicated by septic pulmonary embolism and severe diabetes mellitus. Candidemia likely persisted despite the initiation of intravenous antifungal therapy and control of blood sugar level. Therefore, surgical removal of the urethral foreign bodies was performed, which resulted in resolution of the patient's symptoms. Herein, we report a rare case of candidemia complicated by urinary tract infection that developed due to the long-term presence of urethral foreign bodies. A multidisciplinary therapeutic approach, including surgical removal of the infected foreign bodies, is effective in such cases. This case indicates that long-term presence of foreign bodies and acquired immune dysfunction can be risk factors for candidemia. Therefore, detailed history should be obtained and systemic examination should be performed to identify the complicating risk factors on diagnosis of candidemia.
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http://dx.doi.org/10.1016/j.idcr.2021.e01176DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8196048PMC
June 2021

Secretory Phospholipase A Enzymes in Acute Lung Injury.

Cell Biochem Biophys 2021 Jun 8. Epub 2021 Jun 8.

Division of Pulmonary, Critical Care, Sleep and Allergy, University of Illinois at Chicago, Chicago, IL, USA.

The secretory phospholipase A (sPLA) group of secreted enzymes hydrolyze phospholipids and lead to the production of multiple biologically active lipid mediators. sPLAs and their products (e.g., eicosanoids) play a significant role in the pathophysiology of various inflammatory diseases, including life-threatening lung disorders such as acute lung injury (ALI) and the Acute Respiratory Distress Syndrome (ARDS). The ALI/ARDS spectrum of severe inflammatory conditions is caused by direct (such as bacterial or viral pneumonia) or indirect insults (sepsis) that are associated with high morbidity and mortality. Several sPLA isoforms are upregulated in patients with ARDS as well as in multiple ALI preclinical models, and individual sPLAs exert unique roles in regulating ALI pathophysiology. This brief review will summarize the contributions of specific sPLA isoforms as markers and mediators in ALI, supporting a potential therapeutic role for targeting them in ARDS.
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http://dx.doi.org/10.1007/s12013-021-01003-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8186826PMC
June 2021

Phenotyping Angiotensin-Converting Enzyme in Blood: A Necessary Approach for Precision Medicine.

J Appl Lab Med 2021 Jun 7. Epub 2021 Jun 7.

Medical Center, Moscow University, Moscow, Russia.

Background: Angiotensin-converting enzyme (ACE) metabolizes a number of important peptides participating in blood pressure regulation and vascular remodeling. Elevated ACE expression in tissues (which is generally reflected by ACE in blood) is associated with increased risk of cardiovascular diseases. Elevated ACE in blood is also a marker for granulomatous diseases.

Methods: We applied our novel approach-ACE phenotyping-to characterize serum ACE in 300 unrelated patients and to establish normal values for ACE levels. ACE phenotyping includes (a) determination of ACE activity with 2 substrates (Z-Phe-His-Leu [ZPHL] and Hip-His-Leu [HHL]), (b) calculation of a ratio for hydrolysis of ZPHL and HHL, and (c) quantification of ACE immunoreactive protein levels and ACE conformation with a set of monoclonal antibodies (mAbs) to ACE.

Results: Only a combination of ACE activity determination with 2 substrates and quantification of the amount of ACE immunoreactive protein with mAbs 1G12 and 9B9 allows for the unequivocal detection of the presence of ACE inhibitors in the blood. After excluding such subjects, we were able to establish normal values of ACE in healthy populations: 50%-150% from control pooled serum. This ACE phenotyping approach in screening format with special attention to outliers can also identify patients with various mutations in ACE and may help to identify the as yet unknown ACE secretase or other mechanistic details of precise regulation of ACE expression.

Conclusions: ACE phenotyping is a promising new approach with potential clinical significance to advance precision medicine screening techniques by establishing different risk groups based on ACE phenotype.
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http://dx.doi.org/10.1093/jalm/jfab032DOI Listing
June 2021

Epitope mapping of novel monoclonal antibodies to human angiotensin I-converting enzyme.

Protein Sci 2021 Aug 11;30(8):1577-1593. Epub 2021 May 11.

Department of Medicine, University of Arizona, Tucson, Arizona, USA.

Angiotensin I-converting enzyme (ACE, CD143) plays a crucial role in blood pressure regulation, vascular remodeling, and immunity. A wide spectrum of mAbs to different epitopes on the N and C domains of human ACE have been generated and used to study different aspects of ACE biology, including establishing a novel approach-conformational fingerprinting. Here we characterized a novel set of 14 mAbs, developed against human seminal fluid ACE. The epitopes for these novel mAbs were defined using recombinant ACE constructs with truncated N and C domains, species cross-reactivity, ACE mutagenesis, and competition with the previously mapped anti-ACE mAbs. Nine mAbs recognized regions on the N domain, and 5 mAbs-on the C domain of ACE. The epitopes for most of these novel mAbs partially overlap with epitopes mapped onto ACE by the previously generated mAbs, whereas mAb 8H1 recognized yet unmapped region on the C domain where three ACE mutations associated with Alzheimer's disease are localized and is a marker for ACE mutation T877M. mAb 2H4 could be considered as a specific marker for ACE in dendritic cells. This novel set of mAbs can identify even subtle changes in human ACE conformation caused by tissue-specific glycosylation of ACE or mutations, and can detect human somatic and testicular ACE in biological fluids and tissues. Furthermore, the high reactivity of these novel mAbs provides an opportunity to study changes in the pattern of ACE expression or glycosylation in different tissues, cells, and diseases, such as sarcoidosis and Alzheimer's disease.
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http://dx.doi.org/10.1002/pro.4091DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8284578PMC
August 2021

Genetic and epigenetic regulation of the non-muscle myosin light chain kinase isoform by lung inflammatory factors and mechanical stress.

Clin Sci (Lond) 2021 Apr;135(7):963-977

Department of Medicine, University of Arizona, Tucson, AZ, U.S.A.

Rationale: The myosin light chain kinase gene, MYLK, encodes three proteins via unique promoters, including the non-muscle isoform of myosin light chain kinase (nmMLCK), a cytoskeletal protein centrally involved in regulation of vascular integrity. As MYLK coding SNPs are associated with severe inflammatory disorders (asthma, acute respiratory distress syndrome (ARDS)), we explored clinically relevant inflammatory stimuli and promoter SNPs in nmMLCK promoter regulation.

Methods: Full-length or serially deleted MYLK luciferase reporter promoter activities were measured in human lung endothelial cells (ECs). SNP-containing non-muscle MYLK (nmMYLK) DNA fragments were generated and nmMYLK promoter binding by transcription factors (TFs) detected by protein-DNA electrophoretic mobility shift assay (EMSA). Promoter demethylation was evaluated by 5-aza-2'-deoxycytidine (5-Aza). A preclinical mouse model of lipopolysaccharide (LPS)-induced acute lung injury (ALI) was utilized for nmMLCK validation.

Results: Lung EC levels of nmMLCK were significantly increased in LPS-challenged mice and LPS, tumor necrosis factor-α (TNF-α), 18% cyclic stretch (CS) and 5-Aza each significantly up-regulated EC nmMYLK promoter activities. EC exposure to FG-4592, a prolyl hydroxylase inhibitor that increases hypoxia-inducible factor (HIF) expression, increased nmMYLK promoter activity, confirmed by HIF1α/HIF2α silencing. nmMYLK promoter deletion studies identified distal inhibitory and proximal enhancing promoter regions as well as mechanical stretch-, LPS- and TNFα-inducible regions. Insertion of ARDS-associated SNPs (rs2700408, rs11714297) significantly increased nmMYLK promoter activity via increased transcription binding (glial cells missing homolog 1 (GCM1) and intestine-specific homeobox (ISX), respectively). Finally, the MYLK rs78755744 SNP (-261G/A), residing within a nmMYLK CpG island, significantly attenuated 5-Aza-induced promoter activity.

Conclusion: These findings indicate nmMYLK transcriptional regulation by clinically relevant inflammatory factors and ARDS-associated nmMYLK promoter variants are consistent with nmMLCK as a therapeutic target in severe inflammatory disorders.
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http://dx.doi.org/10.1042/CS20201448DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8047480PMC
April 2021

Endothelial eNAMPT amplifies pre-clinical acute lung injury: efficacy of an eNAMPT-neutralising monoclonal antibody.

Eur Respir J 2021 05 6;57(5). Epub 2021 May 6.

Dept of Medicine, University of Arizona Health Sciences, Tucson, AZ, USA.

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)-c knockout mice (targeted EC 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 (Tc-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 and .

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. human lung EC studies demonstrated eNAMPT-neutralising antibodies (pAb, mAb) to strongly abrogate eNAMPT-induced TLR4 pathway activation and EC barrier disruption. studies in wild-type and EC-c 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 genotyping assays, this offers the opportunity to identify high-risk ARDS subjects for delivery of personalised medicine.
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http://dx.doi.org/10.1183/13993003.02536-2020DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8100338PMC
May 2021

Cigarette Smoke and Nicotine-Containing Electronic-Cigarette Vapor Downregulate Lung WWOX Expression, Which Is Associated with Increased Severity of Murine Acute Respiratory Distress Syndrome.

Am J Respir Cell Mol Biol 2021 01;64(1):89-99

Division of Pulmonary, Critical Care, Sleep and Allergy Medicine, University of Illinois, Chicago, Illinois.

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 CD45cells. 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 or LPS exposure. WWOX KO ECs demonstrated significantly greater declines in barrier function relative to cells from WWOX flox controls during either methicillin-resistant 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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http://dx.doi.org/10.1165/rcmb.2020-0145OCDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7780991PMC
January 2021

Novel ACE mutations mimicking sarcoidosis by increasing blood ACE levels.

Transl Res 2021 04 26;230:5-20. Epub 2020 Jul 26.

Division of Pulmonary and Critical Care Medicine, Albany Medical College, Albany, New York.

An elevated blood angiotensin I-converting enzyme (ACE) supports diagnosis of sarcoidosis and Gaucher disease. However, some ACE mutations increase ACE shedding, and patients with these mutations are therefore at risk of being incorrectly diagnosed with sarcoidosis because of elevated serum ACE levels. We applied a novel approach called "ACE phenotyping" to identify possible ACE mutations in 3 pulmonary clinic patients that had suspected sarcoidosis based on elevated blood ACE levels. Conformational fingerprinting of ACE indicated that these mutations may be localized in the stalk region of the protein and these were confirmed by whole exome sequencing. Index patient 1 (IP1) had a mutation (P1199L) that had been previously identified, while the other 2 patients had novel ACE mutations. IP2 had 2 mutations, T887M and N1196K (eliminating a putative glycosylation site), while IP3 had a stop codon mutation Q1124X (eliminating the transmembrane anchor). We also performed a comprehensive analysis of the existing database of all ACE mutations to estimate the proportion of mutations increasing ACE shedding. The frequency of ACE mutations resulting in increased blood ACE levels may be much higher than previously estimated. ACE phenotyping, together with whole exome sequencing, is a diagnostic approach that could prevent unnecessary invasive and/or costly diagnostic procedures, or potentially harmful treatment for patients misdiagnosed on the basis of elevated blood ACE levels.
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http://dx.doi.org/10.1016/j.trsl.2020.07.010DOI Listing
April 2021

Angiocrine Sphingosine-1-Phosphate Activation of S1PR2-YAP Signaling Axis in Alveolar Type II Cells Is Essential for Lung Repair.

Cell Rep 2020 06;31(13):107828

Department of Pharmacology and Regenerative Medicine, University of Illinois College of Medicine, Chicago, IL 60612, USA. Electronic address:

Lung alveolar epithelium is composed of alveolar type I (AT1) and type II (AT2) cells. AT1 cells mediate gas exchange, whereas AT2 cells act as progenitor cells to repair injured alveoli. Lung microvascular endothelial cells (LMVECs) play a crucial but still poorly understood role in regulating alveolar repair. Here, we studied the role of the LMVEC-derived bioactive lipid sphingosine-1-phosphate (S1P) in promoting alveolar repair using mice with endothelial-specific deletion of sphingosine kinase 1 (Sphk1), the key enzyme promoting S1P generation. These mutant lungs developed airspace-enlargement lesions and exhibited a reduced number of AT1 cells after Pseudomonas-aeruginosa-induced lung injury. We demonstrated that S1P released by LMVECs acted via its receptor, S1PR2, on AT2 cells and induced nuclear translocation of yes-associated protein (YAP), a regulator of AT2 to AT1 transition. Thus, angiocrine S1P released after injury acts via the S1PR2-YAP signaling axis on AT2 cells to promote AT2 to AT1 differentiation required for alveolar repair.
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http://dx.doi.org/10.1016/j.celrep.2020.107828DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7371431PMC
June 2020

Clinical Features of Patients Infected with the 2019 Novel Coronavirus (COVID-19) in Shanghai, China.

medRxiv 2020 Mar 6. Epub 2020 Mar 6.

Background: Since mid-December 2019, a cluster of pneumonia-like diseases caused by a novel coronavirus, now designated COVID-19 by the WHO, emerged in Wuhan city and rapidly spread throughout China. Here we identify the clinical characteristics of COVID-19 in a cohort of patients in Shanghai.

Methods: Cases were confirmed by real-time RT-PCR and were analysed for demographic, clinical, laboratory and radiological features.

Results: Of 198 patients, the median duration from disease onset to hospital admission was 4 days. The mean age of the patients was 50.1 years, and 51.0% patients were male. The most common symptom was fever. Less than half of the patients presented with respiratory systems including cough, sputum production, itchy or sore throat, shortness of breath, and chest congestion. 5.6% patients had diarrhoea. On admission, T lymphocytes were decreased in 45.8% patients. Ground glass opacity was the most common radiological finding on chest computed tomography. 9.6% were admitted to the ICU because of the development of organ dysfunction. Compared with patients not treated in ICU, patients treated in the ICU were older, had longer waiting time to admission, fever over 38.5o C, dyspnoea, reduced T lymphocytes, elevated neutrophils and organ failure.

Conclusions: In this single centre cohort of COVID-19 patients, the most common symptom was fever, and the most common laboratory abnormality was decreased blood T cell counts. Older age, male, fever over 38.5oC, symptoms of dyspnoea, and underlying comorbidity, were the risk factors most associated with severity of disease. Key words: 2019 novel coronavirus; acute respiratory infection; risk factors for disease severity.
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http://dx.doi.org/10.1101/2020.03.04.20030395DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7255784PMC
March 2020

Arg mediates LPS-induced disruption of the pulmonary endothelial barrier.

Vascul Pharmacol 2020 May - Jun;128-129:106677. Epub 2020 Mar 30.

Division of Pulmonary, Critical Care, Sleep and Allergy, University of Illinois at Chicago, Chicago, IL, United States of America. Electronic address:

Acute Respiratory Distress Syndrome (ARDS) is a devastating disease process that involves dysregulated inflammation and decreased alveolar-capillary barrier function. Despite increased understanding of the pathophysiology, no effective targeted therapies exist to treat ARDS. Recent preclinical studies suggest that the multi-tyrosine kinase inhibitor, imatinib, which targets the Abl kinases c-Abl and Arg, has the potential to restore endothelial dysfunction caused by inflammatory agonists. Prior work demonstrates that imatinib attenuates LPS (lipopolysaccharide)-induced vascular leak and inflammation; however, the mechanisms underlying these effects remain incompletely understood. In the current study, we demonstrate that imatinib inhibits LPS-induced increase in the phosphorylation of CrkL, a specific substrate of Abl kinases, in human pulmonary endothelial cells. Specific silencing of Arg, and not c-Abl, attenuated LPS-induced pulmonary vascular permeability as measured by electrical cellular impedance sensing (ECIS) and gap formation assays. In addition, direct activation of Abl family kinases with the small molecule activator DPH resulted in endothelial barrier disruption that was attenuated by Arg siRNA. In complementary studies to characterize the mechanisms by which Arg mediates endothelial barrier function, Arg silencing was found to inhibit LPS-induced disruption of adherens junctions and phosphorylation of myosin light chains (MLC). Overall, these results characterize the mechanisms by which imatinib protects against LPS-induced endothelial barrier disruption and suggest that Arg inhibition may represent a novel strategy to enhance endothelial barrier function.
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http://dx.doi.org/10.1016/j.vph.2020.106677DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7234901PMC
October 2020

Sphingosine-1-phosphate receptor-independent lung endothelial cell barrier disruption induced by FTY720 regioisomers.

Pulm Circ 2020 Jan-Mar;10(1). Epub 2020 Feb 10.

Department of Medicine, The University of Arizona, Tucson, AZ, USA.

Rationale: Vascular permeability is a hallmark of acute respiratory distress syndrome (ARDS) and ventilator-induced lung injury pathobiology; however, the mechanisms underlying this vascular dysregulation remain unclear, thereby impairing the development of desperately needed effective therapeutics. We have shown that sphingosine-1-phosphate (S1P) and 2-amino-2-(2-[4-octylphenyl]ethyl)-1,3-propanediol (FTY720) analogues are useful tools for exploring vascular barrier regulation mechanisms.

Objective: To experimentally define the effects of FTY720 regioisomers on lung endothelial cell barrier regulation.

Methods: Specific barrier-regulatory receptor and kinase inhibitors were utilized to probe signaling mechanisms involved in FTY720 regioisomer-mediated human lung endothelial cell barrier responses (trans-endothelial electrical resistance, TER). Docking simulations with the S1P1 receptor were performed to further evaluate FTY720 regioisomer signaling.

Results: FTY720 regioisomers produced potent endothelial cell barrier disruption reflected by declines in TER alterations. Pharmacologic inhibition of Gi-coupled S1P receptors (S1P1, S1P2, S1P3) failed to alter FTY720 regioisomer-mediated barrier disruption; findings that were corroborated by docking simulations demonstrating FTY720 regiosomers were repelled from S1P1 docking, in contrast to strong S1P1 binding elicited by S1P. Inhibition of either the barrier-disrupting PAR-1 receptor, the VEGF receptor, Rho-kinase, MAPK, NFkB, or PI3K failed to alter FTY720 regioisomer-induced endothelial cell barrier disruption. While FTY720 regioisomers significantly increased protein phosphatase 2 (PP2A) activity, PP2A inhibitors failed to alter FTY720 regioisomer-induced endothelial cell barrier disruption.

Conclusions: Together, these results imply a vexing model of pulmonary vascular barrier dysregulation in response to FTY720-related compounds and highlight the need for further insights into mechanisms of vascular integrity required to promote the development of novel therapeutic tools to prevent or reverse the pulmonary vascular leak central to ARDS outcomes.
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http://dx.doi.org/10.1177/2045894020905521DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7011338PMC
February 2020

Degradation of group V secretory phospholipase A in lung endothelium is mediated by autophagy.

Microvasc Res 2020 05 13;129:103954. Epub 2019 Nov 13.

Division of Pulmonary, Critical Care, Sleep and Allergy, University of Illinois Department of Medicine, Chicago, IL, United States of America. Electronic address:

Group V secretory phospholipase A (gVPLA) is a potent inflammatory mediator in mammalian tissues that hydrolyzes phospholipids and initiates eicosanoid biosynthesis. Previous work has demonstrated that multiple inflammatory stimuli induce its expression and secretion in several cell types, including the lung endothelium. However, little is known about the mechanism(s) by which gVPLA inflammatory signaling is subsequently downregulated. Therefore, in this study we characterized potential clearance mechanisms for gVPLA in lung endothelial cells (EC). We observed that exogenous gVPLA is taken up rapidly by nutrient-starved human pulmonary artery EC (HPAEC) in vitro, and its cellular expression subsequently is reduced over several hours. In parallel experiments performed in pulmonary vascular EC isolated from mice genetically deficient in gVPLA, the degradation of exogenously applied gVPLA occurs in a qualitatively similar fashion. This degradation is significantly attenuated in EC treated with ammonium chloride or chloroquine, which are lysosomal inhibitors that block autophagic flux. In contrast, the proteasomal inhibitor MG132 fails to prevent the clearance of gVPLA. Both immunofluorescence microscopy and proximity ligation assay demonstrate the co-localization of LC3 and gVPLA during this process, indicating the association of gVPLA with autophagosomes. Nutrient starvation, a known inducer of autophagy, is sufficient to stimulate gVPLA degradation. These results suggest that a lysosome-mediated autophagy pathway contributes to gVPLA clearance from lung EC. These novel observations advance our understanding of the mechanism by which this key inflammatory enzyme is downregulated in the lung vasculature.
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http://dx.doi.org/10.1016/j.mvr.2019.103954DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7224701PMC
May 2020

A nonapoptotic endothelial barrier-protective role for caspase-3.

Am J Physiol Lung Cell Mol Physiol 2019 06 25;316(6):L1118-L1126. Epub 2019 Mar 25.

Department of Medicine, Johns Hopkins University School of Medicine , Baltimore, Maryland.

Noncanonical roles for caspase-3 are emerging in the fields of cancer and developmental biology. However, little is known of nonapoptotic functions of caspase-3 in most cell types. We have recently demonstrated a disassociation between caspase-3 activation and execution of apoptosis with accompanying cytoplasmic caspase-3 sequestration and preserved endothelial barrier function. Therefore, we tested the hypothesis that nonapoptotic caspase-3 activation promotes endothelial barrier integrity. Human lung microvascular endothelial cells were exposed to thrombin, a nonapoptotic stimulus, and endothelial barrier function was assessed using electric cell-substrate impedance sensing. Actin cytoskeletal rearrangement and paracellular gap formation were assessed using phalloidin staining. Cell stiffness was evaluated using magnetic twisting cytometry. In addition, cell lysates were harvested for protein analyses. Caspase-3 was inhibited pharmacologically with pan-caspase and a caspase-3-specific inhibitor. Molecular inhibition of caspase-3 was achieved using RNA interference. Cells exposed to thrombin exhibited a cytoplasmic activation of caspase-3 with transient and nonapoptotic decrease in endothelial barrier function as measured by a drop in electrical resistance followed by a rapid recovery. Inhibition of caspases led to a more pronounced and rapid drop in thrombin-induced endothelial barrier function, accompanied by increased endothelial cell stiffness and paracellular gaps. Caspase-3-specific inhibition and caspase-3 knockdown both resulted in more pronounced thrombin-induced endothelial barrier disruption. Taken together, our results suggest cytoplasmic caspase-3 has nonapoptotic functions in human endothelium and can promote endothelial barrier integrity.
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http://dx.doi.org/10.1152/ajplung.00487.2018DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6620669PMC
June 2019

DPP4 inhibition by sitagliptin attenuates LPS-induced lung injury in mice.

Am J Physiol Lung Cell Mol Physiol 2018 11 6;315(5):L834-L845. Epub 2018 Sep 6.

Division of Pulmonary, Critical Care, Sleep, and Allergy, Department of Medicine, University of Illinois at Chicago , Chicago, Illinois.

Acute respiratory distress syndrome (ARDS) is a severe clinical condition marked by acute respiratory failure and dysregulated inflammation. Pulmonary vascular endothelial cells (PVECs) function as an important pro-inflammatory source in ARDS, suggesting that modulation of inflammatory events at the endothelial level may have a therapeutic benefit. Dipeptidyl peptidase-4 (DPP4) inhibitors, widely used for the treatment of diabetes mellitus, have been reported to have possible anti-inflammatory effects. However, the potential anti-inflammatory effects of DPP4 inhibition on PVEC function and ARDS pathophysiology are unknown. Therefore, we evaluated the effects of sitagliptin, a DPP4 inhibitor in wide clinical use, on LPS-induced lung injury in mice and in human lung ECs in vitro. In vivo, sitagliptin reduced serum DPP4 activity, bronchoalveolar lavage protein concentration, cell number, and proinflammatory cytokine levels after LPS and alleviated histological findings of lung injury. LPS decreased the expression levels of CD26/DPP4 on pulmonary epithelial cells and PVECs isolated from mouse lungs, and the effect was partially reversed by sitagliptin. In vitro, human lung microvascular ECs (HLMVECs) expressed higher levels of CD26/DPP4 than human pulmonary arterial ECs. LPS induced the release of TNFα, IL-6, and IL-8 by HLMVECs that were inhibited by sitagliptin. LPS promoted the proliferation of HLMVECs, and sitagliptin suppressed this response. However, sitagliptin failed to reverse LPS-induced permeability in cultured ECs or lung epithelial cells in vitro. In summary, sitagliptin attenuates LPS-induced lung injury in mice and exerts anti-inflammatory effects on HLMVECs. These novel observations indicate DPP4 inhibitors may have potential as therapeutic drugs for ARDS.
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http://dx.doi.org/10.1152/ajplung.00031.2018DOI Listing
November 2018

Myosin light chain kinase ( MYLK) coding polymorphisms modulate human lung endothelial cell barrier responses via altered tyrosine phosphorylation, spatial localization, and lamellipodial protrusions.

Pulm Circ 2018 Apr-Jun;8(2):2045894018764171. Epub 2018 Feb 26.

1 Department of Medicine, University of Arizona Health Sciences, Tucson, AZ, USA.

Sphingosine 1-phosphate (S1P) is a potent bioactive endogenous lipid that signals a rearrangement of the actin cytoskeleton via the regulation of non-muscle myosin light chain kinase isoform (nmMLCK). S1P induces critical nmMLCK Y and Y phosphorylation resulting in translocation of nmMLCK to the periphery where spatially-directed increases in myosin light chain (MLC) phosphorylation and tension result in lamellipodia protrusion, increased cell-cell adhesion, and enhanced vascular barrier integrity. MYLK, the gene encoding nmMLCK, is a known candidate gene in lung inflammatory diseases, with coding genetic variants (Pro21His, Ser147Pro, Val261Ala) that confer risk for inflammatory lung injury and influence disease severity. The functional mechanisms by which these MYLK coding single nucleotide polymorphisms (SNPs) affect biologic processes to increase disease risk and severity remain elusive. In the current study, we utilized quantifiable cell immunofluorescence assays to determine the influence of MYLK coding SNPs on S1P-mediated nmMLCK phosphorylation and translocation to the human lung endothelial cell (EC) periphery . These disease-associated MYLK variants result in reduced levels of S1P-induced Y phosphorylation, a key site for nmMLCK enzymatic regulation and activation. Reduced Y phosphorylation resulted in attenuated nmMLCK protein translocation to the cell periphery. We further conducted EC kymographic assays which confirmed that lamellipodial protrusion in response to S1P challenge was retarded by expression of a MYLK transgene harboring the three MYLK coding SNPs. These data suggest that ARDS/severe asthma-associated MYLK SNPs functionally influence vascular barrier-regulatory cytoskeletal responses via direct alterations in the levels of nmMLCK tyrosine phosphorylation, spatial localization, and lamellipodial protrusions.
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http://dx.doi.org/10.1177/2045894018764171DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5846938PMC
February 2018

Development of ultrasound bioprobe for biological imaging.

Sci Adv 2017 10 25;3(10):e1701176. Epub 2017 Oct 25.

Department of Materials Science and Engineering and NUANCE Center, Northwestern University, Evanston, IL 60208, USA.

We report the development of an ultrasound bioprobe for in vitro molecular imaging. In this method, the phase of the scattered ultrasound wave is mapped to provide in vitro and intracellular imaging with nanometer-scale resolution under physiological conditions. We demonstrated the technique by successfully imaging a magnetic core in silica core shells and the stiffness image of intracellular fibers in endothelial cells that were stimulated with thrombin. The findings demonstrate a significant advancement in high-resolution ultrasound imaging of biological systems with acoustics under physiological conditions. These will open up various applications in biomedical and molecular imaging with subsurface resolution down to the nanometer scale.
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http://dx.doi.org/10.1126/sciadv.1701176DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5656426PMC
October 2017

The ARP 2/3 complex mediates endothelial barrier function and recovery.

Pulm Circ 2017 Mar 1;7(1):200-210. Epub 2017 Feb 1.

Division of Pulmonary, Critical Care, Sleep, and Allergy, University of Illinois Hospital and Health Science System, Chicago, IL, USA.

Pulmonary endothelial cell (EC) barrier dysfunction and recovery is critical to the pathophysiology of acute respiratory distress syndrome. Cytoskeletal and subsequent cell membrane dynamics play a key mechanistic role in determination of EC barrier integrity. Here, we characterizAQe the actin related protein 2/3 (Arp 2/3) complex, a regulator of peripheral branched actin polymerization, in human pulmonary EC barrier function through studies of transendothelial electrical resistance (TER), intercellular gap formation, peripheral cytoskeletal structures and lamellipodia. Compared to control, Arp 2/3 inhibition with the small molecule inhibitor CK-666 results in a reduction of baseline barrier function (1,241 ± 53 vs 988 ± 64 ohm;  < 0.01), S1P-induced barrier enhancement and delayed recovery of barrier function after thrombin (143 ± 14 vs 93 ± 6 min;  < 0.01). Functional changes of Arp 2/3 inhibition on barrier integrity are associated temporally with increased intercellular gap area at baseline (0.456 ± 0.02 vs 0.299 ± 0.02;  < 0.05) and thirty minutes after thrombin (0.885 ± 0.03 vs 0.754 ± 0.03;  < 0.05). Immunofluorescent microscopy reveals reduced lamellipodia formation after S1P and during thrombin recovery in Arp 2/3 inhibited cells. Individual lamellipodia demonstrate reduced depth following Arp 2/3 inhibition vs vehicle at baseline (1.83 ± 0.41 vs 2.55 ± 0.46 µm;  < 0.05) and thirty minutes after S1P treatment (1.53 ± 0.37 vs 2.09 ± 0.36 µm;  < 0.05). These results establish a critical role for Arp 2/3 activity in determination of pulmonary endothelial barrier function and recovery through formation of EC lamellipodia and closure of intercellular gaps.
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http://dx.doi.org/10.1086/690307DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5448540PMC
March 2017

Endothelial Glycocalyx Repair: Building a Wall to Protect the Lung during Sepsis.

Am J Respir Cell Mol Biol 2017 06;56(6):687-688

1 Division of Pulmonary, Critical Care, Sleep and Allergy University of Illinois College of Medicine Chicago, Illinois.

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http://dx.doi.org/10.1165/rcmb.2017-0065EDDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5516297PMC
June 2017

Cortactin in Atherosclerosis: Just Say NO.

Arterioscler Thromb Vasc Biol 2016 12;36(12):2278-2280

From the Department of Medicine, Division of Pulmonary, Critical Care, Sleep and Allergy, University of Illinois College of Medicine, Chicago.

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http://dx.doi.org/10.1161/ATVBAHA.116.308497DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5289296PMC
December 2016

Parkin regulates lipopolysaccharide-induced proinflammatory responses in acute lung injury.

Transl Res 2017 03 13;181:71-82. Epub 2016 Sep 13.

Division of Pulmonary, Critical Care, Sleep, and Allergy, University of Illinois, Chicago, Ill.

The acute respiratory distress syndrome (ARDS) is a serious condition resulting from direct or indirect lung injury that is associated with high mortality and morbidity. A key biological event in the pathogenesis of the acute lung injury (ALI) that causes acute respiratory distress syndrome is activation of the lung endothelium cells (ECs), which is triggered by a variety of inflammatory insults leading to barrier disruption and excessive accumulation of neutrophils. Recently, we demonstrated that imatinib protects against lipopolysaccharide (LPS)-induced EC activation by inhibiting c-Abl kinase. In the present study, we explored the role of parkin, a novel c-Abl substrate, in ALI. Parkin is an E3 ubiquitin ligase originally characterized in the pathogenesis of Parkinson disease; however, its potential role in acute inflammatory processes and lung EC function remains largely unknown. Using parkin deficient (PARK2-/-) mice, we now demonstrate that parkin mediates LPS-induced ALI. After LPS, PARK2-/- mice have reduced total protein and cell levels in bronchoalveolar lavage (BAL) compared to wild type. Moreover, in LPS-treated PARK2-/- lungs, the sequestration and activation of neutrophils and release of inflammatory cytokines (interleukin 6 [IL-6], tumor necrosis factor alpha [TNF-α]) are significantly reduced. The BAL levels of soluble VCAM-1 and ICAM-1 are also decreased in LPS-treated PARK2-/- mice compared to wild type. In cultured human lung endothelial cells, downregulation of parkin by small interfering RNA decreases LPS-induced VCAM-1 expression, IL-8 and IL-6 secretion, and NF-kB phosphorylation. These results suggest a previously unidentified role of parkin in mediating endotoxin-induced endothelial proinflammatory signaling and indicate that it may play a critical role in acute inflammation.
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http://dx.doi.org/10.1016/j.trsl.2016.09.002DOI Listing
March 2017

Regulation of Thrombin-Induced Lung Endothelial Cell Barrier Disruption by Protein Kinase C Delta.

PLoS One 2016 21;11(7):e0158865. Epub 2016 Jul 21.

Institute for Personalized Respiratory Medicine, University of Illinois at Chicago, Chicago, Illinois, United States of America.

Protein Kinase C (PKC) plays a significant role in thrombin-induced loss of endothelial cell (EC) barrier integrity; however, the existence of more than 10 isozymes of PKC and tissue-specific isoform expression has limited our understanding of this important second messenger in vascular homeostasis. In this study, we show that PKCδ isoform promotes thrombin-induced loss of human pulmonary artery EC barrier integrity, findings substantiated by PKCδ inhibitory studies (rottlerin), dominant negative PKCδ construct and PKCδ silencing (siRNA). In addition, we identified PKCδ as a signaling mediator upstream of both thrombin-induced MLC phosphorylation and Rho GTPase activation affecting stress fiber formation, cell contraction and loss of EC barrier integrity. Our inhibitor-based studies indicate that thrombin-induced PKCδ activation exerts a positive feedback on Rho GTPase activation and contributes to Rac1 GTPase inhibition. Moreover, PKD (or PKCμ) and CPI-17, two known PKCδ targets, were found to be activated by PKCδ in EC and served as modulators of cytoskeleton rearrangement. These studies clarify the role of PKCδ in EC cytoskeleton regulation, and highlight PKCδ as a therapeutic target in inflammatory lung disorders, characterized by the loss of barrier integrity, such as acute lung injury and sepsis.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0158865PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4956111PMC
July 2017

Nonmuscle myosin light chain kinase activity modulates radiation-induced lung injury.

Pulm Circ 2016 Jun;6(2):234-9

Department of Medicine and Arizona Respiratory Center, University of Arizona, Tucson, Arizona, USA.

Radiotherapy as a primary treatment for thoracic malignancies induces deleterious effects, such as acute or subacute radiation-induced lung injury (RILI). Although the molecular etiology of RILI is controversial and likely multifactorial, a potentially important cellular target is the lung endothelial cytoskeleton that regulates paracellular gap formation and the influx of macromolecules and fluid to the alveolar space. Here we investigate the central role of a key endothelial cytoskeletal regulatory protein, the nonmuscle isoform of myosin light chain kinase (nmMLCK), in an established murine RILI model. Our results indicate that thoracic irradiation significantly augmented nmMLCK protein expression and enzymatic activity in murine lungs. Furthermore, genetically engineered mice harboring a deletion of the nmMLCK gene (nmMLCK(-/-) mice) exhibited protection from RILI, as assessed by attenuated vascular leakage and leukocyte infiltration. In addition, irradiated wild-type mice treated with two distinct MLCK enzymatic inhibitors, ML-7 and PIK (peptide inhibitor of kinase), also demonstrated attenuated RILI. Taken together, these data suggests a key role for nmMLCK in vascular barrier regulation in RILI and warrants further examination of RILI strategies that target nmMLCK.
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http://dx.doi.org/10.1086/686491DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4869928PMC
June 2016

"Pulmonary Endothelial Cell Barrier Enhancement by Novel FTY720 Analogs: Methoxy-FTY720, Fluoro-FTY720, and β-Glucuronide-FTY720".

Chem Phys Lipids 2016 Jan 29;194:85-93. Epub 2015 Oct 29.

Division of Pulmonary and Critical Care Medicine, Department of Medicine, University of Illinois at Chicago, Chicago, IL, United States. Electronic address:

Effective therapeutic agents are lacking for the prevention and reversal of vascular leak, a frequent pathophysiologic result of inflammatory processes such as acute respiratory distress syndrome (ARDS) and sepsis. We previously demonstrated the potent barrier-enhancing effects of related compounds sphingosine 1-phosphate (S1P), the pharmaceutical agent FTY720, and its analog (S)-FTY720 phosphonate (Tys) in models of inflammatory lung injury. In this study, we characterize additional novel FTY720 analogs for their potential to reduce vascular leak as well as utilize them as tools to better understand the mechanisms by which this class of agents modulates permeability. Transendothelial resistance (TER) and labeled dextran studies demonstrate that (R)-methoxy-FTY720 ((R)-OMe-FTY), (R)/(S)-fluoro-FTY720 (FTY-F), and β-glucuronide-FTY720 (FTY-G) compounds display in vitro barrier-enhancing properties comparable or superior to FTY720 and S1P. In contrast, the (S)-methoxy-FTY720 ((S)-OMe-FTY) analog disrupts lung endothelial cell (EC) barrier integrity in TER studies in association with actin stress fiber formation and robust intracellular calcium release, but independent of myosin light chain or ERK phosphorylation. Additional mechanistic studies with (R)-OMe-FTY, FTY-F, and FTY-G suggest that lung EC barrier enhancement is mediated through lipid raft signaling, Gi-linked receptor coupling to downstream tyrosine phosphorylation events, and S1PR1-dependent receptor ligation. These results provide important mechanistic insights into modulation of pulmonary vascular barrier function by FTY720-related compounds and highlight common signaling events that may assist the development of novel therapeutic tools in the prevention or reversal of the pulmonary vascular leak that characterizes ARDS.
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http://dx.doi.org/10.1016/j.chemphyslip.2015.10.004DOI Listing
January 2016

ATP promotes cell survival via regulation of cytosolic [Ca2+] and Bcl-2/Bax ratio in lung cancer cells.

Am J Physiol Cell Physiol 2016 Jan 21;310(2):C99-114. Epub 2015 Oct 21.

Department of Medicine, Division of Translational and Regenerative Medicine, College of Medicine, The University of Arizona, Tucson, Arizona; Department of Physiology, College of Medicine, The University of Arizona, Tucson, Arizona;

Adenosine triphosphate (ATP) is a ubiquitous extracellular messenger elevated in the tumor microenvironment. ATP regulates cell functions by acting on purinergic receptors (P2X and P2Y) and activating a series of intracellular signaling pathways. We examined ATP-induced Ca(2+) signaling and its effects on antiapoptotic (Bcl-2) and proapoptotic (Bax) proteins in normal human airway epithelial cells and lung cancer cells. Lung cancer cells exhibited two phases (transient and plateau phases) of increase in cytosolic [Ca(2+)] ([Ca(2+)]cyt) caused by ATP, while only the transient phase was observed in normal cells. Removal of extracellular Ca(2+) eliminated the plateau phase increase of [Ca(2+)]cyt in lung cancer cells, indicating that the plateau phase of [Ca(2+)]cyt increase is due to Ca(2+) influx. The distribution of P2X (P2X1-7) and P2Y (P2Y1, P2Y2, P2Y4, P2Y6, P2Y11) receptors was different between lung cancer cells and normal cells. Proapoptotic P2X7 was nearly undetectable in lung cancer cells, which may explain why lung cancer cells showed decreased cytotoxicity when treated with high concentration of ATP. The Bcl-2/Bax ratio was increased in lung cancer cells following treatment with ATP; however, the antiapoptotic protein Bcl-2 demonstrated more sensitivity to ATP than proapoptotic protein Bax. Decreasing extracellular Ca(2+) or chelating intracellular Ca(2+) with BAPTA-AM significantly inhibited ATP-induced increase in Bcl-2/Bax ratio, indicating that a rise in [Ca(2+)]cyt through Ca(2+) influx is the critical mediator for ATP-mediated increase in Bcl-2/Bax ratio. Therefore, despite high ATP levels in the tumor microenvironment, which would induce cell apoptosis in normal cells, the decreased P2X7 and elevated Bcl-2/Bax ratio in lung cancer cells may enable tumor cells to survive. Increasing the Bcl-2/Bax ratio by exposure to high extracellular ATP may, therefore, be an important selective pressure promoting transformation and cancer progression.
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http://dx.doi.org/10.1152/ajpcell.00092.2015DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4719033PMC
January 2016

Imatinib attenuates inflammation and vascular leak in a clinically relevant two-hit model of acute lung injury.

Am J Physiol Lung Cell Mol Physiol 2015 Dec 2;309(11):L1294-304. Epub 2015 Oct 2.

University of Illinois Hospital and Health Sciences System, Division of Pulmonary, Critical Care, Sleep and Allergy, Chicago, Illinois; University of Illinois at Chicago, Department of Pharmacology, Chicago, Illinois;

Acute lung injury/acute respiratory distress syndrome (ALI/ARDS), an illness characterized by life-threatening vascular leak, is a significant cause of morbidity and mortality in critically ill patients. Recent preclinical studies and clinical observations have suggested a potential role for the chemotherapeutic agent imatinib in restoring vascular integrity. Our prior work demonstrates differential effects of imatinib in mouse models of ALI, namely attenuation of LPS-induced lung injury but exacerbation of ventilator-induced lung injury (VILI). Because of the critical role of mechanical ventilation in the care of patients with ARDS, in the present study we pursued an assessment of the effectiveness of imatinib in a "two-hit" model of ALI caused by combined LPS and VILI. Imatinib significantly decreased bronchoalveolar lavage protein, total cells, neutrophils, and TNF-α levels in mice exposed to LPS plus VILI, indicating that it attenuates ALI in this clinically relevant model. In subsequent experiments focusing on its protective role in LPS-induced lung injury, imatinib attenuated ALI when given 4 h after LPS, suggesting potential therapeutic effectiveness when given after the onset of injury. Mechanistic studies in mouse lung tissue and human lung endothelial cells revealed that imatinib inhibits LPS-induced NF-κB expression and activation. Overall, these results further characterize the therapeutic potential of imatinib against inflammatory vascular leak.
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http://dx.doi.org/10.1152/ajplung.00031.2015DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4669340PMC
December 2015

A genetic variant of cortactin linked to acute lung injury impairs lamellipodia dynamics and endothelial wound healing.

Am J Physiol Lung Cell Mol Physiol 2015 Nov 11;309(9):L983-94. Epub 2015 Sep 11.

Department of Chemical and Biomolecular Engineering, University of Illinois, Urbana, Illinois; Department of Chemistry, University of Illinois, Urbana, Illinois

Inflammatory mediators released in acute lung injury (ALI) trigger the disruption of interendothelial junctions, leading to loss of vascular barrier function, protein-rich pulmonary edema, and severe hypoxemia. Genetic signatures that predict patient recovery or disease progression are poorly defined, but recent genetic screening of ALI patients has identified an association between lung inflammatory disease and a single nucleotide polymorphism (SNP) in the gene for the actin-binding and barrier-regulatory protein cortactin. This study investigated the impact of this disease-linked cortactin variant on wound healing processes that may contribute to endothelial barrier restoration. A microfabricated platform was used to quantify wound healing in terms of gap closure speed, lamellipodia dynamics, and cell velocity. Overexpression of wild-type cortactin in endothelial cells (ECs) improved directional cell motility and enhanced lamellipodial protrusion length, resulting in enhanced gap closure rates. By contrast, the cortactin SNP impaired wound closure and cell locomotion, consistent with the observed reduction in lamellipodial protrusion length and persistence. Overexpression of the cortactin SNP in lung ECs mitigated the barrier-enhancing activity of sphingosine 1-phosphate. These findings suggest that this common cortactin variant may functionally contribute to ALI predisposition by impeding endothelial wound healing.
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http://dx.doi.org/10.1152/ajplung.00062.2015DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4628987PMC
November 2015

Pulmonary endothelial cell barrier enhancement by novel FTY720 analogs: methoxy-FTY720, fluoro-FTY720, and β-glucuronide-FTY720.

Chem Phys Lipids 2015 Oct 10;191:16-24. Epub 2015 Aug 10.

Division of Pulmonary and Critical Care Medicine, Department of Medicine, University of Illinois at Chicago, Chicago, IL, United States. Electronic address:

Effective therapeutic agents are lacking for the prevention and reversal of vascular leak, a frequent pathophysiologic result of inflammatory processes such as acute respiratory distress syndrome (ARDS) and sepsis. We previously demonstrated the potent barrier-enhancing effects of related compounds sphingosine 1-phosphate (S1P), the pharmaceutical agent FTY720, and its analog (S)-FTY720 phosphonate (Tys) in models of inflammatory lung injury. In this study, we characterize additional novel FTY720 analogs for their potential to reduce vascular leak as well as utilize them as tools to better understand the mechanisms by which this class of agents modulates permeability. Transendothelial resistance (TER) and labeled dextran studies demonstrate that (R)-methoxy-FTY720 ((R)-OMe-FTY), (R)/(S)-fluoro-FTY720 (FTY-F), and β-glucuronide-FTY720 (FTY-G) compounds display in vitro barrier-enhancing properties comparable or superior to FTY720 and S1P. In contrast, the (S)-methoxy-FTY720 ((S)-OMe-FTY) analog disrupts lung endothelial cell (EC) barrier integrity in TER studies in association with actin stress fiber formation and robust intracellular calcium release, but independent of myosin light chain or ERK phosphorylation. Additional mechanistic studies with (R)-OMe-FTY, FTY-F, and FTY-G suggest that lung EC barrier enhancement is mediated through lipid raft signaling, Gi-linked receptor coupling to downstream tyrosine phosphorylation events, and S1PR1-dependent receptor ligation. These results provide important mechanistic insights into modulation of pulmonary vascular barrier function by FTY720-related compounds and highlight common signaling events that may assist the development of novel therapeutic tools in the prevention or reversal of the pulmonary vascular leak that characterizes ARDS.
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http://dx.doi.org/10.1016/j.chemphyslip.2015.08.004DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4655823PMC
October 2015

Structure-Function Analysis of the Non-Muscle Myosin Light Chain Kinase (nmMLCK) Isoform by NMR Spectroscopy and Molecular Modeling: Influence of MYLK Variants.

PLoS One 2015 25;10(6):e0130515. Epub 2015 Jun 25.

Institute for Personalized Respiratory Medicine, Section of Pulmonary, Critical Care, Sleep, and Allergy, University of Illinois at Chicago, Chicago, Illinois, United States of America; Arizona Respiratory Center and Department of Medicine, University of Arizona, Tucson, Arizona, United States of America.

The MYLK gene encodes the multifunctional enzyme, myosin light chain kinase (MLCK), involved in isoform-specific non-muscle and smooth muscle contraction and regulation of vascular permeability during inflammation. Three MYLK SNPs (P21H, S147P, V261A) alter the N-terminal amino acid sequence of the non-muscle isoform of MLCK (nmMLCK) and are highly associated with susceptibility to acute lung injury (ALI) and asthma, especially in individuals of African descent. To understand the functional effects of SNP associations, we examined the N-terminal segments of nmMLCK by 1H-15N heteronuclear single quantum correlation (HSQC) spectroscopy, a 2-D NMR technique, and by in silico molecular modeling. Both NMR analysis and molecular modeling indicated SNP localization to loops that connect the immunoglobulin-like domains of nmMLCK, consistent with minimal structural changes evoked by these SNPs. Molecular modeling analysis identified protein-protein interaction motifs adversely affected by these MYLK SNPs including binding by the scaffold protein 14-3-3, results confirmed by immunoprecipitation and western blot studies. These structure-function studies suggest novel mechanisms for nmMLCK regulation, which may confirm MYLK as a candidate gene in inflammatory lung disease and advance knowledge of the genetic underpinning of lung-related health disparities.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0130515PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4482139PMC
March 2016
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