Interferon-γ enhances the antifibrotic effects of pirfenidone by attenuating IPF lung fibroblast activation and differentiation.

BACKGROUND: Idiopathic pulmonary fibrosis (IPF) pathogenesis involves multiple pathways, and combined antifibrotic therapy is needed for future IPF therapy. Inhaled interferon-γ (IFN-γ) was recently shown to be safe and without systemic effects in patients with IPF. AIM: To examine the in vitro effects of individual and combined treatment with IFN-γ and pirfenidone (PFD) on normal and IPF fibroblast activation and extracellular matrix remodeling after TGF-ß1 and PDGF-BB stimulation. METHODS: IPF and normal human lung fibroblasts (NHLF) were treated with IFN-γ, PFD or a combination of both drugs in the presence of either TGF-ß1 or PDGF-BB. The effects of TGF-ß1 and PDGF-BB treatment on cell viability, proliferation, differentiation and migration were examined. The expression of collagen 1, matrix metalloproteinases (MMPs) and tissue inhibitors of MMP (TIMPs) was analyzed using qPCR, Western blotting and gelatin zymography. Total collagen content in conditioned media was also measured using a Sircol assay. RESULTS: Compared to that of PFD, the effect of IFN-γ in downregulating normal and IPF lung fibroblast differentiation to myofibroblasts in response to TGF-ß1 was more potent. Importantly, the combination of IFN-γ and PFD had a possibly synergistic/additive effect in inhibiting the TGF-ß1- and PDGF-BB-induced proliferation, migration and differentiation of normal and IPF lung fibroblasts. Furthermore, both drugs reversed TGF-ß1-induced effects on MMP-1, - 2, - 3, - 7, and - 9, while only PFD promoted TIMP-1 and-2 expression and release. CONCLUSIONS: Our findings demonstrate that the antifibrotic effects of IFN-γ and PFD on normal and IPF lung fibroblasts are different and complementary. Combination therapy with inhaled IFN-γ and PFD in IPF is promising and should be further explored in IPF clinical trials.

Extracellular Matrix Metalloproteinase Inducer (EMMPRIN) promotes lung fibroblast proliferation, survival and differentiation to myofibroblasts.

BACKGROUND: Idiopathic pulmonary fibrosis (IPF) is a chronic progressively fatal disease. Extracellular Matrix Metalloproteinase Inducer (EMMPRIN) is a glycosylated transmembrane protein that induces the expression of some matrix metalloproteinase (MMP) in neighboring stromal cells through direct epithelial-stromal interactions. EMMPRIN is highly expressed in type II alveolar epithelial cells at the edges of the fibrotic areas in IPF lung sections. However, the exact role of EMMPRIN in IPF is unknown. METHODS: To determine if EMMPRIN contributes to lung fibroblast proliferation, resistance to apoptosis, and differentiation to myofibroblasts, normal Human lung fibroblasts (NHLF) transiently transfected with either EMMPRIN/GFP or GFP were treated with TGF- ß1 from 0 to 10 ng/ml for 48 h and examined for cell proliferation (thymidine incorporation), apoptosis (FACS analysis and Cell Death Detection ELISA assay), cell migration (Modified Boyden chamber) and differentiation to myofibroblasts using Western blot for α-smooth actin of cell lysates. The effect of EMMPRIN inhibition on NHLF proliferation, apoptosis, migration and differentiation to myofibroblasts after TGF- ß1 treatment was examined using EMMPRIN blocking antibody. We examined the mechanism by which EMMPRIN induces its effects on fibroblasts by studying the ß-catenin/canonical Wnt signaling pathway using Wnt luciferase reporter assays and Western blot for total and phosphorylated ß-catenin. RESULTS: Human lung fibroblasts overexpressing EMMPRIN had a significant increase in cell proliferation and migration compared to control fibroblasts. Furthermore, EMMPRIN promoted lung fibroblasts resistance to apoptosis. Lung fibroblasts overexpressing EMMPRIN showed a significantly increased expression of α- smooth muscle actin, a marker of differentiation to myofibroblasts compared to control cells. TGF-ß1 increased the expression of EMMPRIN in lung fibroblasts in a dose-dependent manner. Attenuation of EMMPRIN expression with the use of an EMMPRIN blocking antibody markedly inhibited TGF-ß1 induced proliferation, migration, and differentiation of fibroblasts to myofibroblasts. EMMPRIN overexpression in lung fibroblasts was found to induce an increase in TOPFLASH luciferase reporter activity when compared with control fibroblasts. CONCLUSION: These findings indicate that TGF-ß1 induces the release of EMMPRIN that activates ß-catenin/canonical Wnt signaling pathway. EMMPRIN overexpression induces an anti-apoptotic and pro-fibrotic phenotype in lung fibroblasts that may contribute to the persistent fibro-proliferative state seen in IPF.

Somatic syndromes, insomnia, anxiety, and stress among sleep disordered breathing patients.

OBJECTIVES: We tested the hypothesis that the prevalence of somatic syndromes, anxiety, and insomnia among sleep disordered breathing (SDB) patients is correlated with their levels of somatic arousal, the symptoms of increased sympathetic nervous system tone under conditions of stress. METHODS: We administered the Body Sensation Questionnaire (BSQ; a 17-item questionnaire with increasing levels of somatic arousal scored 17-85) to 152 consecutive upper airway resistance syndrome (UARS) patients and 150 consecutive obstructive sleep apnea/hypopnea (OSA/H) patients. From medical records, we characterized each patient in terms of the presence of syndromes and symptoms into three categories: somatic syndromes (six syndromes), anxiety (anxiety disorders, nightmares, use of benzodiazepines), and insomnia (sleep onset, sleep maintenance, and use of hypnotics). For the pooled sample of SDB patients, we modeled the correlation of the BSQ score with the presence of each syndrome/symptom parameter within each of the three categories, with adjustment for male vs. female. RESULTS: Mean BSQ scores in females were significantly higher than those in males (32.5 ± 11.1 vs. 26.9 ± 8.2; mean ± SD). Increasing BSQ scores significantly correlated with increasing prevalence rates of somatic syndromes (p < 0.0001), of anxiety (p < 0.0001), and of insomnia (p ≤ 0.0001). In general, females had higher prevalence rates of somatic syndromes and symptoms of anxiety than males at any BSQ score while rates of insomnia were similar. CONCLUSIONS: In patients with SDB, there is a strong association between the level of somatic arousal and the presence of stress-related disorders like somatic syndromes, anxiety, and insomnia.

Somatic arousal and sleepiness/fatigue among patients with sleep-disordered breathing.

OBJECTIVES: In a large clinical sample, we tested the hypothesis that levels of sleepiness and fatigue among upper airway resistance syndrome (UARS) patients are correlated with levels of somatic arousal (SA; reflecting the sympathetic nervous system component of the stress response). We also tested the correlations of post-treatment change in these three parameters, and we extended the investigation to obstructive sleep apnea/hypopnea (OSA/H) patients. METHODS: From 5 years of patient data, we obtained scores on the body sensation questionnaire (BSQ), measuring the level of SA, the fatigue severity scale (FSS), and Epworth sleepiness scale (ESS) for 152 consecutive UARS patients and 150 consecutive OSA/H patients. For each group, we correlated the FSS and ESS scores with the BSQ scores. Among the 45 UARS patients and 49 OSA/H patients treated with nasal CPAP who provided post-treatment data, we correlated change in FSS and ESS scores with change in BSQ scores. RESULTS: Scores on the BSQ, FSS, and ESS for UARS patients and OSA/H patients were comparable. In both UARS and OSA/H patients, both the FSS and ESS scores were positively correlated with the BSQ score. Nasal CPAP use decreased all three questionnaire scores in both patient groups. In the pooled data, changes in FSS were significantly correlated with changes in BSQ. CONCLUSIONS: Our findings confirm our preliminary observations that sleepiness and fatigue among UARS patients are correlated with their level of SA and suggest that the same is true for OSA/H patients. The decrease of SA following treatment suggests that SDB is a cause of SA among patients with UARS and OSA/H.

Effect of intraoperative dobutamine on splanchnic tissue perfusion and outcome after Whipple surgery.

PURPOSE: Splanchnic hypoperfusion during abdominal surgery contributes to postoperative gut sepsis and mortality. Dobutamine is an inotrope with vasodilator properties that improve hepatosplanchnic perfusion. The aim of this study was to examine the effect of intraoperative dobutamine infusion during Whipple surgery on splanchnic perfusion, hemodynamic, and overall postoperative outcome. METHODS: Sixty patients were randomly allocated to receive intraoperatively (3 µg/kg per minute or 5 µg/kg per minute) doses of dobutamine or saline. Baseline measurements included hemodynamic parameters, gastric tonometric parameters, and arterial and mixed venous gases. These patients had a follow-up for development for in-hospital morbidity and mortality. RESULTS: Intraoperative use of dobutamine increased oxygen-derived parameters as evidenced by increased mixed venous oxygen saturation. Tonometered gastric mucosal pH, a surrogate for splanchnic perfusion, increased in patients who received intraoperative dobutamine. Patients in the dobutamine groups demonstrated significant higher heart rates, premature ventricular contraction arrhythmias, and electrocardiographic signs of ischemia. Mean arterial blood pressure demonstrated no significant difference among groups. The overall incidence of postoperative complications was higher in control group 70 % vs 20% to 40% in dobutamine groups. CONCLUSION: Intraoperative use of dobutamine improved global oxygen delivery, splanchnic perfusion, and postoperative outcome after Whipple surgery. These findings may be of clinical importance when the therapeutic goal is to improve gut perfusion.

Angiogenesis is induced by airway smooth muscle strain.

Angiogenesis is an important feature of airway remodeling in both chronic asthma and chronic obstructive pulmonary disease (COPD). Airways in those conditions are exposed to excessive mechanical strain during periods of acute exacerbations. We recently reported that mechanical strain of human airway smooth muscle (HASM) led to an increase in their proliferation and migration. Sustained growth in airway smooth muscle in vivo requires an increase in the nutritional supply to these muscles, hence angiogenesis. In this study, we examined the hypothesis that cyclic mechanical strain of HASM produces factors promoting angiogenic events in the surrounding vascular endothelial cells. Our results show: 1) a significant increase in human lung microvascular endothelial cell (HMVEC-L) proliferation, migration, and tube formation following incubation in conditioned media (CM) from HASM cells exposed to mechanical strain; 2) mechanical strain of HASM cells induced VEGF expression and release; 3) VEGF neutralizing antibodies inhibited the proliferation, migration, and tube formations of HMVEC-L induced by the strained airway smooth muscle CM; 4) mechanical strain of HASM induced a significant increase in hypoxia-inducible factor-1alpha (HIF-1alpha) mRNA and protein, a transcription factor required for VEGF gene transcription; and 5) mechanical strain of HASM induced HIF-1alpha/VEGF through dual phosphatidylinositol 3-kinase (PI3K)/Akt/mammalian target of rapamycin (mTOR) and ERK pathways. In conclusion, exposing HASM cells to mechanical strain induces signal transduction pathway through PI3K/Akt/mTOR and ERK pathways that lead to an increase in HIF-1alpha, a transcription factor required for VEGF expression. VEGF release by mechanical strain of HASM may contribute to the angiogenesis seen with repeated exacerbation of asthma and COPD.

Cyclic mechanical strain-induced proliferation and migration of human airway smooth muscle cells: role of EMMPRIN and MMPs.

Airway smooth muscle (ASM) proliferation and migration are major components of airway remodeling in asthma. Asthmatic airways are exposed to mechanical strain, which contributes to their remodeling. Matrix metalloproteinase (MMP) plays an important role in remodeling. In the present study, we examined if the mechanical strain of human ASM (HASM) cells contributes to their proliferation and migration and the role of MMPs in this process. HASM were exposed to mechanical strain using the FlexCell system. HASM cell proliferation, migration and MMP release, activation, and expression were assessed. Our results show that cyclic strain increased the proliferation and migration of HASM; cyclic strain increased release and activation of MMP-1, -2, and -3 and membrane type 1-MMP; MMP release was preceded by an increase in extracellular MMP inducer; Prinomastat [a MMP inhibitor (MMPI)] significantly decreased cyclic strain-induced proliferation and migration of HASM; and the strain-induced increase in the release of MMPs was accompanied by an increase in tenascin-C release. In conclusion, cyclic mechanical strain plays an important role in HASM cell proliferation and migration. This increase in proliferation and migration is through an increase in MMP release and activation. Pharmacological MMPIs should be considered in the pursuit of therapeutic options for airway remodeling in asthma.

Polymerase chain reaction of pleural biopsy is a rapid and sensitive method for the diagnosis of tuberculous pleural effusion.

BACKGROUND: Tuberculous pleural effusion occurs in 30% of patients with tuberculosis (TB). Rapid diagnosis of a tuberculous pleural effusion would greatly facilitate the management of many patients. Polymerase chain reaction (PCR) has been used to detect Mycobacterium tuberculosis in pleural fluid with highly variable sensitivity. OBJECTIVE: To improve our laboratory diagnosis of tuberculous pleural effusion. METHODS: We applied PCR to detect DNA specific for M tuberculosis in 33 of the studied pleural biopsy specimens using an IS986-based primer that was specific for mycobacterium complex, and compared it to the results of pleural fluid and biopsy cultures performed on either Lowenstein-Jensen (LJ) medium or BACTEC 12B liquid medium (Becton Dickinson Microbiology Systems; Cockeysville, MD), Ziehl-Neelsen (ZN) staining, and histopathology in 45 patients with pleural effusion. RESULTS: Of the 45 patients with pleural effusion who were studied, 26 patients received diagnoses of tuberculous pleural effusion that had been confirmed by either culture and or histopathology, 10 patients received diagnoses of exudative effusion due to causes other than TB, and 9 patients received diagnoses of transudative effusion. Histopathology of the pleural biopsy specimen had a sensitivity of 53.8%. The sensitivity of the ZN staining of pleural fluid and biopsy specimens was 0.0% and 3.8%, respectively. The sensitivity of the culture on both BACTEC 12B liquid medium and LJ medium was higher in pleural biopsy specimens (92.3%) than in pleural fluid specimens (15.4%; p > 0.001). The improvements of the BACTEC culture system improved and shortened the detection time of M tuberculosis in pleural biopsy specimens. PCR of pleural biopsy specimens had 90% sensitivity and 100% specificity. The positive predictive value and the negative predictive value for pleural biopsy specimen cultures were 100% and 90.5% vs 100% and 86.7% for pleural biopsy specimen PCRs. CONCLUSION: The overall accuracy of PCR of pleural biopsy was similar to the results of pleural biopsy culture, however, PCR of the pleural biopsy was much faster in reaching diagnosis. PCR of pleural biopsy is a useful method when used in combination with the BACTEC culture system and histopathologic examination of pleural biopsy to reach a rapid diagnosis of tuberculous pleural effusion.

Nitric oxide and vasoactive intestinal peptide as co-transmitters of airway smooth-muscle relaxation: analysis in neuronal nitric oxide synthase knockout mice.

BACKGROUND: Both vasoactive intestinal peptide (VIP) and nitric oxide (NO) relax airway smooth muscle and are potential co-transmitters of neurogenic airway relaxation. The availability of neuronal NO synthase (nNOS) knockout mice (nNOS-/-) provides a unique opportunity for evaluating NO. OBJECTIVE: To evaluate the relative importance of NO, especially that generated by nNOS, and VIP as transmitters of the inhibitory nonadrenergic, noncholinergic (NANC) system. STUDY DESIGN: In this study, we compared the neurogenic (tetrodotoxin-sensitive) NANC relaxation of tracheal segments from nNOS-/- mice and control wild-type mice (nNOS(+/+)), induced by electrical field stimulation (EFS). We also examined the tracheal contractile response to methacholine and its relaxant response to VIP. RESULTS: EFS (at 60 V for 2 ms, at 10, 15, or 20 Hz) dose-dependently reduced tracheal tension, and the relaxations were consistently smaller (approximately 40%) in trachea from nNOS-/- mice than from control wild-type mice (p < 0.001). VIP (10(- 8) to 10(-6) mol/L) induced concentration-dependent relaxations that were approximately 50% smaller in nNOS-/- tracheas than in control tracheas. Methacholine induced concentration-dependent contractions that were consistently higher in the nNOS-/- tracheas relative to wild-type mice tracheas (p > 0.05). CONCLUSION: Our data suggest that, in mouse trachea, NO is probably responsible for mediating a large (approximately 60%) component of neurogenic NANC relaxation, and a similar (approximately 50%) component of the relaxant effect of VIP. The results imply that NO contributes significantly to neurogenic relaxation of mouse airway smooth muscle, whether due to neurogenic stimulation or to the neuropeptide VIP.