Nobuaki Hirota

Mechanisms of airway remodeling.

Airway remodeling comprises the structural changes of airway walls, induced by repeated injury and repair processes. It is characterized by the changes of tissue, cellular, and molecular composition, affecting airway smooth muscle, epithelium, blood vessels, and extracellular matrix. It occurs in patients with chronic inflammatory airway diseases such as asthma, COPD, bronchiectasis, and cystic fibrosis. Airway remodeling is arguably one of the most intractable problems in these diseases, leading to irreversible loss of lung function. Current therapeutics can ameliorate inflammation, but there is no available therapy proven to prevent or reverse airway remodeling, although reversibility of airway remodeling is suggested by studies in animal models of disease. Airway remodeling is often considered the result of longstanding airway inflammation, but it may be present to an equivalent degree in the airways of children with asthma, raising the necessity for early and specific therapeutic interventions. In this review, we consider the factors that may contribute to airway remodeling and discuss the current and potential therapeutic interventions.

Efficacy of Corticosteroids in the Treatment of Community-Acquired Pneumonia Requiring Hospitalization

BackgroundRecent studies suggested that administration of corticosteroids may improve clinical outcomes in patients with severe pneumonia.ObjectivesThe aim of this study was to assess the effectiveness of corticosteroids as an adjunctive therapy in community-acquired pneumonia (CAP) requiring hospitalization.Design and SettingAn open label, prospective, randomized control study was conducted from September 2003 to February 2004 in a community general hospital in Japan.PatientsThirty-one adult CAP patients who required hospitalization were enrolled.Measurements and ResultsFifteen patients received 40 mg of prednisolone intravenously for 3 days (steroid group). Sixteen patients did not receive prednisolone (control group). Both groups were also evaluated for their adrenal function. The primary endpoint was length of hospital stay. Secondary endpoints were duration of intravenous (IV) antibiotics and time required to stabilize vital signs. Both groups demonstrated similar baseline characteristics and length of hospital stay, and yet a shorter duration of IV antibiotics was observed in the steroid group (p < 0.05). In addition, vital signs were stabilized earlier in the steroid group (p < 0.05). These differences were more prominent in the moderate–severe subgroup but not as significant in the mild–moderate subgroup. The prevalence of relative adrenal insufficiency (RAI) in both groups was high (43%), yet there was no difference in baseline characteristics between patients, with or without RAI. In multiple regression models, RAI seemed to have no influence on clinical courses.ConclusionsIn moderate–severe CAP, administration of corticosteroids promotes resolution of clinical symptoms and reduces the duration of intravenous antibiotic therapy.

Histamine may induce airway remodeling through release of epidermal growth factor receptor ligands from bronchial epithelial cells.

Asthma is a chronic inflammatory disease that is associated with airway remodeling, including hyperplasia of airway epithelial cells and airway smooth muscle cells, and goblet cell differentiation. We wished to address the potential role of histamine, a key biogenic amine involved in allergic reactions, in airway remodeling through the epidermal growth factor receptor (EGFR) pathway. Here, we demonstrate that histamine releases 2 EGFR ligands, amphiregulin and heparin‐binding epidermal growth factor‐like growth factor (HB‐EGF), from airway epithelial cells. Amphiregulin and HB‐EGF were expressed in airway epithelium of patients with asthma. Histamine up‐regulated their mRNA expression (amphiregulin 3.2‐fold, P< 0.001; HB‐EGF 2.3‐fold, P<0.05) and triggered their release (amphiregulin EC50 0.50 μM, 31.2±2.7 pg/ml with 10 μM histamine, P<0.01; HB‐EGF EC50 0.54 μM, 78.5±1.8 pg/ml with 10 μM histamine, P<0.001) compared to vehicle control (amphiregulin 19.3±0.9 pg/ ml; HB‐EGF 60.2± 1.0 pg/ml), in airway epithelial cells. Histamine increased EGFR phosphorylation (2.1‐fold by Western blot analysis) and induced goblet cell differentiation (CLCA1 up‐regulation by real‐time qPCR) in normal human bronchial epithelial (NHBE) cells. Moreover, amphiregulin and HB‐EGF caused proliferation and migration of both NHBE cells and human airway smooth muscle cells. These results suggest that histamine may induce airway remodeling via the epithelial‐derived EGFR ligands amphiregulin and HB‐EGF.—Hirota, N., Risse, P.‐A., Novali, M., McGovern, T., Al‐Alwan, L., McCuaig, S., Proud, D., Hayden, P., Hamid, Q., Martin, J. G. Histamine may induce airway remodeling through release of epidermal growth factor receptor ligands from bronchial epithelial cells. FASEB J. 26, 1704‐1716 (2012). www.fasebj.org

Interleukin-13 inhibits proliferation and enhances contractility of human airway smooth muscle cells without change in contractile phenotype

IL-13 is an important mediator of allergen-induced airway hyperresponsiveness. This Th2 cytokine, produced by activated T cells, mast cells, and basophils, has been described to mediate a part of its effects independently of inflammation through a direct modulation of the airway smooth muscle (ASM). Previous studies demonstrated that IL-13 induces hyperresponsiveness in vivo and enhances calcium signaling in response to contractile agonists in vitro. We hypothesized that IL-13 drives human ASM cells (ASMC) to a procontractile phenotype. We evaluated ASM phenotype through the ability of the cell to proliferate, to contract, and to express contractile protein in response to IL-13. We found that IL-13 inhibits human ASMC proliferation (expression of Ki67 and bromodeoxyuridine incorporation) in response to serum, increasing the number of cells in G0/G1 phase and decreasing the number of cells in G2/M phases of the cell cycle. IL-13-induced inhibition of proliferation was not dependent on signal transducer and activator of transcription-6 but was IL-13Rα2 receptor dependent and associated with a decrease of Kruppel-like factor 5 expression. In parallel, IL-13 increased calcium signaling and the stiffening of human ASMC in response to 1 μM histamine, whereas the stiffening response to 30 mM KCl was unchanged. However, Western blot analysis showed unchanged levels of calponin, smooth muscle α-actin, vinculin, and myosin. We conclude that IL-13 inhibits proliferation via the IL-13Rα2 receptor and induces hypercontractility of human ASMC without change of the phenotypic markers of contractility.

Neutrophils Mediate Airway Hyperresponsiveness after Chlorine-Induced Airway Injury in the Mouse

Chlorine gas (Cl2) inhalation causes oxidative stress, airway epithelial damage, airway hyperresponsiveness (AHR), and neutrophilia. We evaluated the effect of neutrophil depletion on Cl2-induced AHR and its effect on the endogenous antioxidant response, and if eosinophils or macrophages influence Cl2-induced AHR. We exposed male Balb/C mice to 100 ppm Cl2 for 5 minutes. We quantified inflammatory cell populations in bronchoalveolar lavage (BAL), the antioxidant response in lung tissue by quantitative PCR, and nuclear factor (erythroid-derived 2)-like 2 (NRF2) nuclear translocation by immunofluorescence. In vitro, NRF2 nuclear translocation in response to exogenous hypochlorite was assessed using a luciferase assay. Anti-granulocyte receptor-1 antibody or anti-Ly6G was used to deplete neutrophils. The effects of neutrophil depletion on IL-13 and IL-17 were measured by ELISA. Eosinophils and macrophages were depleted using TRFK5 or clodronate-loaded liposomes, respectively. AHR was evaluated with the constant-phase model in response to inhaled aerosolized methacholine. Our results show that Cl2 exposure induced neutrophilia and increased expression of NRF2 mRNA, superoxide dismutase-1, and heme-oxygenase 1. Neutrophil depletion abolished Cl2-induced AHR in large conducting airways and prevented increases in antioxidant gene expression and NRF2 nuclear translocation. Exogenous hypochlorite administration resulted in increased NRF2 nuclear translocation in vitro. After Cl2 exposure, neutrophils occupied 22 ± 7% of the luminal space in large airways. IL-17 in BAL was increased after Cl2, although this effect was not prevented by neutrophil depletion. Neither depletion of eosinophils nor macrophages prevented Cl2-induced AHR. Our data suggest the ability of neutrophils to promote Cl2-induced AHR is dependent on increases in oxidative stress and occupation of luminal space in large airways.

Mesenchymal stem cell-seeded multilayered dense collagen-silk fibroin hybrid for tissue engineering applications

Tissue engineering of multilayered constructs that model complex tissues poses a significant challenge for regenerative medicine. In this study, a three-layered scaffold consisting of an electrospun silk fibroin (SF) mat sandwiched between two dense collagen (DC) layers was designed and characterized. It was hypothesized that the SF layer would endow the DC-SF-DC construct with enhanced mechanical properties (e.g., apparent modulus, tensile strength, and toughness), while the surrounding DC layers provide an extracellular matrix-like environment for mesenchymal stem cell (MSC) growth. MSC-seeded DC-SF-DC hybrids were produced using the plastic compression technique and characterized morphologically, chemically, and mechanically. Moreover, MSC viability was assessed for up to 1 wk in culture. Scaffold analyses confirmed compaction and integration of the meso-scaled multilayered DC-SF-DC hybrid, which was reflected in a significantly higher toughness value when compared to DC and SF alone. MSCs directly incorporated into the DC layers remained viable for up to day 7. The ease of multilayered construct fabrication, enhanced biomechanical properties, along with uniformity of cell distribution confirmed the possibility for the incorporation and segregation of different cell types within distinct layers for the regeneration of complex tissues, such as skin, or central nervous system dura mater.

Helix 8 of leukotriene B4 type-2 receptor is required for the folding to pass the quality control in the endoplasmic reticulum

Many G protein‐coupled receptors (GPCRs) possess a putative cytoplasmic helical domain, termed helix 8 (H8), at the proximal region of the C‐terminal tail. However, the significance of this domain is not fully understood. Here, we demonstrate the requirement of H8 for the proper folding of GPCRs for passage through the quality control in the endoplasmic reticulum (ER). In the human leukotriene B4type‐2 receptor (hBLT2), lack of H8 led to an accumulation of the receptor (KBLT2/AH8) in the ER. Similar results were obtained in two representative human GPCRs, dopaminetype‐1 and lysophosphatidic acidtype‐2 receptors, which were engineered to lack H8. Treatment with the several ligands, which act as pharmacological chaperones, facilitated the surface expression of hBLT2/AH8. The surface‐trafficked hBLT2/AH8 exhibited an agonist‐evoked increase in Ca2+, demonstrating that H8 is not critical for ligand binding and activation of coupled G proteins. Thus, these results suggest that the H8 region of hBLT2 plays an important role in transport‐competent receptor folding.— Yasuda, D., Okuno, T., Yokomizo, T., Hori, T., Hirota, N., Hashidate, .T, Miyano, M., Shimizu, T., Nakamura, M. Helix 8 of leukotriene B4type‐2 receptor is required for the folding to pass the quality control in the endoplasmic reticulum. FASEB J. 23, 1470–1481 (2009)

Amino Acid Residues Critical for Endoplasmic Reticulum Export and Trafficking of Platelet-activating Factor Receptor

Several residues are conserved in the transmembrane domains (TMs) of G-protein coupled receptors. Here we demonstrate that a conserved proline, Pro247, in TM6 of platelet-activating factor receptor (PAFR) is required for endoplasmic reticulum (ER) export and trafficking after agonist-induced internalization. Alanine-substituted mutants of the conserved residues of PAFRs, including P247A, were retained in the ER. Because a PAFR antagonist, Y-24180, acted as a pharmacological chaperone to rescue ER retention, this retention is due to misfolding of PAFR. Methylcarbamyl (mc)-PAF, a PAFR agonist, did not increase the cell surface expression of P247A, even though another ER-retained mutant, D63A, was effectively trafficked. Signaling and accumulation of the receptors in the early endosomes were observed in the mc-PAF-treated P247A-expressing cells, suggesting that P247A was trafficked to the cell surface by mc-PAF, and thereafter disappeared from the surface due to aberrant trafficking, e.g. enhanced internalization, deficiency in recycling, and/or accelerated degradation. The aberrant trafficking was confirmed with a sortase-A-mediated method for labeling cell surface proteins. These results demonstrate that the conserved proline in TM6 is crucial for intracellular trafficking of PAFR.

The presence of LPS in OVA inhalations affects airway inflammation and AHR but not remodeling in a rodent model of asthma

Ovalbumin (OVA) is the most frequently used allergen in animal models of asthma. Lipopolysaccharide (LPS) contaminating commercial OVA may modulate the evoked airway inflammatory response to OVA. However, the effect of LPS in OVA on airway remodeling, especially airway smooth muscle (ASM) has not been evaluated. We hypothesized that LPS in commercial OVA may enhance allergen-induced airway inflammation and remodeling. Brown Norway rats were sensitized with OVA on day 0. PBS, OVA, or endotoxin-free OVA (Ef-OVA) was instilled intratracheally on days 14, 19, 24. Bronchoalveolar lavage (BAL) fluid, lung, and intrathoracic lymph node tissues were collected 48 h after the last challenge. Immunohistochemistry for α-smooth muscle actin, Periodic-Acid-Schiff staining, and real-time qPCR were performed. Airway hyperresponsiveness (AHR) was also measured. BAL fluid macrophages, eosinophils, neutrophils, and lymphocytes were increased in OVA-challenged animals, and macrophages and neutrophils were significantly lower in Ef-OVA-challenged animals. The ASM area in larger airways was significantly increased in both OVA and Ef-OVA compared with PBS-challenged animals. The mRNA expression of IFN-γ and IL-13 in lung tissues and IL-4 in lymph nodes was significantly increased by both OVA and Ef-OVA compared with PBS and were not significantly different between OVA and Ef-OVA. Monocyte chemoattractant protein (MCP)-1 in BAL fluid and AHR were significantly increased in OVA but not in Ef-OVA. LPS contamination in OVA contributes to the influx of macrophages and MCP-1 increase in the airways and to AHR after OVA challenges but does not affect OVA-induced Th1 and Th2 cytokine expression, goblet cell hyperplasia, and ASM remodeling.

The modulation of large airway smooth muscle phenotype and effects of epidermal growth factor receptor inhibition in the repeatedly allergen-challenged rat

Allergen challenges induce airway hyperresponsiveness (AHR) and increased airway smooth muscle (ASM) mass in the sensitized rat. Whether the remodeled ASM changes its phenotype is uncertain. We examined, in sensitized Brown Norway rats, the effects of multiple ovalbumin (Ova) challenges on ASM remodeling and phenotype and the role of the epidermal growth factor receptor (EGFR) in these processes. Rats were sensitized with Ova and challenged three times at 5-day intervals with phosphate-buffered saline or Ova and pretreated with the EGFR inhibitor AG-1478 (5 mg/kg) or its vehicle dimethyl sulfoxide. Ova challenges increased ASM mass in all-sized airways and in large airway mRNA expression of smooth muscle myosin heavy chain (sm-MHC), assessed by laser capture. Myosin light chain kinase and the fast myosin isoform SM-B mRNA expressions were not affected. Ova induced AHR to methacholine, and, based on the constant-phase model, this was largely attributable to the small airways and lung derecruitment at 48 h that recovered by 1 wk. The EGFR ligands amphiregulin and heparin-binding epidermal growth factor (HB-EGF) were increased in bronchoalveolar lavage fluid at 48 h after Ova exposure. AG-1478 inhibited AHR and prevented ASM growth. Epithelial gene expression of EGFR, HB-EGF, matrix metalloproteinase (MMP)-9, Gro-α, and transforming growth factor-β was unaffected by Ova challenges. We conclude that EGFR drives remodeling of ASM, which results from repeated Ova challenge. Furthermore, the latter results in excessive small airway and, to a lesser degree, large airway narrowing to methacholine, and large airway gene expression of contractile protein is conserved.