Pro-Resolving Effects of Resolvin D2 in LTD4 and TNF-α Pre-Treated Human Bronchi.

Inflammation is a major burden in respiratory diseases, resulting in airway hyperresponsiveness. Our hypothesis is that resolution of inflammation may represent a long-term solution in preventing human bronchial dysfunctions. The aim of the present study was to assess the anti-inflammatory effects of RvD2, a member of the D-series resolving family, with concomitant effects on ASM mechanical reactivity. The role and mode of action of RvD2 were assessed in an in vitro model of human bronchi under pro-inflammatory conditions, induced either by 1 µM LTD4 or 10 ng/ml TNF-α pre-treatment for 48h. TNF-α and LTD4 both induced hyperreactivity in response to pharmacological stimuli. Enhanced 5-Lipoxygenase (5-LOX) and cysteinyl leukotriene receptor 1 (CysLTR1) detection was documented in LTD4 or TNF-α pre-treated human bronchi when compared to control (untreated) human bronchi. In contrast, RvD2 treatments reversed 5-LOX/ß-actin and CysLTR1/ß-actin ratios and decreased the phosphorylation levels of AP-1 subunits (c-Fos, c-Jun) and p38-MAP kinase, while increasing the detection of the ALX/FPR2 receptor. Moreover, various pharmacological agents revealed the blunting effects of RvD2 on LTD4 or TNF-α induced hyper-responsiveness. Combined treatment with 300 nM RvD2 and 1 µM WRW4 (an ALX/FPR2 receptor inhibitor) blunted the pro-resolving and broncho-modulatory effects of RvD2. The present data provide new evidence regarding the role of RvD2 in a human model of airway inflammation and hyperrresponsiveness.

MAG-DPA curbs inflammatory biomarkers and pharmacological reactivity in cytokine-triggered hyperresponsive airway models.

Bronchial inflammation contributes to a sustained elevation of airway hyperresponsiveness (AHR) in asthma. Conversely, omega-3 fatty acid derivatives have been shown to resolve inflammation in various tissues. Thus, the effects of docosapentaenoic acid monoacylglyceride (MAG-DPA) were assessed on inflammatory markers and reactivity of human distal bronchi as well as in a cultured model of guinea pig tracheal rings. Human bronchi were dissected and cultured for 48 h with 10 ng/mL TNF-α or IL-13. Guinea pig tracheas were maintained in organ culture for 72 h which was previously shown to trigger spontaneous AHR. All tissues were treated with increasing concentrations of MAG-DPA (0.1, 0.3, and 1 µmol/L). Pharmacomechanical reactivity, Ca2+ sensitivity, and western blot analysis for specific phosphoproteins and transcription factors were performed to assess the effects of both cytokines, alone or in combination with MAG-DPA, on human and guinea pig airway preparations. Although 0.1 µmol/L MAG-DPA did not significantly reduce inflammatory biomarkers, the higher concentrations of MAG-DPA (0.3 and 1 µmol/L) blunted the activation of the TNF-α/NF κB pathway and abolished COX-2 expression in human and guinea pig tissues. Moreover, 0.3 and 1 µmol/L MAG-DPA consistently decreased the Ca2+ sensitivity and pharmacological reactivity of cultured bronchial explants. Furthermore, in human bronchi, IL-13-stimulated phosphorylation of CPI-17 was reversed by 1 µmol/L MAG-DPA. This effect was further amplified in the presence of 100 µmol/L aspirin. MAG-DPA mediates antiphlogistic effects by increasing the resolution of inflammation, while resetting Ca2+ sensitivity and contractile reactivity.

Resolvin E1 normalizes contractility, Ca2+ sensitivity and smooth muscle cell migration rate in TNF-α- and IL-6-pretreated human pulmonary arteries.

Pulmonary hypertension (PH) is a rare disease in which pathophysiology is characterized by an increase in proinflammatory mediators, chronic endothelial dysfunctions, and a high migration rate of smooth muscle cells (SMC). Over the course of the last decade, various treatments have been proposed to relax the pulmonary arteries, none of which have been effective in resolving PH. Our hypothesis is that artery-relaxing drugs are not the long-term solution, but rather the inhibition of tissue inflammation, which underlies human pulmonary artery (HPA) dysfunctions that lead to abnormal vasoconstriction. The goal of the present study was to assess the anti-inflammatory effects of resolvin E1 (RvE1) with concomitant effects on SMC migration and on HPA reactivity. The role and mode of action of RvE1 and its precursor, monoacylglyceride eicosapentaenoic acid were assessed on HPA under proinflammatory conditions, involving a combined pretreatment with 10 ng/ml TNF-α and 10 ng/ml IL-6. Our results show that TNF-α and IL-6 treatment induced hyperreactivity and Ca(2+) hypersensitivity in response to pharmaco-mechanical stimuli, including 80 mM KCl, 1 µM phorbol 12-13-dibutyrate, and 30 nM U-46619. Furthermore, the proinflammatory treatment increased the migration rate of SMC isolated from HPA. The phosphorylation level of regulatory contractile proteins (CPI-17, MYPT-1), and proinflammatory signaling pathways (c-Fos, c-Jun, NF-κB) were also significantly increased compared with control conditions. Conversely, 300 nM RvE1 was able to normalize all of the above abnormal events triggered by proinflammation. In conclusion, RvE1 can resolve human arterial hyperreactivity via the resolution of inflammatory markers.

Reversal of IL-13-induced inflammation and Ca(2+) sensitivity by resolvin and MAG-DHA in association with ASA in human bronchi.

The aim of this study was to investigate the effects of resolvin D1 (RvD1), as well as the combined treatment of docosahexaenoic acid monoglyceride (MAG-DHA) and acetylsalicylic acid (ASA), on the resolution of inflammation markers and Ca(2+) sensitivity in IL-13-pretreated human bronchi (HB). Tension measurements performed with 300 nM RvD1 largely abolished (50%) the over-reactivity triggered by 10 ng/ml IL-13 pretreatment and reversed hyper Ca(2+) sensitivity. Addition of 300 nM 17(S)-HpDoHE, the metabolic intermediate between DHA and RvD1, displayed similar effects. In the presence of 100 µM ASA (a COX inhibitor), the inhibitory effect of 1 µM MAG-DHA on muscarinic tone was further amplified, but not in the presence of Ibuprofen. Western blot analysis revealed that the combined treatment of MAG-DHA and ASA upregulated GPR-32 expression and downregulated cytosolic TNFα detection, hence preventing IκBα degradation and p65-NFκB phosphorylation. The Ca(2+) sensitivity of HB was also quantified on ß-escin permeabilized preparations. The presence of ASA potentiated the inhibitory effects of MAG-DHA in reducing the Ca(2+) hypersensitivity triggered by IL-13 by decreasing the phosphorylation levels of the PKC-potentiated inhibitor protein-17 regulatory protein (CPI-17). In summary, MAG-DHA combined with ASA, as well as exogenously added RvD1, may represent valuable assets against critical AHR disorder.

Proresolving Action of Docosahexaenoic Acid Monoglyceride in Lung Inflammatory Models Related to Cystic Fibrosis.

Cystic fibrosis (CF) is a hereditary, chronic disease of the exocrine glands, characterized by the production of viscid mucus that obstructs the pancreatic ducts and bronchi, leading to infection and fibrosis. ω3 fatty acid supplementations are known to improve the essential fatty acid deficiency as well as reduce inflammation in CF. The objective of this study was to determine the effects of docosahexaenoic acid monoacylglyceride (MAG-DHA) on mucin overproduction and resolution of airway inflammation in two in vitro models related to CF. Isolated human bronchi reverse permeabilized with CF transmembrane conductance regulator (CFTR) silencing (si) RNA and stable Calu3 cells expressing a short hairpin (sh) RNA directed against CFTR (shCFTR) were used. Lipid analyses revealed that MAG-DHA increased DHA/arachidonic acid (AA) ratio in shCFTR Calu-3 cells. MAG-DHA treatments, moreover, resulted in a decreased activation of Pseudomonas aeruginosa LPS-induced NF-κB in CF and non-CF Calu-3 cells. Data also revealed a reduction in MUC5AC, IL-6, and IL-8 expression levels in MAG-DHA-treated shCFTR cells stimulated, or not, with LPS. Antiinflammatory properties of MAG-DHA were also investigated in a reverse-permeabilized human bronchi model with CFTR siRNA. After MAG-DHA treatments, messenger RNA transcript levels for MUC5AC, IL-6, and IL-8 were markedly reduced in LPS-treated CFTR siRNA bronchi. MAG-DHA displays antiinflammatory properties and reduces mucin overexpression in Calu-3 cells and human bronchi untreated or treated with P. aeruginosa LPS, a finding consistent with the effects of resolvinD1, a known antiinflammatory mediator.

Resolvin D1 reverses reactivity and Ca2+ sensitivity induced by ET-1, TNF-α, and IL-6 in the human pulmonary artery.

Pulmonary hypertension (PH) is a rare and progressive disease characterized by an inflammatory status and vessel wall remodeling, resulting in increased pulmonary artery resistance. During the last decade, treatments have been proposed; most of them target the endothelial pathways that stimulate smooth muscle cell relaxation. However, PH remains associated with significant morbidity. We hypothesized that inflammation plays a crucial role in the severity of the abnormal vasoconstriction in PH. The goal of this study was to assess the effects of resolvin D1 (RvD1), a potent anti-inflammatory agent, on the pharmacological reactivity of human pulmonary arteries (HPAs) via an in vitro model of induced hyperreactivity. The effects of RvD1 and monoacylglyceride compounds were measured on contractile activity and Ca(2+) sensitivity developed by HPAs that had been pretreated (or not) under proinflammatory conditions with either 10 ng/ml TNF-α or 10 ng/ml IL-6 or under hyperreactive conditions with 5 nM endothelin-1. The results demonstrated that, compared with controls, 24-h pretreatment with TNF-α, IL-6, or endothelin-1 increased reactivity and Ca(2+) sensitivity of HPAs as revealed by agonist challenges with 80 mM KCl, 1 µM serotonin (5-hydroxytryptamine), 30 nM U-46619, and 1 µM phorbol 12,13-dibutyrate. However, 300 nM RvD1 as well as 1 µM monoacylglyceride-docosapentaenoic acid monoglyceride strongly reversed the overresponsiveness induced by both proinflammatory and hyperreactive treatments. In pretreated pulmonary artery smooth muscle cells, Western blot analyses revealed that RvD1 treatment decreased the phosphorylation level of CPI-17 and expression of transmembrane protein member 16A while increasing the detection of G protein-coupled receptor 32. The present data demonstrate that RvD1, a trihydroxylated docosahexaenoic acid derivative, decreases induced overreactivity in HPAs via a reduction in CPI-17 phosphorylation and transmembrane protein member 16A expression.

Lipoxin generation is related to soluble epoxide hydrolase activity in severe asthma.

RATIONALE: Severe asthma is characterized by airway inflammatory responses associated with aberrant metabolism of arachidonic acid. Lipoxins (LX) are arachidonate-derived pro-resolving mediators that are decreased in severe asthma, yet mechanisms for defective LX biosynthesis and a means to increase LXs in severe asthma remain to be established. OBJECTIVES: To determine if oxidative stress and soluble epoxide hydrolase (sEH) activity are linked to decreased LX biosynthesis in severe asthma. METHODS: Aliquots of blood, sputum, and bronchoalveolar lavage fluid were obtained from asthma subjects for mediator determination. Select samples were exposed to t-butyl-hydroperoxide or sEH inhibitor (sEHI) before activation. Peripheral blood leukocyte-platelet aggregates were monitored by flow cytometry, and bronchial contraction was determined with cytokine-treated human lung sections. MEASUREMENTS AND MAIN RESULTS: 8-Isoprostane levels in sputum supernatants were inversely related to LXA4 in severe asthma (r = -0.55; P = 0.03) and t-butyl-hydroperoxide decreased LXA4 and 15-epi-LXA4 biosynthesis by peripheral blood leukocytes. LXA4 and 15-epi-LXA4 levels were inversely related to sEH activity in sputum supernatants and sEHIs significantly increased 14,15-epoxy-eicosatrienoic acid and 15-epi-LXA4 generation by severe asthma whole blood and bronchoalveolar lavage fluid cells. The abundance of peripheral blood leukocyte-platelet aggregates was related to asthma severity. In a concentration-dependent manner, LXs significantly inhibited platelet-activating factor-induced increases in leukocyte-platelet aggregates (70.8% inhibition [LXA4 100 nM], 78.3% inhibition [15-epi-LXA4 100 nM]) and 15-epi-LXA4 markedly inhibited tumor necrosis factor-α-induced increases in bronchial contraction. CONCLUSIONS: LX levels were decreased by oxidative stress and sEH activity. Inhibitors of sEH increased LXs that mediated antiphlogistic actions, suggesting a new therapeutic approach for severe asthma. Clinical trial registered with www.clinicaltrials.gov (NCT 00595114).

Closure of the uterine incision with one or two layers after caesarean section: a randomized controlled study in sheep.

OBJECTIVE: To compare the quality of the uterine scar with one or two layer closure after caesarean section by studying biomechanical and pathological properties of the scar. METHODS: A randomized controlled trial performed on eight term pregnant ewes assigned into two groups during caesarean according to type of uterine closure: single-layer or double-layer. Hysterectomy was performed 8 months after caesarean delivery. Tensile strength of all scars and of unscarred myometrium was measured. Pathological properties of the scars were analyzed histologically. RESULTS: The force required to reach the yield point was similar between scarred and unscarred myometrium (p=0.96), and between the scars in single-layer and double-layer closure groups (p=0.65). There was a significant increase in fibrosis width on the superficial part of the uterus in the double-layer closure group compared to the single-layer group (p=0.02). CONCLUSIONS: Double-layer uterine closure modified wound healing without significant change in biomechanical properties.

Relationship between bradykinin-induced relaxation and endogenous epoxyeicosanoid synthesis in human bronchi.

Epoxyeicosanoids (EETs) are produced by cytochrome P-450 epoxygenase; however, it is not yet known what triggers their endogenous production in epithelial cells. The relaxing effects of bradykinin are known to be related to endogenous production of epithelial-derived hyperpolarizing factors (EpDHF). Because of their effects on membrane potential, EETs have been reported to be EpDHF candidates (Benoit C, Renaudon B, Salvail D, Rousseau E. Am J Physiol Lung Cell Mol Physiol 280: L965-L973, 2001.). Thus, we hypothesized that bradykinin (BK) may stimulate endogenous EET production in human bronchi. To test this hypothesis, the relaxing and hyperpolarizing effects of BK and 14,15-EET were quantified on human bronchi, as well as the effects of various enzymatic inhibitors on these actions. One micromolar BK or 1 µM 14,15-EET induced a 45% relaxation on the tension induced by 30 nM U-46619 [a thromboxane-prostanoid (TP)-receptor agonist]. These BK-relaxing effects were reduced by 42% upon addition of 10 nM iberiotoxin [a large-conductance Ca(2+)-sensitive K(+) (BK(Ca)) channel blocker], by 27% following addition of 3 µM 14,15-epoxyeicosa-5(Z)-enoic acid (an EET antagonist), and by 32% with 3 µM N-methanesulfonyl-6-(2-propargyloxyphenyl)hexanamide (MS-PPOH, an epoxygenase inhibitor). Hence, BK and 14,15-EET display net hyperpolarizing effects on airway smooth muscle cells that are related to the activation of BK(Ca) channels and ultimately yielding to relaxation. Data also indicate that 3 µM MS-PPOH reduced the hyperpolarizing effects of BK by 43%. Together, the present data support the current hypothesis suggesting a direct relationship between BK and the production of EET regioisomers. Because of its potent anti-inflammatory and relaxing properties, epoxyeicosanoid signaling may represent a promising target in asthma and chronic obstructive pulmonary disease.

Anti-cancer effects of a new docosahexaenoic acid monoacylglyceride in lung adenocarcinoma.

Lung cancer is the leading cause of cancer-related deaths worldwide. Despite advances in research, diagnosis and treatment, lung cancer remains a highly lethal disease, often diagnosed at advanced stages and with a very poor prognosis. Therefore, new strategies for the prevention and treatment of lung cancer are urgently needed. The aim of the present study was to determine the anti-tumorigenic effects of docosahexaenoic acid monoacylglyceride (MAG-DHA), a newly patented DHA derivative in lung adenocarcinoma. Our results demonstrate that MAG-DHA treatments decreased cell proliferation and induced apoptosis in A549 human lung carcinoma cells whereas MAG-DHA treatment did not induce apoptosis of normal bronchial epithelial BEAS-2B cells. MAG-DHA decreased NFκB activation leading to a reduction in COX-2 expression level in both A549 cells and lung adenocarcinoma tissues. Furthermore, MAG-DHA treatment increased PTEN expression and activation concomitant with a decrease in AKT phosphorylation levels and enhanced apoptosis. Oral administration of MAG-DHA significantly reduced tumor growth in a mouse A549 xenograft model. Lastly, MAG-DHA markedly decreased COX-2 and enhanced PTEN protein expression in tumor tissue sections. Altogether, these data provide new evidence regarding the mode of action of MAG-DHA and strongly suggest that this compound could be of clinical interest in cancer treatment.

Huge malignant solitary fibrous tumor of the pleura.

INTRODUCTION: Solitary fibrous pleural tumors are rare mesenchymal tumors that can be mistaken for mesothelioma. A positive staining of vimentin, negative staining of cytoplasmic keratin, and expression of the CD34 antigen can confirm their diagnosis. These tumors should be surgically excised although they are benign because they have malignant potential and tendency for recurrence. PATIENTS AND METHODS: We present here a 63-year-old patient who had an inconclusive biopsy of a huge right intrathoracic lesion. Further testing after radical surgical resection revealed a malignant solitary fibrous pleural tumor. Surgery was complicated with injury of the thoracic duct. Patient was re-operated upon for ligation of the thoracic duct. He is doing well with no recurrence after 18 months of follow-up. CONCLUSION: In conclusion, definitive treatment for solitary fibrous tumor is radical surgical resection with close follow-up for the recurrences. Follow-up of this tumor is essential especially when it showed malignant features on pathological examination.

7-tesla MR imaging of non-melanoma skin cancer samples: correlation with histopathology.

PURPOSE: The aims of this study were to compare in vitro magnetic resonance imaging (MRI) characteristics of keratinocytic skin cancer assessed by a 7-tesla (T) MRI with histopathology, and to describe MRI features of skin tumors. METHODS: This prospective study included 30 skin tumors treated by surgery. MR images of skin samples were acquired on a 7-T MR scanner using a fast spin-echo T(2)-weighted and an isotropic 3D gradient-echo T(1)-weighted sequence. Length, width, Breslow index and margins of the lesions were measured. The presence or absence of the following was noted: healthy margins, ulceration of the dermis, in situ lesions, superficial and deep dermis involvement, subcutaneous involvement, superficial and intratumoral keratin. MR results were compared to histopathology. RESULTS: Interclass correlation coefficient (ICC) was very good for the evaluation of the width (ICC = 0.86) and Breslow index (ICC = 0.87). The ICC was good for the evaluation of the margins (ICC = 0.70) but for length, ICC was lower (ICC = 0.67). Mean bias between MRI and histopathology was inferior to 1 mm for width, Breslow index and margin. CONCLUSION: In vitro 7-T MRI of keratinocytic skin cancer allows delineation of lesions with good correlation with histopathology. After in vivo confirmation it could have a diagnostic role regarding the delineation of surgical margins but its actual limitations prevent its practical adoption at this time.

Relaxing effects of 17(18)-EpETE on arterial and airway smooth muscles in human lung.

Human cytochrome P-450 epoxygenase enzymes metabolize eicosapentaenoic acid (EPA), an omega-3-polyunsaturated fatty acid (PUFA), and leads to the production of 17(18)-epoxyeicosatetraenoic acid, or 17(18)-EpETE. The aim of the present study was to delineate the mode of action of 17(18)-EpETE on human pulmonary artery (HPA) and distal bronchi. Isometric tension measurements demonstrated that 17(18)-EpETE induced concentration-dependent relaxing effects in pulmonary artery and airway smooth muscles. Iberiotoxin (IbTx) and glyburide (Glyb), known BK(Ca) and K(ATP) channel inhibitors, respectively, reversed the relaxation induced by 17(18)-EpETE on both tissues types. Microelectrode measurements showed that exogenous addition of 17(18)-EpETE hyperpolarized the membrane potential of HPA and bronchial smooth muscle cells. These induced electrophysiological effects were reversed by the addition of 10 nM IbTx and 10 muM Glyb. Complementary experiments performed on human bronchi, using the planar lipid bilayer reconstitution technique, demonstrated that 17(18)-EpETE activated reconstituted BK(Ca) channels at low free Ca(2+) concentration. Moreover, in bronchi, the relaxing responses induced by 17(18)-EpETE were also related to reduced Ca(2+) sensitivity of the myofilaments, since free Ca(2+) concentration-response curves, performed on beta-escin-permeabilized cultured explants, were shifted toward higher Ca(2+). Together, these results provide new insight into the mode of action of 17(18)-EpETE in lung tissues and highlight this eicosanoid as a potent modulator of tone on both HPA and distal bronchi in vitro, which may be of clinical relevance in the pathophysiology of pulmonary hypertension and airway diseases.