Novel air-liquid interface culture model to investigate stiffness-dependent behaviors of alveolar epithelial cells.

Pulmonary alveoli are functional units in gas exchange in the lung, and their dysfunctions in lung diseases such as interstitial pneumonia are accompanied by fibrotic changes in structure, elevating the stiffness of extracellular matrix components. The present study aimed to test the hypothesis that such changes in alveoli stiffness induce functional alteration of epithelial cell functions, exacerbating lung diseases. For this, we have developed a novel method of culturing alveolar epithelial cells on polyacrylamide gel with different elastic modulus at an air-liquid interface. It was demonstrated that A549 cells on soft gels, mimicking the modulus of a healthy lung, upregulated mRNA expression and protein synthesis of surfactant protein C (SFTPC). By contrast, the cells on stiff gels, mimicking the modulus of the fibrotic lung, exhibited upregulation of SFTPC gene expression but not at the protein level. Cell morphology, as well as cell nucleus volume, were also different between the two types of gels.

A Triply Linked Porphyrin-Norcorrole Hybrid with Singlet Diradical Character.

Norcorrole Ni(II) complexes have recently received considerable attention because they are readily accessible antiaromatic molecules. Their high stability under ambient conditions and ease of synthesis have enabled the exploration of the intrinsic properties of antiaromatic molecules. Here, we report the synthesis and properties of meso-meso singly linked porphyrin-norcorrole hybrids and a triply linked porphyrin-norcorrole hybrid. The singly linked and triply linked porphyrin-norcorrole hybrids were fully characterized, including an X-ray structural analysis. Due to their orthogonal conformation, the singly linked hybrids maintain the individual electronic properties of their porphyrin and norcorrole subunits, while the triply linked hybrid shows a significantly smaller electrochemical HOMO-LUMO gap (0.45 eV) than that of Ni(II) dimesitylnorcorrole (1.08 eV). Furthermore, the triply linked hybrid exhibits singlet diradical characteristics, as confirmed by VT NMR, ESR, and SQUID experiments.

Guanine nucleotide exchange factor DOCK11-binding peptide fused with a single chain antibody inhibits hepatitis B virus infection and replication.

Hepatitis B virus (HBV) infection is a major global health problem with no established cure. Dedicator of cytokinesis 11 (DOCK11), known as a guanine nucleotide exchange factor (GEF) for Cdc42, is reported to be essential for the maintenance of HBV. However, potential therapeutic strategies targeting DOCK11 have not yet been explored. We have previously developed an in vitro virus method as a more efficient tool for the analysis of proteomics and evolutionary protein engineering. In this study, using the in vitro virus method, we screened and identified a novel antiasialoglycoprotein receptor (ASGR) antibody, ASGR3-10M, and a DOCK11-binding peptide, DCS8-42A, for potential use in HBV infection. We further constructed a fusion protein (10M-D42AN) consisting of ASGR3-10M, DCS8-42A, a fusogenic peptide, and a nuclear localization signal to deliver the peptide inside hepatocytes. We show using immunofluorescence staining that 10M-D42AN was endocytosed into early endosomes and released into the cytoplasm and nucleus. Since DCS8-42A shares homology with activated cdc42-associated kinase 1 (Ack1), which promotes EGFR endocytosis required for HBV infection, we also found that 10M-D42AN inhibited endocytosis of EGFR and Ack1. Furthermore, we show 10M-D42AN suppressed the function of DOCK11 in the host DNA repair system required for covalently closed circular DNA synthesis and suppressed HBV proliferation in mice. In conclusion, this study realizes a novel hepatocyte-specific drug delivery system using an anti-ASGR antibody, a fusogenic peptide, and DOCK11-binding peptide to provide a novel treatment for HBV.

Overcoming challenges in acute care hospitalizations for patients with opioid use disorder.

BACKGROUND: Hospitalized patients with opioid use disorder (OUD) present unique challenges and opportunities for inpatient medical teams. Having the ability to initiate medications for opioid use disorder (MOUD) and linkage to outpatient treatment are key to improve inpatient care of patients with OUD. OBJECTIVE: This study aimed to describe the process taken by a multidisciplinary work group to improve the acute care management of patients with OUD. PRACTICE DESCRIPTION: In 2018, we identified that inpatient care teams at the University of Chicago Medicine (UCM) lacked a standardized approach to the management of hospitalized patients with OUD and that the care typically did not include evidence-based therapies. Herein, we describe the process taken to develop the OUD workgroup and the work completed by the workgroup. PRACTICE INNOVATION: The OUD workgroup spearheaded the development of an OUD consult service, formulary revisions, education for health care workers (inpatient nurse training and X-waiver training for prescribers), and outpatient partnerships. Pharmacy-led initiatives included formulary management, electronic medication orders, naloxone co-prescribing decision support, and MOUD education. EVALUATION METHODS: The OUD consult service was granted an Institutional Review Board exemption for quality improvement analysis through UCM. A data analytics dashboard was built to track consult service volumes and outcomes. RESULTS: From July 2020 to April 2021, 296 OUD consults occurred. In total, 103 consult patients (35%) received and were discharged with buprenorphine. An additional 118 patients (40%) were managed with methadone and linked to outpatient care. Naloxone dispensing at discharge increased to over 65%, which did not include patients who opted out or were discharged to a facility. CONCLUSION: The ongoing OUD epidemic presents a need for the development of services to improve management of patients with OUD in the acute care setting. The OUD workgroup has improved the management of patients admitted with OUD. Pharmacy-based initiatives are key to the development of safe and effective management of OUD in hospitalized patients.

[DRUG-INDUCED LUNG INJURY DUE TO OTSU-JI-TO FOLLOWED BY DRUG-INDUCED LIVER INJURY: A CASE REPORT].

A 69-year-old woman who had been treated with Otsu-ji-to for fourteen days developed liver dysfunction. She continued to take Otsu-ji-to and was admitted to our hospital due to respiratory failure with extensive ground-glass opacities on chest computed tomography 22 days after starting to take Otsuji-to. Although she developed severe respiratory failure, her condition was improved by discontinuation of Otsu-ji-to and high-dose corticosteroid pulse therapy. The lymphocyte stimulation test was positive for Otsu-ji-to. Finally, we diagnosed drug-induced lung injury due to Otsu-ji-to. As in this case, severe herbal medicine-induced lung injury may be developed secondary to preceding liver injury. When a patient prescribed ou-gon-containing herbal medicines such as Otsu-ji-to develops liver dysfunction, due to herbal medicines containing ou-gon such as Otsu-ji-to, it is important to evaluate lung injury and discontinue the Kampo drug.

Preoperative six-minute walk distance as a predictor of postoperative complication in patients with esophageal cancer.

Surgery for esophageal cancer is associated with high morbidity and mortality. Reduced pulmonary functions and exercise capacity are known as risk factors for complications after esophagectomy. The 6-minute walk distance (6MWD) measured by the 6-minute walk test (6MWT) is a simple field test that can be used to evaluate the functional exercise capacity of patients who undergo thoracic surgery. The aim of this study was to evaluate the association of the preoperative 6MWD with postoperative complications in patients with esophageal cancer. Records of a total of 111 patients who underwent thoracic surgery followed by postoperative rehabilitation from January 2013 to December 2015 were retrospectively reviewed. Data of patients who experienced Clavien-Dindo grade II or severer (grade ≥ II) complications were compared with those who experienced grade ≤I complications. The 6MWD was significantly correlated with age, serum albumin concentration, hemoglobin concentration, and hand grip strength. A total of 42 patients experienced grade ≥II. The 6MWD of patients with grade ≥ II complications was significantly shorter than that of those with grade ≤I complications. In receiver operating characteristic analysis, 6MWD ≤ 454 m was a threshold for predicting grade ≥II complications with 71.0% sensitivity and 54.8% specificity. The incidence of grade ≥II complications led to delayed ambulation and longer stays in hospital. In the multiple regression analysis, the preoperative risk factors for incidence of grade ≥II complications included lower levels of preoperative 6MWD and % of the predicted value of forced expiratory volume in 1 second. Our results indicate that the 6MWT is useful to assess preoperative physical status in patients with esophageal cancer.

High TLR4 expression predicts a poor prognosis after esophagectomy for advanced thoracic esophageal squamous cell carcinoma.

BACKGROUND: Poor oral health is an independent risk factor for upper aerodigestive tract cancers, including esophageal squamous cell carcinoma (ESCC). The pattern recognition receptor Toll-like receptor 4 (TLR4) recognizes lipopolysaccharide in the cell walls of Gram-negative periodontal pathogens associated with the development and progression of ESCC. It is, therefore, plausible that TLR4 plays a crucial role in the pathogenesis of ESCC. METHODS: We used an ESCC tissue microarray to confirm expression of TLR4 in patients with ESCC and to determine whether TLR4 expression status correlates with the clinicopathological features of these patients or their prognosis after esophagectomy. We also tested whether the combined expression statuses of TLR4 and TLR3 better correlate with prognosis in these patients than either parameter alone. RESULTS: Clinical ESCC samples from all 177 patients tested showed expression of TLR4. Moreover, high TLR4 expression (3 + and 2 +) correlated with poorer 5-year overall survival after esophagectomy than lower TLR4 expression (1 +) (p = 0.0491). Patients showing high TLR4 expression tended to have a poorer prognosis whether treated with surgery alone or with surgery and adjuvant chemotherapy. Univariate and multivariate analyses showed TLR4 expression status to be an independent prognostic factor affecting 5-year overall survival. Patients exhibiting high TLR4 expression with low TLR3 expression had a much poorer prognosis than other patients (p = < 0.0001). CONCLUSION: High TLR4 expression predicts a poor prognosis in advanced thoracic ESCC patients after esophagectomy.

Titania-Catalyzed H2O2 Thermal Oxidation of Styrenes to Aldehydes.

We investigated the selective oxidation of styrenes to benzaldehydes by using a non-irradiated TiO2-H2O2 catalytic system. The oxidation promotes multi-step reactions from styrenes, including the cleavage of a C=C double bond and the addition of an oxygen atom selectively and stepwise to provide the corresponding benzaldehydes in good yields (up to 72%). These reaction processes were spectroscopically shown by fluorescent measurements under the presence of competitive scavengers. The absence of the signal from OH radicals indicates the participation of other oxidants such as hydroperoxy radicals (•OOH) and superoxide radicals (•O2-) into the selective oxidation from styrene to benzaldehyde.

Regulation of PD-L1 expression by matrix stiffness in lung cancer cells.

Expression of programmed death-ligand 1 (PD-L1) in tumor cells such as lung cancer cells plays an important role in mechanisms underlying evasion of an immune check point system. Lung cancer tissue with increased deposition of extracellular matrix is much stiffer than normal lung tissue. There is emerging evidence that the matrix stiffness of cancer tissue affects the phenotypes and properties of cancer cells. Nevertheless, the effects of substrate rigidity on expression of PD-L1 in lung cancer cells remain elusive. We evaluated the effects of substrate stiffness on PD-L1 expression in HCC827 lung adenocarcinoma cells by using polyacrylamide hydrogels with stiffnesses of 2 and 25 kPa. Expression of PD-L1 protein was higher on the stiffer substrates (25 kPa gel and plastic dish) than on the soft 2 kPa gel. PD-L1 expression was reduced by detachment of cells adhering to the substrate. Interferon-γ enhanced expression of PD-L1 protein cultured on stiff (25 kPa gel and plastic dishes) and soft (2 kPa gel) substrates and in the cell adhesion-free condition. As the stiffness of substrates increased, formation of actin stress fiber and cell growth were enhanced. Transfection of the cells with short interfering RNA for PD-L1 inhibited cell growth without affecting stress fiber formation. Treatment of the cells with cytochalasin D, an inhibitor of actin polymerization, significantly reduced PD-L1 protein levels. Taken together, a stiff substrate enhanced PD-L1 expression via actin-dependent mechanisms in lung cancer cells. It is suggested that stiffness as a tumor environment regulates PD-L1 expression, which leads to evasion of the immune system and tumor growth.

Adenomatoid tumour of the uterus is frequently associated with iatrogenic immunosuppression.

AIMS: Uterine adenomatoid tumour (AT) is a benign proliferation of cells showing mesothelial differentiation within the myometrium that usually presents as a single nodule. Rare diffuse uterine ATs have been reported, often in patients undergoing immunosuppressive therapy. Herein, we aimed to elucidate the general association between the incidence of uterine AT and iatrogenic immunosuppression by cohort analysis. METHODS AND RESULTS: We analysed 611 consecutive hysterectomy specimens to determine the incidence of AT and its correlation with the immunosuppressive status. Mesothelial lineage, p16 expression, mismatch repair (MMR) protein alterations, and the possible integration of tumorigenic viruses were examined by in situ hybridizasion and immunohistochemistry. ATs were detected in 14 of 611 hysterectomy cases (2.3%). The incidence of AT was significantly higher in the immunosuppressed (IS) group (5/20, 25.0%) than in the non-IS group (9/591, 1.52%), with a relative risk of 16.4. Of the five ATs in the IS group, three were multifocal or diffuse. Latent uterine AT was detected, by in toto sectioning, in one of four immunosuppressed autopsy cases. The tumor cells of ATs commonly expressed calretinin and podoplanin. Characteristic block-type (≥90%) positivity for p16 was observed in most ATs. None of the ATs were positive for human herpes virus type 8, Merkel cell polyomavirus, SV40 large T antigen, Epstein-Barr virus, and human papilloma virus, and the MMR proteins were retained. A TRAF7 mutation was identified from macrodissected tissue in one of 12 ATs by Sanger sequencing. CONCLUSION: Uterine AT is an immunosuppression-associated mesothelial lesion characterised by p16 overexpression.

Combination of anti-CD4 antibody treatment and donor lymphocyte infusion ameliorates graft-versus-host disease while preserving graft-versus-tumor effects in murine allogeneic hematopoietic stem cell transplantation.

Allogeneic hematopoietic stem cell transplantation (allo-HSCT) is not only a well-established immunotherapy for hematologic malignancies, but is potentially useful for treating solid tumors refractory to available therapies. However, application of allo-HSCT to solid tumors is limited, despite the beneficial antitumor effects, by the risk of graft-versus-host disease (GVHD). CD4+ T cells have been implicated in several aspects of GVHD, and suppress antitumor CD8+ T-cell responses. In the present study, we investigated clinically applicable allo-HSCT protocols designed to maximize antitumor effects while reducing the risk of GVHD. We used a mouse model of allo-HSCT with s.c. tumors. We found that myeloablative conditioning was associated with better inhibition of tumor growth but with severe acute GVHD. Early treatment with anti-CD4 mAb substantially ameliorated GVHD while preserving antitumor effects, leading to improved survival in myeloablative allo-HSCT. Late treatment with anti-CD4 mAb also ameliorated GVHD to some extent. Donor lymphocyte infusion in GVHD mice treated with anti-CD4 mAb further suppressed tumor growth without exacerbating GVHD. Collectively, our results suggest that myeloablative allo-HSCT followed by anti-CD4 mAb treatment and donor lymphocyte infusion could be a potent and safe immunotherapy for patients with cancers refractory to available therapies.

Pirfenidone as salvage treatment for refractory bleomycin-induced lung injury: a case report of seminoma.

BACKGROUND: Bleomycin-induced lung injury, a major complication of chemotherapy for germ cell tumors, occasionally fails to respond to the standard treatment with corticosteroids and develops into severe respiratory insufficiency. Little is known about salvage treatment for refractory cases. CASE PRESENTATION: A 63-year-old man who had been diagnosed with stage I seminoma and undergone a high orchiectomy 1 year previously developed swelling of his left iliac lymph node and was diagnosed with a recurrence of the seminoma. He was administered a standard chemotherapy regimen of cisplatin, etoposide, and bleomycin. At the end of second cycle, he developed a dry cough and fever that was accompanied by newly-identified bilateral infiltrates on chest X-ray. Despite initiation of oral prednisolone, his exertional dyspnea and decline in pulmonary functions continued to be aggravated. High-dose pulse treatment with methylprednisolone was introduced and improved his symptoms and radiologic findings. However, the maintenance dose of oral prednisolone allowed reactivation of the disease with evidence of newly-developed bilateral lung opacities on high-resolution CT scans. Considering his glucose intolerance and cataracts as complications of corticosteroid treatment, administration of pirfenidone was initiated with the patient's consent. Pirfenidone at 1800 mg/day was well tolerated, and resolved his symptoms and abnormal opacities on a chest CT scan. Subsequently, the dose of prednisolone was gradually tapered without worsening of the disease. At the most recent follow-up, he was still in complete remission of seminoma with a successfully tapered combination dose of prednisolone and pirfenidone. CONCLUSIONS: Pirfenidone, a novel oral agent with anti-inflammatory and -fibrotic properties, should be considered as a salvage drug for refractory cases of bleomycin-induced lung injury.

Prostaglandin E-major Urinary Metabolite as a Biomarker for Pediatric Ulcerative Colitis Activity.

OBJECTIVES: Prostaglandin E-major urinary metabolite (PGE-MUM) is a useful biomarker for adult ulcerative colitis (UC) activity. In the present study, we evaluated whether PGE-MUM can also be a biomarker of pediatric UC activity and compared its efficacy in predicting UC activity with that of C-reactive protein and erythrocyte sedimentation rate. METHODS: Twenty-nine pediatric patients with UC (8-18 years) and 29 healthy age- and sex-matched subjects were enrolled. UC activity was evaluated using the Pediatric Ulcerative Colitis Activity Index, highest Mayo endoscopic scoring (Mayo), and Matts grading (Matts) for histologic scoring, and the sum of Mayo (total of 6 segments) and Matts in all patients with UC. PGE-MUM levels were measured using a radioimmunoassay. RESULTS: PGE-MUM levels were elevated in endoscopically and histologically active UC patients, but not in patients with endoscopic and histologic remission or controls. PGE-MUM levels positively and significantly correlated with UC activity. PGE-MUM levels were positively correlated with Pediatric Ulcerative Colitis Activity Index (r = 0.594), highest Mayo (r = 0.462), the sum of Mayo (r = 0.694), and the sum of Matts (r = 0.613), but not with highest Matt (r = 0.352). The sum of Mayo and the sum of Matts, which reflect total colon inflammation, showed highest correlation with PGE-MUM. C-reactive protein levels did not correlate with any UC activity scores. Erythrocyte sedimentation rate exhibited correlation (r = 0.490) with the sum of Mayo only. CONCLUSIONS: PGE-MUM is a reliable biomarker that reflects both the endoscopic and histologic activity of the entire colon in pediatric UC.

Exercise hypoxaemia as a predictor of pulmonary hypertension in COPD patients without severe resting hypoxaemia.

BACKGROUND AND OBJECTIVE: Pulmonary hypertension (PH) in COPD is associated with morbidity and mortality. Previous studies showed a relationship between resting hypoxaemia and PH, but little is known about the relationship between exercise hypoxaemia and PH in COPD without resting hypoxaemia. METHODS: A retrospective observational study of COPD patients without resting hypoxaemia was conducted to evaluate the relationships between exercise hypoxaemia and pulmonary haemodynamics. Clinical characteristics, pulmonary function, blood gas analysis, 6-min walk distance (6MWD) and oxygen saturation of peripheral artery (SpO2 ) at the end of the 6-min walk test (6MWT) were reviewed. Correlation analysis and stepwise regression analysis were performed to identify the predictor of mean pulmonary artery pressure (mPAP). RESULTS: Eighty-four consecutive patients with a mean predicted forced expiratory volume in 1 s (FEV1 ) of 47 ± 21% were evaluated. In univariate analysis, mPAP had negative correlations with age (r = -0.27, P < 0.05), arterial partial pressure of oxygen (PaO2 , r = -0.24, P < 0.05), % predicted forced vital capacity (FVC, r = -0.28, P < 0.05), % predicted FEV1 (r = -0.40, P < 0.001), FEV1 /FVC ratio (r = -0.33, P < 0.005), % predicted diffusion capacity for carbon monoxide (DLCO , r = -0.40, P < 0.001), 6MWD (r = -0.40, P < 0.001) and SpO2 at the end of the 6MWT (r = -0.74, P < 0.001). In stepwise regression analysis, SpO2 at the end of the 6MWT and 6MWD remained as independent predictors of mPAP (R2 = 0.60). In receiver operating characteristic (ROC) analysis, SpO2 at the end of the 6MWT presented an area under the curve of 0.896 for the prediction of PH, with a sensitivity of 0.86 and specificity of 0.84 for the cut-off point of 81%. CONCLUSION: In addition to 6MWD, exercise hypoxaemia indicates PH in patients with COPD without resting hypoxaemia.

Prostaglandin E-Major Urinary Metabolite as a Biomarker for Inflammation in Ulcerative Colitis: Prostaglandins Revisited.

With the development of new therapeutic approaches, the ultimate goal of ulcerative colitis (UC) treatment is not only clinical remission but also mucosal healing. Successful mucosal healing has been associated with a dramatic risk reduction in UC recurrence and colitis-associated cancer development, which are the most critical complications of UC. However, invasive tests such as colonoscopy and biopsy are required to evaluate mucosal healing. Therefore, frequent examinations are unsuitable for UC patients. Mucosal inflammation of the colon and prostaglandin E2 production are assumed to be correlated; therefore, we considered that prostaglandin E-major urinary metabolite (PGE-MUM; 7-hydroxy-5,11-diketotetranor-prosta-1,16-dioic acid) may be a surrogate biomarker of UC activity. In this review, we propose that PGE-MUM levels reflect the colonoscopic and histological appearance of UC, suggesting that it is a more sensitive biomarker than those previously utilized for UC-related mucosal inflammation. According to the 'organ-specific chronic inflammation-carcinoma sequence' theory, by measuring PGE-MUM periodically, it would be possible to control inflammation, with subsequent prevention of UC recurrence and colitis-associated cancer development. The measurement of urine samples for PGE-MUM - a simple, noninvasive method - can reduce the patient burden as well as medical costs, suggesting its potential for application in routine practice.

Mean pulmonary arterial pressure as a prognostic indicator in connective tissue disease associated with interstitial lung disease: a retrospective cohort study.

BACKGROUND: Pulmonary hypertension (PH) can develop in connective tissue disease associated interstitial lung disease (CTD-ILD), and contributes to increased morbidity and mortality. However, except for systemic sclerosis and mixed connective tissue disease, the impact of mean pulmonary arterial pressure (MPAP) on survival in CTD-ILD has not been sufficiently elucidated. We hypothesized that pulmonary arterial pressure may be a prognostic factor in CTD-ILDs regardless of the kind of CTD. METHODS: We evaluated the survival impact of MPAP, which is measured using right heart catheterization, on survival of patients with CTD-ILD with various CTD backgrounds. We retrospectively analyzed data of consecutive CTD-ILD patients undergoing a pulmonary function test and right-heart-catheterization at the initial evaluation. RESULTS: We studied 74 patients (33 men and 41 women, mean age 62.8 ± 9.6, 24 with rheumatoid arthritis, 14 with systemic sclerosis, 14 with polymyositis/dermatomyositis, 11 with primary Sjögren's syndrome, and 11 with other diagnoses). Six patients exhibited pulmonary hypertension (MPAP ≥ 25 mmHg), and 16 (21.6 %) had mild elevation of MPAP (≥20 mmHg). The mean MPAP was 17.2 ± 5.5 mmHg. We did not observe a significant difference in MPAP among various CTDs. A univariate Cox proportional hazard model showed that MPAP has a significant impact on survival, while the type of CTD did not contribute to survival in our cohort. A multivariate Cox proportional hazard model showed MPAP (HR = 1.087; 95 % CI 1.008-1.172; p = 0.030) to be the sole independent determinant of survival. CONCLUSIONS: Mild elevation of MPAP is relatively common in CTD-ILD patients with various CTD backgrounds. A higher MPAP at the initial evaluation was a significant independent predictor of survival in CTD-ILD. MPAP evaluation provides additional information on disease status and will help physicians predict mortality in CTD-ILD.

Synthesis of highly twisted and fully π-conjugated porphyrinic oligomers.

Highly twisted π-conjugated molecules have been attractive but challenging targets. We report here an efficient synthesis of highly twisted diporphyrins with 126° and 136° twist angles that involves an oxidative fusion reaction of planar aminoporphyrin precursors at room temperature. Repeated amination-oxidative fusion sequences provide a unidirectionally twisted tetramer. The twisting angle of the tetramer is 298°.

Real-time imaging of ATP release induced by mechanical stretch in human airway smooth muscle cells.

Airway smooth muscle (ASM) cells within the airway walls are continually exposed to mechanical stimuli, and exhibit various functions in response to these mechanical stresses. ATP acts as an extracellular mediator in the airway. Moreover, extracellular ATP is considered to play an important role in the pathophysiology of asthma and chronic obstructive pulmonary disease. However, it is not known whether ASM cells are cellular sources of ATP secretion in the airway. We therefore investigated whether mechanical stretch induces ATP release from ASM cells. Mechanical stretch was applied to primary human ASM cells cultured on a silicone chamber coated with type I collagen using a stretching apparatus. Concentrations of ATP in cell culture supernatants measured by luciferin-luciferase bioluminescence were significantly elevated by cyclic stretch (12 and 20% strain). We further visualized the stretch-induced ATP release from the cells in real time using a luminescence imaging system, while acquiring differential interference contrast cell images with infrared optics. Immediately after a single uniaxial stretch for 1 second, strong ATP signals were produced by a certain population of cells and spread to surrounding spaces. The cyclic stretch-induced ATP release was significantly reduced by inhibitors of Ca(2+)-dependent vesicular exocytosis, 1,2-bis(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid tetraacetoxymethyl ester, monensin, N-ethylmaleimide, and bafilomycin. In contrast, the stretch-induced ATP release was not inhibited by a hemichannel blocker, carbenoxolone, or blockade of transient receptor potential vanilloid 4 by short interfering RNA transfection or ruthenium red. These findings reveal a novel property of ASM cells: mechanically induced ATP release may be a cellular source of ATP in the airway.

Ca2+ influx and ATP release mediated by mechanical stretch in human lung fibroblasts.

One cause of progressive pulmonary fibrosis is dysregulated wound healing after lung inflammation or damage in patients with idiopathic pulmonary fibrosis and severe acute respiratory distress syndrome. The mechanical forces are considered to regulate pulmonary fibrosis via activation of lung fibroblasts. In this study, the effects of mechanical stretch on the intracellular Ca(2+) concentration ([Ca(2+)]i) and ATP release were investigated in primary human lung fibroblasts. Uniaxial stretch (10-30% in strain) was applied to fibroblasts cultured in a silicone chamber coated with type I collagen using a stretching apparatus. Following stretching and subsequent unloading, [Ca(2+)]i transiently increased in a strain-dependent manner. Hypotonic stress, which causes plasma membrane stretching, also transiently increased the [Ca(2+)]i. The stretch-induced [Ca(2+)]i elevation was attenuated in Ca(2+)-free solution. In contrast, the increase of [Ca(2+)]i by a 20% stretch was not inhibited by the inhibitor of stretch-activated channels GsMTx-4, Gd(3+), ruthenium red, or cytochalasin D. Cyclic stretching induced significant ATP releases from fibroblasts. However, the stretch-induced [Ca(2+)]i elevation was not inhibited by ATP diphosphohydrolase apyrase or a purinergic receptor antagonist suramin. Taken together, mechanical stretch induces Ca(2+) influx independently of conventional stretch-sensitive ion channels, the actin cytoskeleton, and released ATP.

Airway smooth muscle in asthma: linking contraction and mechanotransduction to disease pathogenesis and remodelling.

Asthma is an obstructive airway disease, with a heterogeneous and multifactorial pathogenesis. Although generally considered to be a disease principally driven by chronic inflammation, it is becoming increasingly recognised that the immune component of the pathology poorly correlates with the clinical symptoms of asthma, thus highlighting a potentially central role for non-immune cells. In this context airway smooth muscle (ASM) may be a key player, as it comprises a significant proportion of the airway wall and is the ultimate effector of acute airway narrowing. Historically, the contribution of ASM to asthma pathogenesis has been contentious, yet emerging evidence suggests that ASM contractile activation imparts chronic effects that extend well beyond the temporary effects of bronchoconstriction. In this review article we describe the effects that ASM contraction, in combination with cellular mechanotransduction and novel contraction-inflammation synergies, contribute to asthma pathogenesis. Specific emphasis will be placed on the effects that ASM contraction exerts on the mechanical properties of the airway wall, as well as novel mechanisms by which ASM contraction may contribute to more established features of asthma such as airway wall remodelling.