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author:("Mathew, fiji")
1.  Novel Role of the Mu Opioid Receptor in Lung Cancer Progression: A Laboratory Study 
Anesthesia and analgesia  2010;112(3):558-567.
Background
The possibility that mu opioid agonists can influence cancer recurrence is a subject of recent interest. Epidemiologic studies suggested that there were differences in cancer recurrence in breast and prostate cancer contingent on anesthetic regimens. In this study, we identify a possible mechanism for these epidemiologic findings based on mu opioid receptor (MOR) regulation of Lewis lung carcinoma (LLC) tumorigenicity in cell and animal models.
Methods
We utilized human lung tissue and human non-small cell lung cancer (NSCLC) cell lines and evaluated MOR expression using immunoblot and immunohistochemical analysis. LLC cells were treated with the peripheral opioid antagonist methylnalnaltrexone (MNTX) or MOR shRNA and evaluated for proliferation, invasion and soft agar colony formation in vitro and primary tumor growth and lung metastasis in C57BL/6 and MOR knockout mice using Visen FMT imaging and immunohistochemical analysis.
Results
We provide several lines of evidence that the MOR may be a potential target for lung cancer, a disease with high mortality and few treatment options. We first observed that there is ~5 to 10 fold increase in MOR expression in lung samples from patients with NSCLC and in several human NSCLC cell lines. The MOR agonists morphine and DAMGO increased in vitro LLC cell growth. Treatment with MNTX or silencing MOR expression inhibited LLC invasion and anchorage-independent growth by 50–80%. Injection of MOR silenced LLC lead to a ~65% reduction in mouse lung metastasis. In addition, MOR knockout mice do not develop significant tumors when injected with LLC as compared to wildtype controls. Finally, continuous infusion of the peripheral opioid antagonist methylnaltrexone attenuates primary LLC tumor growth and reduces lung metastasis.
Conclusions
Taken together, our data suggests a possible direct effect of opiates on lung cancer progression, and provides a plausible explanation for the epidemiologic findings. Our observations further suggest a possible therapeutic role for opioid antagonists.
doi:10.1213/ANE.0b013e31820568af
PMCID: PMC4327979  PMID: 21156980
2.  Role of Migratory Inhibition Factor in Age-Related Susceptibility to Radiation Lung Injury via NF-E2–Related Factor–2 and Antioxidant Regulation 
Microvascular injury and increased vascular leakage are prominent features of radiation-induced lung injury (RILI), and often follow cancer-associated thoracic irradiation. Our previous studies demonstrated that polymorphisms in the gene (MIF) encoding macrophage migratory inhibition factor (MIF), a multifunctional pleiotropic cytokine, confer susceptibility to acute inflammatory lung injury and increased vascular permeability, particularly in senescent mice. In this study, we exposed wild-type and genetically engineered mif−/− mice to 20 Gy single-fraction thoracic radiation to investigate the age-related role of MIF in murine RILI (mice were aged 8 wk, 8 mo, or 16 mo). Relative to 8-week-old mice, decreased MIF was observed in bronchoalveolar lavage fluid and lung tissue of 8- to 16-month-old wild-type mice. In addition, radiated 8- to 16-month-old mif−/− mice exhibited significantly decreased bronchoalveolar lavage fluid total antioxidant concentrations with progressive age-related decreases in the nuclear expression of NF-E2–related factor–2 (Nrf2), a transcription factor involved in antioxidant gene up-regulation in response to reactive oxygen species. This was accompanied by decreases in both protein concentrations (NQO1, GCLC, and heme oxygenase–1) and mRNA concentrations (Gpx1, Prdx1, and Txn1) of Nrf2-influenced antioxidant gene targets. In addition, MIF-silenced (short, interfering RNA) human lung endothelial cells failed to express Nrf2 after oxidative (H2O2) challenge, an effect reversed by recombinant MIF administration. However, treatment with an antioxidant (glutathione reduced ester), but not an Nrf2 substrate (N-acetyl cysteine), protected aged mif−/− mice from RILI. These findings implicate an important role for MIF in radiation-induced changes in lung-cell antioxidant concentrations via Nrf2, and suggest that MIF may contribute to age-related susceptibility to thoracic radiation.
doi:10.1165/rcmb.2012-0291OC
PMCID: PMC3824032  PMID: 23526214
radiation pneumonitis; lung vascular permeability; macrophage migratory inhibition factor; Nrf2; antioxidant system; aging
3.  Sphingosine-1–Phosphate, FTY720, and Sphingosine-1–Phosphate Receptors in the Pathobiology of Acute Lung Injury 
Acute lung injury (ALI) attributable to sepsis or mechanical ventilation and subacute lung injury because of ionizing radiation (RILI) share profound increases in vascular permeability as a key element and a common pathway driving increased morbidity and mortality. Unfortunately, despite advances in the understanding of lung pathophysiology, specific therapies do not yet exist for the treatment of ALI or RILI, or for the alleviation of unremitting pulmonary leakage, which serves as a defining feature of the illness. A critical need exists for new mechanistic insights that can lead to novel strategies, biomarkers, and therapies to reduce lung injury. Sphingosine 1–phosphate (S1P) is a naturally occurring bioactive sphingolipid that acts extracellularly via its G protein–coupled S1P1–5 as well as intracellularly on various targets. S1P-mediated cellular responses are regulated by the synthesis of S1P, catalyzed by sphingosine kinases 1 and 2, and by the degradation of S1P mediated by lipid phosphate phosphatases, S1P phosphatases, and S1P lyase. We and others have demonstrated that S1P is a potent angiogenic factor that enhances lung endothelial cell integrity and an inhibitor of vascular permeability and alveolar flooding in preclinical animal models of ALI. In addition to S1P, S1P analogues such as 2-amino-2-(2-[4-octylphenyl]ethyl)-1,3-propanediol (FTY720), FTY720 phosphate, and FTY720 phosphonates offer therapeutic potential in murine models of lung injury. This translational review summarizes the roles of S1P, S1P analogues, S1P-metabolizing enzymes, and S1P receptors in the pathophysiology of lung injury, with particular emphasis on the development of potential novel biomarkers and S1P-based therapies for ALI and RILI.
doi:10.1165/rcmb.2012-0411TR
PMCID: PMC3727889  PMID: 23449739
sphingolipids; S1P receptors; sphingosine kinase; S1P lyase; sepsis
4.  Hydrogen Sulfide Attenuates Particulate Matter–Induced Human Lung Endothelial Barrier Disruption via Combined Reactive Oxygen Species Scavenging and Akt Activation 
Exposure to particulate air pollution is associated with increased cardiopulmonary morbidity and mortality, although the pathogenic mechanisms are poorly understood. We previously demonstrated that particulate matter (PM) exposure triggers massive oxidative stress in vascular endothelial cells (ECs), resulting in the loss of EC integrity and lung vascular hyperpermeability. We investigated the protective role of hydrogen sulfide (H2S), an endogenous gaseous molecule present in the circulation, on PM-induced human lung EC barrier disruption and pulmonary inflammation. Alterations in EC monolayer permeability, as reflected by transendothelial electrical resistance (TER), the generation of reactive oxygen species (ROS), and murine pulmonary inflammatory responses, were studied after exposures to PM and NaSH, an H2S donor. Similar to N-acetyl cysteine (5 mM), NaSH (10 μM) significantly scavenged PM-induced EC ROS and inhibited the oxidative activation of p38 mitogen-activated protein kinase. Concurrent with these events, NaSH (10 μM) activated Akt, which helps maintain endothelial integrity. Both of these pathways contribute to the protective effect of H2S against PM-induced endothelial barrier dysfunction. Furthermore, NaSH (20 mg/kg) reduced vascular protein leakage, leukocyte infiltration, and proinflammatory cytokine release in bronchoalveolar lavage fluids in a murine model of PM-induced lung inflammation. These data suggest a potentially protective role for H2S in PM-induced inflammatory lung injury and vascular hyperpermeability.
doi:10.1165/rcmb.2011-0248OC
PMCID: PMC3488621  PMID: 22592920
particulate matter; hydrogen sulfide; endothelial permeability; Akt
5.  A Sphingosine 1–Phosphate 1 Receptor Agonist Modulates Brain Death–Induced Neurogenic Pulmonary Injury 
Lung transplantation remains the only viable therapy for patients with end-stage lung disease. However, the full utilization of this strategy is severely compromised by a lack of donor lung availability. The vast majority of donor lungs available for transplantation are from individuals after brain death (BD). Unfortunately, the early autonomic storm that accompanies BD often results in neurogenic pulmonary edema (NPE), producing varying degrees of lung injury or leading to primary graft dysfunction after transplantation. We demonstrated that sphingosine 1–phosphate (S1P)/analogues, which are major barrier-enhancing agents, reduce vascular permeability via the S1P1 receptor, S1PR1. Because primary lung graft dysfunction is induced by lung vascular endothelial cell barrier dysfunction, we hypothesized that the S1PR1 agonist, SEW-2871, may attenuate NPE when administered to the donor shortly after BD. Significant lung injury was observed after BD, with increases of approximately 60% in bronchoalveolar lavage (BAL) total protein, cell counts, and lung tissue wet/dry (W/D) weight ratios. In contrast, rats receiving SEW-2871 (0.1 mg/kg) 15 minutes after BD and assessed after 4 hours exhibited significant lung protection (∼ 50% reduction, P = 0.01), as reflected by reduced BAL protein/albumin, cytokines, cellularity, and lung tissue wet/dry weight ratio. Microarray analysis at 4 hours revealed a global impact of both BD and SEW on lung gene expression, with a differential gene expression of enriched immune-response/inflammation pathways across all groups. Overall, SEW served to attenuate the BD-mediated up-regulation of gene expression. Two potential biomarkers, TNF and chemokine CC motif receptor-like 2, exhibited gene array dysregulation. We conclude that SEW-2871 significantly attenuates BD-induced lung injury, and may serve as a potential candidate to improve human donor availability.
doi:10.1165/rcmb.2010-0267OC
PMCID: PMC3262681  PMID: 21617203
neurogenic pulmonary edema; lung injury; sphingosine 1–phosphate; sphingolipids; lung transplant donors
6.  Particulate matter air pollution disrupts endothelial cell barrier via calpain-mediated tight junction protein degradation 
Background
Exposure to particulate matter (PM) is a significant risk factor for increased cardiopulmonary morbidity and mortality. The mechanism of PM-mediated pathophysiology remains unknown. However, PM is proinflammatory to the endothelium and increases vascular permeability in vitro and in vivo via ROS generation.
Objectives
We explored the role of tight junction proteins as targets for PM-induced loss of lung endothelial cell (EC) barrier integrity and enhanced cardiopulmonary dysfunction.
Methods
Changes in human lung EC monolayer permeability were assessed by Transendothelial Electrical Resistance (TER) in response to PM challenge (collected from Ft. McHenry Tunnel, Baltimore, MD, particle size >0.1 μm). Biochemical assessment of ROS generation and Ca2+ mobilization were also measured.
Results
PM exposure induced tight junction protein Zona occludens-1 (ZO-1) relocation from the cell periphery, which was accompanied by significant reductions in ZO-1 protein levels but not in adherens junction proteins (VE-cadherin and β-catenin). N-acetyl-cysteine (NAC, 5 mM) reduced PM-induced ROS generation in ECs, which further prevented TER decreases and atteneuated ZO-1 degradation. PM also mediated intracellular calcium mobilization via the transient receptor potential cation channel M2 (TRPM2), in a ROS-dependent manner with subsequent activation of the Ca2+-dependent protease calpain. PM-activated calpain is responsible for ZO-1 degradation and EC barrier disruption. Overexpression of ZO-1 attenuated PM-induced endothelial barrier disruption and vascular hyperpermeability in vivo and in vitro.
Conclusions
These results demonstrate that PM induces marked increases in vascular permeability via ROS-mediated calcium leakage via activated TRPM2, and via ZO-1 degradation by activated calpain. These findings support a novel mechanism for PM-induced lung damage and adverse cardiovascular outcomes.
doi:10.1186/1743-8977-9-35
PMCID: PMC3489700  PMID: 22931549
Calpain; Endothelial permeability; Particulate matter; ROS; TRPM2
7.  Simvastatin Attenuates Radiation-Induced Murine Lung Injury and Dysregulated Lung Gene Expression 
Novel therapies are desperately needed for radiation-induced lung injury (RILI), which, despite aggressive corticosteroid therapy, remains a potentially fatal and dose-limiting complication of thoracic radiotherapy. We assessed the utility of simvastatin, an anti-inflammatory and lung barrier–protective agent, in a dose- and time-dependent murine model of RILI (18–(25 Gy). Simvastatin reduced multiple RILI indices, including vascular leak, leukocyte infiltration, and histological evidence of oxidative stress, while reversing RILI-associated dysregulated gene expression, including p53, nuclear factor–erythroid-2–related factor, and sphingolipid metabolic pathway genes. To identify key regulators of simvastatin-mediated RILI protection, we integrated whole-lung gene expression data obtained from radiated and simvastatin-treated mice with protein–protein interaction network analysis (single-network analysis of proteins). Topological analysis of the gene product interaction network identified eight top-prioritized genes (Ccna2a, Cdc2, fcer1 g, Syk, Vav3, Mmp9, Itgam, Cd44) as regulatory nodes within an activated RILI network. These studies identify the involvement of specific genes and gene networks in RILI pathobiology, and confirm that statins represent a novel strategy to limit RILI.
doi:10.1165/rcmb.2010-0122OC
PMCID: PMC3095940  PMID: 20508068
radiation pneumonitis; lung vascular permeability; simvastatin; gene dysregulation; protein–protein interaction
8.  Differential Effects of Sphingosine 1–Phosphate Receptors on Airway and Vascular Barrier Function in the Murine Lung 
The therapeutic options for ameliorating the profound vascular permeability, alveolar flooding, and organ dysfunction that accompanies acute inflammatory lung injury (ALI) remain limited. Extending our previous finding that the intravenous administration of the sphingolipid angiogenic factor, sphingosine 1–phosphate (S1P), attenuates inflammatory lung injury and vascular permeability via ligation of S1PR1, we determine that a direct intratracheal or intravenous administration of S1P, or a selective S1P receptor (S1PR1) agonist (SEW-2871), produces highly concentration-dependent barrier-regulatory responses in the murine lung. The intratracheal or intravenous administration of S1P or SEW-2871 at < 0.3 mg/kg was protective against LPS-induced murine lung inflammation and permeability. However, intratracheal delivery of S1P at 0.5 mg/kg (for 2 h) resulted in significant alveolar–capillary barrier disruption (with a 42% increase in bronchoalveolar lavage protein), and produced rapid lethality when delivered at 2 mg/kg. Despite the greater selectivity for S1PR1, intratracheally delivered SEW-2871 at 0.5 mg/kg also resulted in significant alveolar–capillary barrier disruption, but was not lethal at 2 mg/kg. Consistent with the S1PR1 regulation of alveolar/vascular barrier function, wild-type mice pretreated with the S1PR1 inverse agonist, SB-649146, or S1PR1+/− mice exhibited reduced S1P/SEW-2871–mediated barrier protection after challenge with LPS. In contrast, S1PR2−/− knockout mice as well as mice with reduced S1PR3 expression (via silencing S1PR3-containing nanocarriers) were protected against LPS-induced barrier disruption compared with control mice. These studies underscore the potential therapeutic effects of highly selective S1PR1 receptor agonists in reducing inflammatory lung injury, and highlight the critical role of the S1P delivery route, S1PR1 agonist concentration, and S1PR1 expression in target tissues.
doi:10.1165/rcmb.2009-0223OC
PMCID: PMC2951871  PMID: 19749179
SEW-2871; LPS; SB-649146; S1P; lung edema
9.  Methylnaltrexone Potentiates the Anti-Angiogenic Effects of mTOR Inhibitors 
Background
Recent cancer therapies include drugs that target both tumor growth and angiogenesis including mammalian target of rapamycin (mTOR) inhibitors. Since mTOR inhibitor therapy is associated with significant side effects, we examined potential agents that can reduce the therapeutic dose.
Methods
Methylnaltrexone (MNTX), a peripheral mu opioid receptor (MOR) antagonist, in combination with the mTOR inhibitors temsirolimus and/or rapamycin, was evaluated for inhibition of VEGF-induced human pulmonary microvascular endothelial cell (EC) proliferation and migration as well as in vivo angiogenesis (mouse Matrigel plug assay).
Results
MNTX inhibited VEGF-induced EC proliferation and migration with an IC50 of ~100 nM. Adding 10 nM MNTX to EC shifted the IC50 of temsirolimus inhibition of VEGF-induced proliferation and migration from ~10 nM to ~1 nM and from ~50 to ~10 nM respectively. We observed similar effects with rapamycin. On a mechanistic level, we observed that MNTX increased EC plasma membrane-associated tyrosine phosphate activity. Inhibition of tyrosine phosphatase activity (3,4-dephostatin) blocked the synergy between MNTX and temsirolimus and increased VEGF-induced tyrosine phosphorylation of Src with enhanced PI3 kinase and mTOR Complex 2-dependent phosphorylation of Akt and subsequent activation of mTOR Complex 1 (rapamycin and temsirolimus target), while silencing Src, Akt or mTOR complex 2 components blocked VEGF-induced angiogenic events.
Conclusions
Our data indicate that MNTX exerts a synergistic effect with rapamycin and temsirolimus on inhibition of VEGF-induced human EC proliferation and migration and in vivo angiogenesis. Therefore, addition of MNTX could potentially lower the dose of mTOR inhibitors which could improve therapeutic index.
doi:10.1186/2040-2384-2-5
PMCID: PMC2831839  PMID: 20298531
10.  Inhibitory κB Kinase 2 Activates Airway Epithelial Cells to Stimulate Bone Marrow Macrophages 
It has not been resolved whether macrophages or airway epithelial cells primarily respond to infectious and inflammatory stimuli and initiate a cell-to-cell inflammatory interaction within the airways. We hypothesized that the airway epithelial cells are primary responders that activate macrophages in response to environmental stimuli. To investigate the unilateral contribution of airway epithelial cells in the activation of macrophages, we developed an in vitro system in which the primary mouse tracheal epithelial cells (MTEC) and primary bone marrow–derived macrophages (BMDM) were incubated together for a brief period of time in a Transwell culture plate. MTEC were transfected with adenoviral vectors that express a constitutively active form of IKK2 (Ad-cIKK2), Ad-β-Gal, or PBS for 48 h before incubating with the macrophages. Macrophage activation was determined by measuring surface expression of CD11b, activation of NF-κB, phagocytic activity and production of reactive oxygen species, and cyclooxygenase (COX)-2 gene expression and production of prostaglandins. Macrophage adherence to epithelial layer was confirmed by CD68 immunostaining and scanning electron microscopy. MTEC cells transfected with Ad-cIKK2 produced increased amounts of IL-6, mouse GRO-α, TNF-α, and prostaglandin (PG)E2. Exposure of BMDM to MTEC, transfected with Ad-cIKK2, led to an increase in the CD11b expression and increased adherence of macrophages to the epithelial cell layer. NF-κB activation, COX-2 gene expression, and PGD2 synthesis were also increased in BMDM that were incubated with MTEC transfected with Ad-cIKK2. These data suggest that airway epithelial cells potentially play a primary role in generating inflammatory signals that result in activation of macrophages.
doi:10.1165/rcmb.2006-0245OC
PMCID: PMC1899334  PMID: 17204585
airway epithelium; macrophages; lung; inflammation

Results 1-10 (10)