Effect of airway acidosis and alkalosis on airway vascular smooth muscle responsiveness to albuterol.

BACKGROUND: In vitro and animal experiments have shown that the transport and signaling of ß2-adrenergic agonists are pH-sensitive. Inhaled albuterol, a hydrophilic ß2-adrenergic agonist, is widely used for the treatment of obstructive airway diseases. Acute exacerbations of obstructive airway diseases can be associated with changes in ventilation leading to either respiratory acidosis or alkalosis thereby affecting albuterol responsiveness in the airway. The purpose of this study was to determine if airway pH has an effect on albuterol-induced vasodilation in the airway. METHODS: Ten healthy volunteers performed the following respiratory maneuvers: quiet breathing, hypocapnic hyperventilation, hypercapnic hyperventilation, and eucapnic hyperventilation (to dissociate the effect of pH from the effect of ventilation). During these breathing maneuvers, exhaled breath condensate (EBC) pH and airway blood flow response to inhaled albuterol (ΔQ̇aw) were assessed. RESULTS: Mean ± SE EBC pH (units) and ΔQ̇aw (µl.min(-1).mL(-1)) were 6.4 ± 0.1 and 16.8 ± 1.9 during quiet breathing, 6.3 ± 0.1 and 14.5 ± 2.4 during eucapnic hyperventilation, 6.6 ± 0.2 and -0.2 ± 1.8 during hypocapnic hyperventilation (p = 0.02 and <0.01 vs. quiet breathing), and 5.9 ± 0.1 and 2.0 ± 1.5 during hypercapnic hyperventilation (p = 0.02 and <0.02 vs quiet breathing). CONCLUSIONS: Albuterol responsiveness in the airway as assessed by ΔQ̇aw is pH sensitive. The breathing maneuver associated with decreased and increased EBC pH both resulted in a decreased responsiveness independent of the level of ventilation. These findings suggest an attenuated response to hydrophilic ß2-adrenergic agonists during airway disease exacerbations associated with changes in pH. TRIAL REGISTRATION: Registered at clinicaltrials.gov: NCT01216748 .

Hyaluronan and layilin mediate loss of airway epithelial barrier function induced by cigarette smoke by decreasing E-cadherin.

Cigarette smoke (CigS) exposure is associated with increased bronchial epithelial permeability and impaired barrier function. Primary cultures of normal human bronchial epithelial cells exposed to CigS exhibit decreased E-cadherin expression and reduced transepithelial electrical resistance. These effects were mediated by hyaluronan (HA) because inhibition of its synthesis with 4-methylumbelliferone prevented these effects, and exposure to HA fragments of <70 kDa mimicked these effects. We show that the HA receptor layilin is expressed apically in human airway epithelium and that cells infected with lentivirus expressing layilin siRNAs were protected against increased permeability triggered by both CigS and HA. We identified RhoA/Rho-associated protein kinase (ROCK) as the signaling effectors downstream layilin. We conclude that HA fragments generated by CigS bind to layilin and signal through Rho/ROCK to inhibit the E-cadherin gene and protein expression, leading to a loss of epithelial cell-cell contact. These studies suggest that HA functions as a master switch protecting or disrupting the epithelial barrier in its high versus low molecular weight form and that its depolymerization is a first and necessary step triggering the inflammatory response to CigS.

MCP-1/CCR2B-dependent loop upregulates MUC5AC and MUC5B in human airway epithelium.

Cigarette smoke represents a major risk factor for the development of chronic obstructive pulmonary disease (COPD), a respiratory condition associated with airflow obstruction, mucus hypersecretion, chronic inflammation, and upregulation of inflammatory mediators such as the monocyte chemotactic protein-1 (MCP-1). MCP-1 through its receptor CCR2 induces chemotaxis and activates (44/42)MAPK, a kinase known to play a key role in mucin regulation in bronchial epithelium. In the present study we used differentiated primary cultures of normal human bronchial epithelial (NHBE) cells to test whether MCP-1 through its receptor CCR2 induces mucin upregulation. We have provided evidence that NHBE cells release MCP-1 to the epithelial surface and express the CCR2B isoform of the receptor mainly at the apical pole. In addition, we found that MCP-1 has a novel function in airway epithelium, increasing the two major airway mucins MUC5AC and MUC5B, an effect mediated, at least in part, by a cascade of events initiated by interaction of its receptor CCR2B with G(q) subunits in caveolae, followed by PLCß, PKC, and (44/42)MAPK activation. We also have shown that MCP-1 is able to induce its own expression using the same receptor but through a different pathway that involves RhoA GTPase. Furthermore, we found that a single exposure to MCP-1 is enough to induce MCP-1 secretion and sustained mucin upregulation up to 7 days after initial exposure, an effect mediated by CCR2B as confirmed using short hairpin RNA. These results agree with our data in smoker's airway epithelium, where CCR2B is present in MUC5AC- and MUC5B-expressing cells and augmented MCP-1 expression is associated with increased MUC5AC and MUC5B immunolabeling, suggesting that the mechanisms described in primary cell cultures in the present study are operative in vivo. Therefore, therapeutic approaches targeting MCP-1/CCR2B may be useful in preventing not only influx of inflammatory cells to the airways but also mucus hypersecretion and goblet cell hyperplasia.

Reactive oxygen species and hyaluronidase 2 regulate airway epithelial hyaluronan fragmentation.

Hyaluronidase 2 (Hyal2) is a hyaluronan (HA)-degrading enzyme found intracellularly or/and anchored to the plasma membrane through glycosylphosphatidylinositol (GPI). Normal human bronchial epithelial cells (NHBE) grown at the air-liquid interphase (ALI), treated with PI-specific phospholipase C (PI-PLC), exhibited increased Hyal activity in secretions and decreased protein and activity on the apical membrane, confirming that GPI-anchored Hyal2 is expressed in NHBE cells and it remains active in its soluble form. We have reported that HA degradation was mediated by reactive oxygen species (ROS) in human airways. Here we show that ROS increase Hyal2 expression and activity in NHBE cells and that the p38MAPK signaling pathway is involved in this effect. Hyal2 induction was confirmed by using small interfering RNA (siRNA) expressing lentivirus. These in vitro findings correlated in vivo with smokers, where increased Hyal2 immunoreactivity in the epithelium was associated with augmented levels of HA and the appearance of low molecular mass HA species in bronchial secretions. In summary, this work provides evidence that ROS induce Hyal2, suggesting that Hyal2 is likely responsible for the sustained HA fragmentation in the airway lumen observed in inflammatory conditions associated with oxidative stress.

Hyaluronan fragments/CD44 mediate oxidative stress-induced MUC5B up-regulation in airway epithelium.

Mucus hypersecretion with elevated MUC5B mucin production is a pathologic feature in many airway diseases associated with oxidative stress. In the present work, we evaluated MUC5B expression in airways and in primary cultures of normal human bronchial epithelial (NHBE) cells, as well as the mechanisms involved in its regulation. We found that oxidative stress generated by cigarette smoke or reactive oxygen species (ROS) induces MUC5B up-regulation in airway epithelium from smokers and in NHBE cells, respectively. We have previously shown that ROS-induced MUC5AC expression in NHBE cells is dependent on hyaluronan depolymerization and epidermal growth factor receptor (EGFR)/mitogen-activated protein kinase (MAPK) activation. Since hyaluronan fragments can activate MAPK through the hyaluronan receptor CD44, and CD44 heterodimerizes with EGFR, we tested whether ROS and/or hyaluronan fragments induce MUC5B mRNA and protein expression through CD44/EGFR. We found that ROS promotes CD44/EGFR interaction, EGFR/MAPK activation, and MUC5B up-regulation that are prevented by blocking CD44 and/or EGFR. These results were mimicked by hyaluronan fragments. In summary, our results show that oxidative stress in vivo (cigarette smoke) or in vitro (ROS) induces MUC5B up-regulation. This ROS-induced MUC5B expression requires CD44 as well as EGFR and MAPK activation. In addition, we also provide evidence that hyaluronan fragments are sufficient to induce CD44/EGFR interaction and downstream signaling that results in MUC5B up-regulation, suggesting that hyaluronan depolymerization during inflammatory responses could be directly involved in the induction of mucus hypersecretion.

Epidermal growth factor receptor activation by epidermal growth factor mediates oxidant-induced goblet cell metaplasia in human airway epithelium.

Mucus overproduction in inflammatory and obstructive airway diseases is associated with goblet cell (GC) metaplasia in airways. Although the mechanisms involved in GC metaplasia and mucus hypersecretion are not completely understood, association with oxidative stress and epidermal growth factor receptor (EGFR) signaling has been reported. To explore the mechanisms involved in oxidative stress-induced GC metaplasia, cultures of differentiated normal human bronchial epithelial cells grown at the air-liquid interface were exposed to reactive oxygen species (ROS) generated by xanthine/xanthine oxidase. EGFR activation and signaling was assessed by measuring EGF and transforming growth factor-alpha release and EGFR and (44/42)MAPK phosphorylation. The GC population was evaluated by confocal microscopy. ROS-induced EGFR activation resulted in GC proliferation and increased MUC5AC gene and protein expression. Signaling was due to pro-EGF processing by tissue kallikrein (TK), which was activated by ROS-induced hyaluronan breakdown. It was inhibited by catalase, a TK inhibitor, and EGF-blocking antibodies. Exposure to recombinant TK mimicked the ROS effects, increasing the expression of MUC5AC and lactoperoxidase. In addition, ROS induced the antiapoptotic factor Bcl-2 in a TK-dependent fashion. In conclusion, ROS-induced GC metaplasia in normal human bronchial epithelial cells is associated with HA depolymerization and EGF processing by TK followed by EGFR signaling, suggesting that increases in TK activity could contribute to GC metaplasia and mucus hypersecretion in diseases such as asthma and chronic bronchitis. The data also suggest that increases in GC population could be sustained by the associated upregulation of Bcl-2 in airway epithelial cells.

Identification of glycosaminoglycans in human airway secretions.

Glycosaminoglycans (GAGs), known to be present in airway mucus, are macromolecules with a variety of structural and biological functions. In the present work, we used fluorophore-assisted carbohydrate electrophoresis (FACE) to identify and relatively quantify GAGs in human tracheal aspirates (HTA) obtained from healthy volunteers. Primary cultures of normal human bronchial epithelial (NHBE) and submucosal gland (SMG) cells were used to assess their differential contribution to GAGs in mucus. Distribution was further assessed by immunofluorescence in human trachea tissue sections and in cell cultures. HTA samples contained keratan sulfate (KS), chondroitin/dermatan sulfate (CS/DS), and hyaluronan (HA), whereas heparan sulfate (HS) was not detected. SMG cultures secreted CS/DS and HA, CS/DS being the most abundant GAGs in these cultures. NHBE cells synthesized KS, HA, and CS/DS. Confocal microscopy showed that KS was exclusively found at the apical border of NHBE cells and on the apical surface of ciliated epithelial cells in tracheal tissues. CS/DS and HA were present in both NHBE and SMG cells. HS was only found in the extracellular matrix in trachea tissue sections. In summary, HTA samples contain KS, CS/DS, and HA, mirroring a mixture of secretions originated in surface epithelial cells and SMGs. We conclude that surface epithelium is responsible for most HA and all KS present in secretions, whereas glands secrete most of CS/DS. These data suggest that, in diseases where the contribution to secretions of glands versus epithelial cells is altered, the relative concentration of individual GAGs, and therefore their biological activities, will also be affected.

Ghrelin increases anxiety-like behavior and memory retention in rats.

Ghrelin is a peptide found in the hypothalamus and stomach that stimulates food intake and whose circulating concentrations are affected by nutritional state. Very little is known about other central behavioral effects of ghrelin, and thus, we investigated the effects of ghrelin on anxiety and memory retention. The peptide was injected intracerebroventricularly in rats and we performed open-field, plus-maze, and step-down tests (inhibitory avoidance). The administration of ghrelin increased freezing in the open field and decreased the number of entries into the open spaces and the time spent on the open arms in the plus-maze, indicating an anxiogenic effect. Moreover, the peptide increased in a dose-dependent manner the latency time in the step-down test. A rapid and prolonged increase in food intake was also observed. Our results indicate that ghrelin induces anxiogenesis in rats. Moreover, we show for the first time that ghrelin increases memory retention, suggesting that the peptide may influence processes in the hippocampus.