Gustavo Juan

Effect of carbon dioxide on diaphragmatic function in human beings

We studied the effects of acute changes in the partial pressure of arterial carbon dioxide on diaphragmatic contractility and performance in four normal men. To study contractility we measured the ability of the diaphragm to generate pressure at a given level of excitation by determining the relation between the electrical activity of the diaphragm and transdiaphragmatic pressure during a voluntary quasi-isometric inspiratory effort carried out at different levels of end-tidal carbon dioxide. Our results show that contractility was reduced with hypercapnia (when end-tidal carbon dioxide was 7.5 per cent or higher), although hypocapnia (end-tidal carbon dioxide, 3 per cent) had no effect on diaphragmatic contractility. We also studied the development of diaphragmatic fatigue before and during carbon dioxide breathing. Subjects were studied at the same diaphragmatic tension-time index, a value analogous to the more familiar myocardial tension-time index, while the same inspiratory flow was maintained. Electromyographic signs of fatigue appeared at a lower tension-time index during hypercapnia than during normocapnia, indicating that endurance is diminished during hypercapnia. These findings show that acute respiratory acidosis equivalent to an arterial carbon dioxide tension of about 54 mm Hg decreases the contractility and endurance time of the diaphragm in human beings.

Sphingosine-1-phosphate is increased in patients with idiopathic pulmonary fibrosis and mediates epithelial to mesenchymal transition

Background Idiopathic pulmonary fibrosis (IPF) is characterised by the aberrant epithelial to mesenchymal transition (EMT) and myofibroblast accumulation. Sphingosine-1-phosphate (S1P) and sphingosine kinase 1 (SPHK1) have been implicated in lung myofibroblast transition, but their role in EMT and their expression in patients with IPF is unknown. Methods and results S1P levels were measured in serum (n=27) and bronchoalveolar lavage (BAL; n=15) from patients with IPF and controls (n=30 for serum and n=15 for BAL studies). SPHK1 expression was measured in lung tissue from patients with IPF (n=12) and controls (n=15). Alveolar type II transformation into mesenchymal cells was studied in response to S1P (10−9–10−5 M). The median (IQR) of S1P serum levels was increased in patients with IPF (1.4 (0.4) μM) versus controls (1 (0.26) μM; p<0.0001). BAL S1P levels were increased in patients with IPF (1.12 (0.53) μM) versus controls (0.2 (0.5); p<0.0001) and correlated with diffusion capacity of the lung for carbon monoxide, forced expiratory volume in 1 s and forced vital capacity (Spearmans r=−0.87, −0.72 and −0.68, respectively) in patients with IPF. SPHK1 was upregulated in lung tissue from patients with IPF and correlated with α-smooth muscle actin, vimentin and collagen type I (Spearmans r=0.82, 0.85 and 0.72, respectively). S1P induced EMT in alveolar type II cells by interacting with S1P2 and S1P3, as well as by the activation of p-Smad3, RhoA-GTP, oxidative stress and transforming growth factor-β1 (TGF-β1) release. Furthermore, TGF-β1-induced EMT was partially conducted by the S1P/SPHK1 activation, suggesting crosstalk between TGF-β1 and the S1P/SPHK1 axis. Conclusions S1P is elevated in patients with IPF, correlates with the lung function and mediates EMT.

Cigarette smoke-induced pulmonary endothelial dysfunction is partially suppressed by sildenafil.

Cigarette smoke mediated oxidative stress and endothelial dysfunction are important processes in the pathogenesis of several lung disorders. In this study we evaluated the effect of PDE5 inhibition on pulmonary artery endothelial dysfunction induced by cigarette smoke in vitro. Human pulmonary artery endothelial cells (HPAEC) were incubated in the absence or presence of PDE5 inhibitor sildenafil (10 nM-1 microM), PKG agonist 8-Br-cGMP (1mM), or the antioxidants dyphenyleneiodonium (DPI 1 microM) and N-acetylcysteine (NAC 1mM) for 30 min. Then, cigarette smoke extract (CSE) was added for 24h. CSE (2.5-10%)-induced ROS generation was suppressed by DPI, and partially reversed by sildenafil and 8-Br-cGMP. Decreases in intracellular levels of cGMP and extracellular NO induced by CSE were reversed by sildenafil and DPI. Furthermore, CSE-induced pg91(phox) and PDE5 mRNA overexpression were suppressed by both sildenafil and DPI. CSE (2.5-10%) induced upregulation of IL-6, IL-8 and Ang-2, and decreased Ang-1 expression in parallel to apoptosis which were partially suppressed by sildenafil, 8-Br-cGMP, DPI and NAC. This study demonstrates that PDE5 inhibition attenuates the oxidant burden and the inflammatory and remodeling effects of CSE in human HPAEC which may contribute to the therapeutic value of PDE5 inhibitors for pulmonary disorders coursing with endothelial dysfunction.

Cigarette smoke exposure up‐regulates endothelin receptor B in human pulmonary artery endothelial cells: molecular and functional consequences

BACKGROUND AND PURPOSE Pulmonary arteries from smokers and chronic obstructive pulmonary disease patients show abnormal endothelium‐dependent vascular reactivity. We studied the effect of cigarette smoke extract (CSE) on endothelin receptor B (ETB) expression in human pulmonary artery endothelial cells (HPAECs) and its role in endothelial dysfunction.

Neutrophil Activation in Severe, Early-Onset COPD Patients versus Healthy Non-Smoker Subjects in vitro: Effects of Antioxidant Therapy

Background: Neutrophils and oxidative stress have been implicated in the pathogenesis of COPD. Severe, early-onset COPD is characterized by a rapid decline in the lung function at an early age; however, nothing is known about neutrophil activation in COPD patients. Objectives: The aim of this study was to evaluate peripheral blood neutrophil activation in severe, early-onset COPD patients versus healthy non-smokers and the effect of N-acetyl-L-cysteine (NAC) on neutrophil activation in vitro. Methods: Neutrophils were isolated from 15 severe, early-onset COPD patients and 15 age-matched healthy subjects and stimulated with N-formyl- Met-Leu-Phe (fMLP) in the presence or absence of NAC (10 µM to 10 mM). Neutrophil chemotaxis, elastase release, reactive oxygen species (ROS), intracellular thiols and apoptosis were measured by Boyden chamber, spectrofluorometry, CMFDA and H2DCF-DA dyes and by annexin V-FITC, respectively. Results: Chemotaxis of peripheral blood neutrophils from COPD patients in response to fMLP was 30% more increased than that observed in healthy subjects. Elastase release in response to fMLP was 2-fold higher in neutrophils from COPD patients versus healthy subjects. Intracellular thiol levels were 30% lower in COPD and ROS was approximately 30% higher in COPD versus healthy neutrophils. Spontaneous apoptosis showed no differences in both groups of patients and fMLP-induced apoptosis was higher in COPD. Pre-treatment with the antioxidant NAC effectively attenuated neutrophil chemotaxis, elastase release and ROS as well as effectively increased thiol levels in COPD. Conclusions: Neutrophils in severe, early-onset COPD patients are highly activated and this is alleviated by NAC in vitro.

Direct effect of cigarette smoke on human pulmonary artery tension

The effect of chronic cigarette smoke on pulmonary artery (PA) tension has been studied extensively; nevertheless, the direct effect of cigarette smoke is poorly understood. We investigated the direct effect of cigarette smoke extract (CSE) on PA tension in non-smokers, smokers, and COPD patients in vitro. PA samples from 35 patients who underwent lung resection were examined by measuring isometric tension in response to increasing serotonin concentrations. CSE dose dependently inhibited the response to serotonin in smokers and COPD patients, and to a lesser extent in non-smokers. CSE-induced relaxation was similarly inhibited by the nonspecific nitric oxide synthase (NOS) inhibitor l-NOARG and the specific inducible NOS (iNOS) inhibitor l-NIL, mainly in non-smokers and smokers, and to a lesser extent in COPD patients. Immunostaining of iNOS in PA samples was greater for smokers and COPD patients compared with non-smokers, which explains the lesser effect of CSE on PA tension in non-smokers. Moreover, CSE induced the release of nitrite via iNOS in human PA smooth muscle cells. In conclusion, CSE inhibition of serotonin-induced PA contraction was mediated mainly by iNOS in non-smokers, smokers, and COPD patients, but in different ways, which may be explained by differential iNOS expression in the PA of these patients.

Modulatory effects of N-acetyl-l-cysteine on human eosinophil apoptosis

Eosinophils are oxidant-sensitive cells considered relevant in allergic inflammation. The present study aimed to examine the effects of the antioxidant N-acetyl-l-cysteine (NAC) on constitutive and cytokine-delayed apoptosis in human isolated eosinophils. Human eosinophils were purified from the blood of healthy donors by a magnetic separation system. Apoptosis and cellular glutathione were assessed by cytofluorometric analysis and nuclear factor (NF)-κB binding activity assessed by electrophoresis mobility shift assay. The rate of spontaneous apoptosis of human eosinophils after 24 h culture, as assessed by annexin-V-positive staining, was mean±sem 48.2±1.4%, n = 5. Granulocyte-macrophage colony-stimulating factor (GM-CSF; 10 ng·mL−1) decreased apoptosis to 19.4±1.8%, n = 5. NAC (5 mM) inhibited spontaneous apoptosis (33.6±2.7%, n = 5) but augmented apoptosis in the presence of GM-CSF (30.9±1.5%, n = 5). NAC (5 mM) also increased the rate of apoptosis in the presence of tumour necrosis factor (TNF)-α (10 ng·mL−1) and interleukin-5 (5 ng·mL−1). NAC (5 mM) increased eosinophil glutathione content. The increase in eosinophil NF-κB binding activity induced by GM-CSF and TNF-α was suppressed by NAC. In conclusion, N-acetylcysteine modulates eosinophil apoptosis by inhibiting constitutive apoptosis but reversing the survival effect produced by inflammatory cytokines in human eosinophils.

Bosentan inhibits cigarette smoke-induced endothelin receptor expression in pulmonary arteries

The endothelin (ET) system contributes to lung vascular tension and remodelling in smokers and chronic obstructive pulmonary disease (COPD) patients. This study examined the effect of cigarette smoke (CS) on ET receptor A (ETA) and B (ETB) expression in human pulmonary artery smooth muscle cells (HPASMCs) and human small intrapulmonary arteries, as well as their functional consequences. CS extract (CSE) increased ETA and ETB expression in HPASMCs and small intrapulmonary arteries, which was attenuated by bosentan, the ETA antagonist BQ123 and the ETB antagonist BQ788, and by blocking ET-1 with a monoclonal antibody against ET-1, suggesting a feed-forward mechanism mediated by ET-1 release. ET receptor (ETR) antagonism attenuated the CSE-induced HPASMC proliferation. Furthermore, CSE exposure increased the acute ET-1-induced small intrapulmonary artery contraction, which was attenuated by bosentan, BQ123 and BQ788. Pulmonary arteries from smokers and COPD patients showed a higher expression of ETA and ETB than those of nonsmoker patients. These results show a novel mechanism by which ETR blockade attenuates CS-induced ETR overexpression and, subsequently, small intrapulmonary artery tension. These data may be of potential value to explain therapeutic effects of bosentan in some forms of disproportionate pulmonary hypertension in COPD patients.

Diaphragmatic paralysis following minor cervical trauma

Two asthmatic patients developed unilateral diaphragmatic paralysis from phrenic nerve injury, in one case following cervical chiropractic manipulation and in the other after a motorcycle accident. Both presented with increased dyspnea and orthopnea. Diagnosis, severity, and level of the lesion were established by neurophysiological methods, which are preferred to chest radiography and diaphragmatic ultrasonography. In spite of only partial electrophysiological recovery of the nerve, both patients were asymptomatic 1 year later. Muscle Nerve, 2007

PGC-1α induction in pulmonary arterial hypertension.

Idiopathic Pulmonary arterial hypertension (IPAH) is characterized by the obstructive remodelling of pulmonary arteries, and a progressive elevation in pulmonary arterial pressure (PAP) with subsequent right-sided heart failure and dead. Hypoxia induces the expression of peroxisome proliferator activated receptor γ coactivator-1α (PGC-1α) which regulates oxidative metabolism and mitochondrial biogenesis. We have analysed the expression of PGC-1α, cytochrome C (CYTC), superoxide dismutase (SOD), the total antioxidant status (TAS) and the activity of glutathione peroxidase (GPX) in blood samples of IPAH patients. Expression of PGC-1α was detected in IPAH patients but not in healthy volunteers. The mRNA levels of SOD were lower in IPAH patients compared to controls (3.93 ± 0.89 fold change). TAS and GPX activity were lower too in patients compared to healthy donors, (0.13 ± 0.027 versus 0.484 ± 0.048 mM and 56.034 ± 10.37 versus 165.46 ± 11.38 nmol/min/mL, resp.). We found a negative correlation between expression levels of PGC-1α and age, PAP and PVR, as well as a positive correlation with CI, PaO2, mRNA levels of CYTC and SOD, TAS and GPX activity. These results taken together are indicative of the possible role of PGC-1α as a potential biomarker of the progression of IPAH.