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In JoVE (1)
Other Publications (6)
- Journal of Applied Physiology (Bethesda, Md. : 1985)
- American Journal of Physiology. Lung Cellular and Molecular Physiology
- American Journal of Physiology. Lung Cellular and Molecular Physiology
- American Journal of Physiology. Lung Cellular and Molecular Physiology
- Journal of Aerosol Medicine and Pulmonary Drug Delivery
- Canadian Respiratory Journal : Journal of the Canadian Thoracic Society
Articles by Asma Yaghi in JoVE
JoVE General
Primary Human Bronchial Epithelial Cells Grown from Explants
Medicine, Faculty of Health Sciences, McMaster University
Here we describe a detailed method for growing primary human bronchial epithelial cells from explants of human bronchial airway tissue including differentiated growth on an air-liquid interface. This method provides an abundant source of primary cells for investigating the role of the airway epithelium in human lung health and disease.
Other articles by Asma Yaghi on PubMed
Delayed Rectifier Potassium Channels Contribute to the Depressed Pulmonary Artery Contractility in Pneumonia
We investigated the role of K(+) channels in the attenuated pulmonary artery (PA) contractility characteristic of acute Pseudomonas pneumonia. Contractility of PA rings from the lungs of control or pneumonia rats was assessed in vitro by obtaining cumulative concentration-response curves to the contractile agonists KCl, phenylephrine, or PGF(2 alpha) on PA rings before and after treatment with K(+) channel blockers. In rings from pneumonia rats, paxilline (10 microM), tetraethylammonium (2 mM) (blockers of large-conductance Ca(2+)-activated K(+) channels), and glybenclamide (ATP-sensitive K(+) channel blocker, 80 microM) had no significant effect on the attenuated contractile responses to KCl, phenylephrine, and PGF(2 alpha). However, 4-aminopyridine (2 mM), a blocker of voltage-gated K(+) channels (delayed rectifier K(+) channel) reversed this depressed contractility. Therefore, large-conductance Ca(2+)-activated K(+) and ATP-sensitive K(+) channels do not contribute to the attenuated PA contractility observed in this model of acute pneumonia. In contrast, 4-aminopyridine enhances contraction in PA rings from pneumonia lungs, consistent with involvement of a voltage-gated K(+) channel in the depressed PA contractility in acute pneumonia. Unraveling the precise mechanism of attenuated contractility in pneumonia could lead to innovative therapies for the pulmonary vascular abnormalities associated with this disease.
Pulmonary Cytochrome P-450 2J4 is Reduced in a Rat Model of Acute Pseudomonas Pneumonia
We previously reported that the levels of epoxyeicosatrienoic acids (EETs) and 20-hydroxyeicosatetraenoic acid (20-HETE) are depressed in microsomes prepared from lungs of rats with acute Pseudomonas pneumonia. We also showed a potential role for cytochrome P-450 (CYP) metabolites of arachidonic acid (AA) in contractile responses of both normal pulmonary arteries and pulmonary arteries from rats with pneumonia. The CYP2J subfamily enzymes (endogenous source of EETs and HETEs) are constitutively expressed in human and rat lungs where they are localized in vascular smooth muscle and endothelium. The purpose of this study was to determine if CYP2J proteins are modified in pneumonia. Pseudomonas organisms were injected via a tracheostomy in the lungs of rats. Later (44 h), lungs were frozen, and microsomes were prepared from pneumonia and control rat lung homogenates. Lung microsomal proteins were then immunoblotted with anti-CYP2B1/2B2, anti-CYP4A, anti-CYP2J9pep2 (which reacts with rat CYP2J3), anti-CYP2J6pep1 (which reacts with rat CYP2J4), anti-CYP2J2pep4, or anti-CYP2J2pep3 (both of which react with all known CYP2J isozymes). Western blotting revealed a prominent 55-kDa band with anti-CYP2J2pep3, anti-CYP2J2pep4, and anti-CYP2J6pep1 (but not anti-CYP2J9pep2) that was reduced in pneumonia compared with control lung microsomes. The CYP2B bands (51-52 kDa) were less prominent and not different between pneumonia and control lungs. CYP4A proteins (20-HETE sources) were not detected in rat lung microsomes. Therefore, rat lung contains a protein with immunological characteristics similar to CYP2J4, and this CYP is reduced after pneumonia. We speculate that CYP2J (but not CYP2B) enzymes and their AA metabolic products (EETs) are involved in the modulation of pulmonary vascular tone in pneumonia in rats.
Excess Nitric Oxide Decreases Cytochrome P-450 2J4 Content and P-450-dependent Arachidonic Acid Metabolism in Lungs of Rats with Acute Pneumonia
Recently, we demonstrated that pulmonary CYP2J4 content, a prominent source of EETs and HETEs formation in rat lungs, is reduced in pneumonia. Therefore, the purpose of this study was to determine the role of iNOS-derived NO in reduced pulmonary CYP2J4 protein content and decreased CYP metabolites in pneumonia. Rats were randomized to control, control plus 1400W (iNOS inhibitor), pneumonia, and pneumonia plus 1400W groups. Pseudomonas organisms were injected into lungs of pneumonia rats. At 40 h after surgery, rats were treated with either saline or 1400W for 4 h before death. Venous plasma samples were obtained for measuring nitrites/nitrates (NOx). There was no significant effect of 1400W on blood pressure measured in control or pneumonia rats, whereas 1400W reduced the elevated plasma NOx levels in pneumonia rats by half. CYP primary metabolites of AA formed at significantly lower rates in pulmonary microsomes from pneumonia rats compared with control rats. Treatment of pneumonia rats with 1400W resulted in a significant increase in the rate of formation of pulmonary EETs and omega-terminal HETEs compared with untreated pneumonia rats. The reduction in CYP2J4 protein content in pneumonia lung microsomes was also partially prevented by 1400W. Therefore, excess NO from iNOS decreases the pulmonary production of EETs and omega-HETEs in acute pneumonia. Inhibition of iNOS restores CYP2J4 protein content and CYP activity in acute pneumonia, indicating an important NO-CYP interaction in pulmonary responses to infection. We speculate CYP2J4 and its AA metabolites are involved in the modulation of pulmonary function in health and disease.
Constrictor-induced Translocation of NFAT3 in Human and Rat Pulmonary Artery Smooth Muscle
The transcription factor nuclear factor of activated T cells (NFAT) resides in the cytoplasm in resting cells and upon stimulation is dephosphorylated, translocates to the nucleus, and becomes transcriptionally active. NFAT is commonly activated by stimulation of receptors coupled to Ca(2+) mobilization; however, little is known about the regulation of NFAT in pulmonary vascular smooth muscle. The aim of this study was to investigate regulation of NFAT in human and rat intralobar pulmonary artery by two constrictors: phenylephrine (PE) and 20-hydroxyeicosatetraenoic acid (20-HETE), a cytochrome P-450 metabolite formed endogenously in lungs. Immunostaining of smooth muscle cells revealed cytoplasmic localization of NFAT in untreated cells, and PE or 20-HETE induced translocation to the nucleus, with maximal effect at 30 min. Cyclosporin A and FK-506 (both 1 microM) inhibited NFAT translocation, indicating involvement of calcineurin. Moreover, the Rho-kinase blocker Y-27632 prevented translocation. Translocation of NFAT was confirmed by Western blots, with NFAT3 the prominent isoform in pulmonary artery. Constrictors caused calcineurin-sensitive translocation of NFAT to nuclei in intact arteries, demonstrating regulation in native tissue. To investigate a role for Ca(2+), cells were loaded with fura-2. Whereas PE caused an acute transient rise of [Ca(2+)](i), 20-HETE caused a prolonged low amplitude rise of [Ca(2+)](i). The involvement of Rho-kinase in PE- and 20-HETE-induced NFAT3 translocation in pulmonary artery suggests a level of control not previously recognized in smooth muscle. Constrictors of the pulmonary vasculature not only cause acute responses but also activate NFAT, which may alter gene expression in pulmonary health and disease.
The Direct Effect of Hyperosmolar Agents on Ciliary Beating of Human Bronchial Epithelial Cells
Abstract Background: Inhalation of hypertonic saline and mannitol improve mucociliary clearance in patients with bronchiectasis, but little is known about how the relative osmotic strengths of these compounds affect ciliary beat frequency (CBF) of ciliated human bronchial epithelial cells (HBEC). Our aim was to compare in vitro the direct effects of osmotically equivalent solutions on CBF of HBEC. Methods: HBEC were acutely (10, 30 min) exposed to comparable osmolar solutions of saline (0.03-0.48%), mannitol (0.19-3%) and dextran (10%-39.39%). Effects of higher % solutions, reversibility of responses, and prolonged treatments (15-20 h) were also compared. CBF was measured using digital videomicroscopy at baseline and at all time points. Results: CBF of HBEC increased significantly after acute exposure to mannitol (0.19%, 0.38%), decreased with dextran and remained unchanged with saline. Prolonged exposure to mannitol at high (3%, 6%) osmolar concentrations reversibly suppressed CBF. In comparison, acute and prolonged treatment with 39.39% dextran (equivalent to 3% mannitol) reduced CBF irreversibly. Furthermore, acute and prolonged treatment with 1% saline (equivalent to 6% mannitol) suppressed CBF with only the acute effect being reversible. Conclusions: Mannitol had a direct osmolarity-independent cilio-stimulatory effect at lower % solutions and a reversible cilio-inhibitory effect at higher % solutions, and prolonged exposure to mannitol inhibited CBF reversibly. Both the acute and prolonged effects of mannitol compared to dextran and saline on CBF of HBEC in vitro, imply a unique mechanism of action for mannitol on ciliary beating and might contribute to the improved clearance observed in mannitol-treated patients.
Cystic Fibrosis Transmembrane Conductance Regulator Gene Abnormalities in Patients with Asthma and Recurrent Neutrophilic Bronchitis
The present case series describes four patients with asthma, airway hyperresponsiveness and neutrophilic bronchitis who harboured abnormal cystic fibrosis transmembrance conductance regulator (CFTR) gene mutations. It serves both to alert clinicians to consider CFTR-related disease in both young and elderly patients with persistent neutrophilic bronchitis, and to highlight the potential utility of future genetic testing for CFTR abnormalities in patients with asthma and recurrent bronchitis or pansinusitis, and the role of nebulized hypertonic saline as a therapeutic option in these patients.
