Nitric oxide decreases cytokine-induced endothelial activation. Nitric oxide selectively reduces endothelial expression of adhesion molecules and proinflammatory cytokines

To test the hypothesis that nitric oxide (NO) limits endothelial activation, we treated cytokine-stimulated human saphenous vein endothelial cells with several NO donors and assessed their effects on the inducible expression of vascular cell adhesion molecule-1 (VCAM-1). In a concentration-dependent manner, NO inhibited interleukin (IL)-1 alpha-stimulated VCAM-1 expression by 35-55% as determined by cell surface enzyme immunoassays and flow cytometry. This inhibition was paralleled by reduced monocyte adhesion to endothelial monolayers in nonstatic assays, was unaffected by cGMP analogues, and was quantitatively similar after stimulation by either IL-1 alpha, IL-1 beta, IL-4, tumor necrosis factor (TNF alpha), or bacterial lipopolysaccharide. NO also decreased the endothelial expression of other leukocyte adhesion molecules (E-selectin and to a lesser extent, intercellular adhesion molecule-1) and secretable cytokines (IL-6 and IL-8). Inhibition of endogenous NO production by L-N-monomethyl-arginine also induced the expression of VCAM-1, but did not augment cytokine-induced VCAM-1 expression. Nuclear run-on assays, transfection studies using various VCAM-1 promoter reporter gene constructs, and electrophoretic mobility shift assays indicated that NO represses VCAM-1 gene transcription, in part, by inhibiting NF-kappa B. We propose that NO's ability to limit endothelial activation and inhibit monocyte adhesion may contribute to some of its antiatherogenic and antiinflammatory properties within the vessel wall

Role of Nox4 and Nox2 in Hyperoxia-Induced Reactive Oxygen Species Generation and Migration of Human Lung Endothelial Cells

Abstract In vascular endothelium, the major research focus has been on reactive oxygen species (ROS) derived from Nox2. The role of Nox4 in endothelial signal transduction, ROS production, and cytoskeletal reorganization is not well defined. In this study, we show that human pulmonary artery endothelial cells (HPAECs) and human lung microvascular endothelial cells (HLMVECs) express higher levels of Nox4 and p22phox compared to Nox1, Nox2, Nox3, or Nox5. Immunofluorescence microscopy and Western blot analysis revealed that Nox4 and p22phox, but not Nox2 or p47phox, are localized in nuclei of HPAECs. Further, knockdown of Nox4 with siRNA decreased Nox4 nuclear expression significantly. Exposure of HPAECs to hyperoxia (3-24h) enhanced mRNA and protein expression of Nox4, and Nox4 siRNA decreased hyperoxia-induced ROS production. Interestingly, Nox4 or Nox2 knockdown with siRNA upregulated the mRNA and protein expression of the other, suggesting activation of compensatory mechanisms. A similar upregulation of Nox4 mRNA was observed in Nox2 2/ko mice. Downregulation of Nox4, or pretreatment with N-acetylcysteine, attenuated hyperoxia-induced cell migration and capillary tube formation, suggesting that ROS generated by Nox4 regulate endothelial cell motility. These results indicate that Nox4 and Nox2 play a physiological role in hyperoxia-induced ROS production and migration of ECs. Antioxid. Redox Signal. 11, 747-764.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/78121/1/ars.2008.2203.pd

Fibroblastic Foci in Usual Interstitial Pneumonia: Idiopathic versus Collagen Vascular Disease

A histologic feature of usual interstitial pneumonia is the presence of fibroblastic foci. As some patients with usual interstitial pneumonia and an underlying collagen vascular disease have a better prognosis, we hypothesized that they would have fewer fibroblastic foci. Pathologists reviewed surgical lung biopsies from 108 patients with usual interstitial pneumonia (nine with collagen vascular disease) and assigned a score (absent 0, mild 1, moderate 2, and marked 3) for fibroblastic foci. Patients with idiopathic usual interstitial pneumonia had a higher median profusion of fibroblastic foci (1.75 vs. 1.0, p = 0.003). Baseline characteristics were similar, although patients with a collagen vascular disease were younger, had a shorter duration of symptoms, and had a higher percentage of predicted total lung capacity. Profusion of fibroblastic foci was the most discriminative feature for separating idiopathic from collagen vascular disease–associated usual interstitial pneumonia (odds ratio 8.31; 95% confidence interval, 1.98, 59.42; p = 0.002 for a one-unit increase in fibroblastic foci score). No deaths were noted in the collagen vascular disease–associated usual interstitial pneumonia group; 52 deaths occurred in the idiopathic usual interstitial pneumonia group (log rank; p = 0.005). We conclude that patients with collagen vascular disease–associated usual interstitial pneumonia have fewer fibroblastic foci and improved survival.Supported in part by National Institutes of Health National Heart, Lung, and Blood Institute grant #P50HL46487, NIH/NCRR 3 MO1 RR00042–33S3, NIH/NIA P60 AG08808–06, NHLBI 1 K24 HL04212, and 1 K23 HL68713.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/91974/1/2003 AJRCCM - Fibroblastic Foci in Usual Interstitial Pneumonia -Idiopathic versus Collagen Vascular Disease.pd

Lung Cancer in Pulmonary Fibrosis: Tales of Epithelial Cell Plasticity

Lung epithelial cells exhibit a high degree of plasticity. Alterations to lung epithelial cell function are critically involved in several chronic lung diseases such as pulmonary fibrosis. Pulmonary fibrosis is characterized by repetitive injury and subsequent impaired repair of epithelial cells, which leads to aberrant growth factor activation and fibroblast accumulation. Increased proliferation and hyper- and metaplasia of epithelial cells upon injury have also been observed in pulmonary fibrosis; this epithelial cell activation might represent the basis for lung cancer development. Indeed, several studies have provided histopathological evidence of an increased incidence of lung cancer in pulmonary fibrosis. The mechanisms involved in the development of cancer in pulmonary fibrosis, however, remain poorly understood. This review highlights recently uncovered molecular mechanisms shared between lung cancer and fibrosis, which extend the current evidence of a common trait of cancer and fibrosis, as provided by histopathological observations. Copyright (C) 2011 S. Karger AG, Base

Increased Immune Complexes of Hypocretin Autoantibodies in Narcolepsy

International audienceBACKGROUND: Hypocretin peptides participate in the regulation of sleep-wake cycle while deficiency in hypocretin signaling and loss of hypocretin neurons are causative for narcolepsy-cataplexy. However, the mechanism responsible for alteration of the hypocretin system in narcolepsy-cataplexy and its relevance to other central hypersomnias remain unknown. Here we studied whether central hypersomnias can be associated with autoantibodies reacting with hypocretin-1 peptide present as immune complexes. METHODOLOGY: Serum levels of free and dissociated (total) autoantibodies reacting with hypocretin-1 peptide were measured by enzyme-linked immunosorbent assay and analyzed with regard to clinical parameters in 82 subjects with narcolepsy-cataplexy, narcolepsy without cataplexy or idiopathic hypersomnia and were compared to 25 healthy controls. PRINCIPAL FINDINGS: Serum levels of total but not free IgG autoantibodies against hypocretin-1 were increased in narcolepsy-cataplexy. Increased levels of complexed IgG autoantibodies against hypocretin-1 were found in all patients groups with a further increase in narcolepsy-cataplexy. Levels of total IgM hypocretin-1 autoantibodies were also elevated in all groups of patients. Increased levels of anti-idiotypic IgM autoantibodies reacting with hypocretin-1 IgG autoantibodies affinity purified from sera of subjects with narcolepsy-cataplexy were found in all three groups of patients. Disease duration correlated negatively with serum levels of hypocretin-1 IgG and IgM autoantibodies and with anti-idiotypic IgM autoantibodies. CONCLUSION: Central hypersomnias and particularly narcolepsy-cataplexy are characterized by higher serum levels of autoantibodies directed against hypocretin-1 which are present as immune complexes most likely with anti-idiotypic autoantibodies suggesting their relevance to the mechanism of sleep-wake cycle regulation

Idiopathic interstitial pneumonia: Do community and academic physicians agree on diagnosis?

Rationale: Treatment and prognoses of diffuse parenchymal lung diseases (DPLDs) varies by diagnosis. Obtaining a uniform diagnosis among observers is difficult. Objectives: Evaluate diagnostic agreement between academic and community-based physicians for patients with DPLDs, and determine if an interactive approach between clinicians, radiologists, and pathologists improved diagnostic agreement in community and academic centers. Methods: Retrospective review of 39 patients with DPLD. A total of 19 participants reviewed cases at 2 community locations and 1 academic location. Information from the history, physical examination, pulmonary function testing, high-resolution computed tomography, and surgical lung biopsy was collected. Data were presented in the same sequential fashion to three groups of physicians on separate days. Measurements and Main Results: Each observer’s diagnosis was coded into one of eight categories. A statistic allowing formultiple raters was used to assess agreement in diagnosis. Interactions between clinicians, radiologists, and pathologists improved interobserver agreement at both community and academic sites; however, final agreement was better within academic centers (Kappa= 0.55–0.71) than within community centers (Kappa=0.32–0.44). Clinically significant disagreement was present between academic and communitybased physicians (Kappa=0.11–0.56). Community physicians were more likely to assign a final diagnosis of idiopathic pulmonary fibrosis compared with academic physicians. Conclusions: Significant disagreement exists in the diagnosis of DPLD between physicians based in communities compared with those in academic centers. Wherever possible, patients should be referred to centers with expertise in diffuse parenchymal lung disorders to help clarify the diagnosis and provide suggestions regarding treatment options.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/91941/1/2007 AJRCCM Idiopathic interstitial pneumonia - Do community and academic physicians agree on diagnosis.pd

Acute and Long-Term Effects of Hyperthermia in B16-F10 Melanoma Cells

OBJECTIVE: Hyperthermia uses exogenous heat induction as a cancer therapy. This work addresses the acute and long-term effects of hyperthermia in the highly metastatic melanoma cell line B16-F10. MATERIALS AND METHODS: Melanoma cells were submitted to one heat treatment, 45°C for 30 min, and thereafter were kept at 37°C for an additional period of 14 days. Cultures maintained at 37°C were used as control. Cultures were assessed for the heat shock reaction. RESULTS: Immediately after the heat shock, cells began a process of fast degradation, and, in the first 24 h, cultures showed decreased viability, alterations in cell morphology and F-actin cytoskeleton organization, significant reduction in the number of adherent cells, most of them in a process of late apoptosis, and an altered gene expression profile. A follow-up of two weeks after heat exposure showed that viability and number of adherent cells remained very low, with a high percentage of early apoptotic cells. Still, heat-treated cultures maintained a low but relatively constant population of cells in S and G(2)/M phases for a long period after heat exposure, evidencing the presence of metabolically active cells. CONCLUSION: The melanoma cell line B16-F10 is susceptible to one hyperthermia treatment at 45°C, with significant induced acute and long-term effects. However, a low but apparently stable percentage of metabolically active cells survived long after heat exposure

PDGF-Rα gene expression predicts proliferation, but PDGF-A suppresses transdifferentiation of neonatal mouse lung myofibroblasts

<p>Abstract</p> <p>Background</p> <p>Platelet-derived growth factor A (PDGF-A) signals solely through PDGF-Rα, and is required for fibroblast proliferation and transdifferentiation (fibroblast to myofibroblast conversion) during alveolar development, because <it>pdgfa</it>-null mice lack both myofibroblasts and alveoli. However, these PDGF-A-mediated mechanisms remain incompletely defined. At postnatal days 4 and 12 (P4 and P12), using mouse lung fibroblasts, we examined (a) how PDGF-Rα correlates with ki67 (proliferation marker) or alpha-smooth muscle actin (αSMA, myofibroblast marker) expression, and (b) whether PDGF-A directly affects αSMA or modifies stimulation by transforming growth factor beta (TGFβ).</p> <p>Methods</p> <p>Using flow cytometry we examined PDGF-Rα, αSMA and Ki67 in mice which express green fluorescent protein (GFP) as a marker for PDGF-Rα expression. Using real-time RT-PCR we quantified αSMA mRNA in cultured Mlg neonatal mouse lung fibroblasts after treatment with PDGF-A, and/or TGFβ.</p> <p>Results</p> <p>The intensity of GFP-fluorescence enabled us to distinguish three groups of fibroblasts which exhibited absent, lower, or higher levels of PDGF-Rα. At P4, more of the higher than lower PDGF-Rα + fibroblasts contained Ki67 (Ki67+), and Ki67+ fibroblasts predominated in the αSMA + but not the αSMA- population. By P12, Ki67+ fibroblasts comprised a minority in both the PDGF-Rα + and αSMA+ populations. At P4, most Ki67+ fibroblasts were PDGF-Rα + and αSMA- whereas at P12, most Ki67+ fibroblasts were PDGF-Rα- and αSMA-. More of the PDGF-Rα + than - fibroblasts contained αSMA at both P4 and P12. In the lung, proximate αSMA was more abundant around nuclei in cells expressing high than low levels of PDGF-Rα at both P4 and P12. Nuclear SMAD 2/3 declined from P4 to P12 in PDGF-Rα-, but not in PDGF-Rα + cells. In Mlg fibroblasts, αSMA mRNA increased after exposure to TGFβ, but declined after treatment with PDGF-A.</p> <p>Conclusion</p> <p>During both septal eruption (P4) and elongation (P12), alveolar PDGF-Rα may enhance the propensity of fibroblasts to transdifferentiate rather than directly stimulate αSMA, which preferentially localizes to non-proliferating fibroblasts. In accordance, PDGF-Rα more dominantly influences fibroblast proliferation at P4 than at P12. In the lung, TGFβ may overshadow the antagonistic effects of PDGF-A/PDGF-Rα signaling, enhancing αSMA-abundance in PDGF-Rα-expressing fibroblasts.</p

Cholinergic Modulation of Narcoleptic Attacks in Double Orexin Receptor Knockout Mice

To investigate how cholinergic systems regulate aspects of the sleep disorder narcolepsy, we video-monitored mice lacking both orexin (hypocretin) receptors (double knockout; DKO mice) while pharmacologically altering cholinergic transmission. Spontaneous behavioral arrests in DKO mice were highly similar to those reported in orexin-deficient mice and were never observed in wild-type (WT) mice. A survival analysis revealed that arrest lifetimes were exponentially distributed indicating that random, Markovian processes determine arrest lifetime. Low doses (0.01, 0.03 mg/kg, IP), but not a high dose (0.08 mg/kg, IP) of the cholinesterase inhibitor physostigmine increased the number of arrests but did not alter arrest lifetimes. The muscarinic antagonist atropine (0.5 mg/kg, IP) decreased the number of arrests, also without altering arrest lifetimes. To determine if muscarinic transmission in pontine areas linked to REM sleep control also influences behavioral arrests, we microinjected neostigmine (50 nl, 62.5 µM) or neostigmine + atropine (62.5 µM and 111 µM respectively) into the nucleus pontis oralis and caudalis. Neostigmine increased the number of arrests in DKO mice without altering arrest lifetimes but did not provoke arrests in WT mice. Co-injection of atropine abolished this effect. Collectively, our findings establish that behavioral arrests in DKO mice are similar to those in orexin deficient mice and that arrests have exponentially distributed lifetimes. We also show, for the first time in a rodent narcolepsy model, that cholinergic systems can regulate arrest dynamics. Since perturbations of muscarinic transmission altered arrest frequency but not lifetime, our findings suggest cholinergic systems influence arrest initiation without influencing circuits that determine arrest duration