Brent E. McParland

Airway smooth muscle dynamics: a common pathway of airway obstruction in asthma

Excessive airway obstruction is the cause of symptoms and abnormal lung function in asthma. As airway smooth muscle (ASM) is the effecter controlling airway calibre, it is suspected that dysfunction of ASM contributes to the pathophysiology of asthma. However, the precise role of ASM in the series of events leading to asthmatic symptoms is not clear. It is not certain whether, in asthma, there is a change in the intrinsic properties of ASM, a change in the structure and mechanical properties of the noncontractile components of the airway wall, or a change in the interdependence of the airway wall with the surrounding lung parenchyma. All these potential changes could result from acute or chronic airway inflammation and associated tissue repair and remodelling. Anti-inflammatory therapy, however, does not “cure” asthma, and airway hyperresponsiveness can persist in asthmatics, even in the absence of airway inflammation. This is perhaps because the therapy does not directly address a fundamental abnormality of asthma, that of exaggerated airway narrowing due to excessive shortening of ASM. In the present study, a central role for airway smooth muscle in the pathogenesis of airway hyperresponsiveness in asthma is explored.

Comparison of gel contraction mediated by airway smooth muscle cells from patients with and without asthma

Backgrounds: Exaggerated bronchial constriction is the most significant and life threatening response of patients with asthma to inhaled stimuli. However, few studies have investigated the contractility of airway smooth muscle (ASM) from these patients. The purpose of this study was to establish a method to measure contraction of ASM cells by embedding them into a collagen gel, and to compare the contraction between subjects with and without asthma. Methods: Gel contraction to histamine was examined in floating gels containing cultured ASM cells from subjects with and without asthma following overnight incubation while unattached (method 1) or attached (method 2) to casting plates. Smooth muscle myosin light chain kinase protein levels were also examined. Results: Collagen gels containing ASM cells reduced in size when stimulated with histamine in a concentration-dependent manner and reached a maximum at a mean (SE) of 15.7 (1.2) min. This gel contraction was decreased by inhibitors for phospholipase C (U73122), myosin light chain kinase (ML-7) and Rho kinase (Y27632). When comparing the two patient groups, the maximal decreased area of gels containing ASM cells from patients with asthma was 19 (2)% (n = 8) using method 1 and 22 (3)% (n = 6) using method 2, both of which were greater than that of cells from patients without asthma: 13 (2)% (n = 9, p = 0.05) and 10 (4)% (n = 5, p = 0.024), respectively. Smooth muscle myosin light chain kinase levels were not different between the two groups. Conclusion: The increased contraction of asthmatic ASM cells may be responsible for exaggerated bronchial constriction in asthma.

Isolating vessels from the mouse brain for gene expression analysis using laser capture microdissection

Studies of gene expression often examine a pool of RNA extracted from the diverse cell types making up a tissue. We have developed a method for isolating vessels from the brain in order to understand the changes occurring in the vessels during the pathogenesis of cerebral malaria. Vessels were visualised by incubating sections of mouse brain with a substrate for alkaline phosphatase. Vessels were collected by laser capture microdissection and the specificity was monitored by measuring the expression of cell-specific markers. RNA from the captured vessels was highly enriched in mRNA for genes associated with endothelial cells and pericytes. Measurement of indoleamine 2,3-dioxygenase mRNA indicated it was possible to detect changes in gene expression, due to malaria infection, occurring specifically within the vessels. Laser capture microdissection can be used to study changes in gene expression occurring at the blood-brain barrier.

Structural basis for exaggerated airway narrowing.

Airway hyperresponsiveness, particularly the ability of airways to narrow excessively in response to stimuli that normally cause little airway narrowing in nonasthmatic subjects, is a characteristic feature of asthma and the basis of its symptoms. Although airway hyperresponsiveness may be partly the result of alterations in the contractile phenotype of the airway smooth muscle, there is evidence that it may also be caused by structural changes in the airway wall, collectively termed airway remodeling. Airway remodeling is defined as changes in composition, quantity, and (or) organization of cellular and molecular constituents of the airway wall. Airway wall remodeling that occurs in asthma can result in functional alterations because of quantitative changes in airway wall compartments, and (or) because of changes in the biochemical composition or material properties of the various constituents of the airway wall. This brief review summarizes the quantitative changes in the dimensions and organization of the airway wall compartments that have been described and explains how structural alterations may lead to the exaggerated airway narrowing.

Deep inspirations protect against airway closure in nonasthmatic subjects

The mechanism by which deep inspirations protect against increased airway responsiveness in nonasthmatic subjects is not known. The aim was to investigate the role of airway closure and airway narrowing in deep inspiration bronchoprotection. Twelve nonasthmatic and nine asthmatic subjects avoided deep inspirations (DI) for 20 min, then took five DI expired to functional residual capaciy (DI-FRC) or, on a separate day, no DI (no DI) before inhaling a single dose of methacholine. On another day, eight nonasthmatic subjects took five DI expired to residual volume (DI-RV). Peripheral airway function was measured by respiratory system reactance (Xrs), using the forced oscillation technique, and by forced vital capacity (FVC) as an index of airway closure. Respiratory system resistance (Rrs) and forced expiratory volume in 1 s (FEV1)/FVC were measured as indexes of airway narrowing. In nonasthmatic subjects, DI-FRC reduced the response measured by FEV1 (P=0.019), Xrs (P=0.02), and FVC (P=0.0005) but not by Rrs (P=0.15) or FEV1/FVC (P=0.52) compared with no DI. DI-RV had a less protective effect than DI-FRC on response measured by FEV1 (P=0.04) and FVC (P=0.016). There was no difference between all protocols when the response was measured by Xrs (P=0.20), Rrs (P=0.88), or FEV1/FVC (P=0.88). DI had no effect on methacholine response in asthmatic subjects. DI protect against airway responsiveness through an effect on peripheral airways involving reduced airway closure. The protective effect of DI on FEV1 and FVC was abolished by expiration to residual volume. We speculate that the reduced airway closure is due to reduced baseline ventilation heterogeneity and/or reduced airway surface tension.

Rhinovirus-induced exacerbations of asthma : How is the β2-adrenoceptor implicated?

Rhinovirus (RV) infections are the major cause of asthma exacerbations in children and adults. Under normal circumstances, asthmatic airway obstruction improves spontaneously or characteristically briskly in response to inhaled beta(2)-adrenergic receptor (beta(2)AR) agonists. During virus-associated exacerbations, an impaired response to beta(2)AR agonists is observed; the reason for this is not known. The objective of this study was to determine the effect of RV infection on airway smooth muscle beta(2)AR function. The human cell line Beas-2B and primary human bronchial epithelial cells (HBECs) were infected with RV (multiplicity of infection = 1). After 1 or 5 days for primary and Beas-2B cells, respectively, cell culture supernatants were harvested, UV-irradiated to inactivate RV, and applied to human airway smooth muscle cells for 3 days to assess modifications of beta(2)AR function. RV conditioned medium from Beas-2B and HBECs decreased beta(2)AR agonist-induced cAMP by 50 and 65%, respectively (n = 5; P < 0.05). When cAMP was induced independently of the beta(2)AR using forskolin, no impairment was found. Using flow cytometry, we demonstrated that this decrease was likely the result of beta(2)AR desensitization because membrane but not total cell receptor beta(2)AR was decreased. Pretreatment of HBECs and Beas-2B cells but not human airway smooth muscle cells with the corticosteroids dexamethasone or fluticasone abolished virus-mediated beta(2)AR loss of function. This study shows that epithelial infection with RV induces a decrease of beta(2)AR function on airway smooth muscle cells, potentially explaining the clinical observation of loss of beta(2)AR agonist function during RV-induced asthma exacerbations.

Characterising the mechanism of airway smooth muscle β2 adrenoceptor desensitization by rhinovirus infected bronchial epithelial cells

Rhinovirus (RV) infections account for approximately two thirds of all virus-induced asthma exacerbations and often result in an impaired response to β2 agonist therapy. Using an in vitro model of RV infection, we investigated the mechanisms underlying RV-induced β2 adrenoceptor desensitization in primary human airway smooth muscle cells (ASMC). RV infection of primary human bronchial epithelial cells (HBEC) for 24 hours produced conditioned medium that caused β2 adrenoceptor desensitization on ASMCs without an effect on ASMCs viability. Less than 3 kDa size fractionation together with trypsin digestion of RV-induced conditioned medium did not prevent β2 adrenoceptor desensitization, suggesting it could potentially be mediated by a small peptide or lipid. RV infection of BECs, ASMCs and fibroblasts produced prostaglandins, of which PGE2, PGF2α and PGI2 had the ability to cause β2 adrenoceptor desensitization on ASMCs. RV-induced conditioned medium from HBECs depleted of PGE2 did not prevent ASMC β2 adrenoceptor desensitization; however this medium induced PGE2 from ASMCs, suggesting that autocrine prostaglandin production may be responsible. Using inhibitors of cyclooxygenase and prostaglandin receptor antagonists, we found that β2 adrenoceptor desensitization was mediated through ASMC derived COX-2 induced prostaglandins. Since ASMC prostaglandin production is unlikely to be caused by RV-induced epithelial derived proteins or lipids we next investigated activation of toll-like receptors (TLR) by viral RNA. The combination of TLR agonists poly I:C and imiquimod induced PGE2 and β2 adrenoceptor desensitization on ASMC as did the RNA extracted from RV-induced conditioned medium. Viral RNA but not epithelial RNA caused β2 adrenoceptor desensitization confirming that viral RNA and not endogenous human RNA was responsible. It was deduced that the mechanism by which β2 adrenoceptor desensitization occurs was by pattern recognition receptor activation of COX-2 induced prostaglandins.

Airway mechanics and methods used to visualize smooth muscle dynamics in vitro

Contraction of airway smooth muscle (ASM) is regulated by the physiological, structural and mechanical environment in the lung. We review two in vitro techniques, lung slices and airway segment preparations, that enable in situ ASM contraction and airway narrowing to be visualized. Lung slices and airway segment approaches bridge a gap between cell culture and isolated ASM, and whole animal studies. Imaging techniques enable key upstream events involved in airway narrowing, such as ASM cell signalling and structural and mechanical events impinging on ASM, to be investigated.

Diagnosis of oligodendroglioma: Molecular and classical histological assessment in the twenty‐first century

Advances in molecular genetics are currently challenging the traditional morphological categorization of gliomas. Recurrent molecular and cytogenetic aberrations add prognostic and predictive information over and above that provided by standard histomorphological techniques and may influence decisions to re‐operate or observe, to deliver radiation or not, or to administer chemotherapy to glioma patients. The importance of routine hematoxylin and eosin (H–E pathological stains cannot be underestimated, especially in resource‐poor areas and developing countries where there is likely to be a significant economic opportunity cost for molecular diagnosis services. New research tools for image analyses of histological H–E slides, such as the precise measures of cell area, curvature and nuclear roundness, may provide an increased ability to provide an accurate classification for an inherently subjective process of histological assessment. We discuss the current trends, limitations and impact of molecular classification in this mini review.

Latrophilin receptors: novel bronchodilator targets in asthma

Background Asthma affects 300 million people worldwide. In asthma, the major cause of morbidity and mortality is acute airway narrowing, due to airway smooth muscle (ASM) hypercontraction, associated with airway remodelling. However, little is known about the transcriptional differences between healthy and asthmatic ASM cells. Objectives To investigate the transcriptional differences between asthmatic and healthy airway smooth muscle cells (ASMC) in culture and investigate the identified targets using in vitro and ex vivo techniques. Methods Human asthmatic and healthy ASMC grown in culture were run on Affymetrix_Hugene_1.0_ST microarrays. Identified candidates were confirmed by PCR, and immunohistochemistry. Functional analysis was conducted using in vitro ASMC proliferation, attachment and contraction assays and ex vivo contraction of mouse airways. Results We suggest a novel role for latrophilin (LPHN) receptors, finding increased expression on ASMC from asthmatics, compared with non-asthmatics in vivo and in vitro, suggesting a role in mediating airway function. A single nucleotide polymorphism in LPHN1 was associated with asthma and with increased LPHN1 expression in lung tissue. When activated, LPHNs regulated ASMC adhesion and proliferation in vitro, and promoted contraction of mouse airways and ASMC. Conclusions Given the need for novel inhibitors of airway remodelling and bronchodilators in asthma, the LPHN family may represent promising novel targets for future dual therapeutic intervention.