Diminished sarco/endoplasmic reticulum Ca2+ ATPase (SERCA) expression contributes to airway remodelling in bronchial asthma.

Phenotypic modulation of airway smooth muscle (ASM) is an important feature of airway remodeling in asthma that is characterized by enhanced proliferation and secretion of pro-inflammatory chemokines. These activities are regulated by the concentration of free Ca(2+) in the cytosol ([Ca(2+)](i)). A rise in [Ca(2+)](i) is normalized by rapid reuptake of Ca(2+) into sarcoplasmic reticulum (SR) stores by the sarco/endoplasmic reticulum Ca(2+) (SERCA) pump. We examined whether increased proliferative and secretory responses of ASM from asthmatics result from reduced SERCA expression. ASM cells were cultured from subjects with and without asthma. SERCA expression was evaluated by western blot, immunohistochemistry and real-time PCR. Changes in [Ca(2+)](i), cell spreading, cellular proliferation, and eotaxin-1 release were measured. Compared with control cells from healthy subjects, SERCA2 mRNA and protein expression was reduced in ASM cells from subjects with moderately severe asthma. SERCA2 expression was similarly reduced in ASM in vivo in subjects with moderate/severe asthma. Rises in [Ca(2+)](i) following cell surface receptor-induced SR activation, or inhibition of SERCA-mediated Ca(2+) re-uptake, were attenuated in ASM cells from asthmatics. Likewise, the return to baseline of [Ca](i) after stimulation by bradykinin was delayed by approximately 50% in ASM cells from asthmatics. siRNA-mediated knockdown of SERCA2 in ASM from healthy subjects increased cell spreading, eotaxin-1 release and proliferation. Our findings implicate a deficiency in SERCA2 in ASM in asthma that contributes to its secretory and hyperproliferative phenotype in asthma, and which may play a key role in mechanisms of airway remodeling.

Repeated allergen inhalation induces cytoskeletal remodeling in smooth muscle from rat bronchioles.

Airway hyperresponsiveness (AHR) is associated with airway wall structural remodeling and alterations in airway smooth muscle (ASM) function. Previously, in bronchioles from Brown Norway rats challenged by repeated ovalbumin (OVA) inhalation, we have reported increased force generation and depletion of smooth muscle contractile proteins. Here, we investigated if cytoskeletal changes in smooth muscle could account for this paradox. Sensitized rats (n = 5/group) were repeatedly challenged with OVA or saline, and the lungs were removed 24 h after the last challenge. Levels of globular (G) and filamentous (F) actin in bronchioles were determined by DNase I inhibition and contraction assessed in intact small bronchioles using a myograph. DNase I inhibition assays showed that G-actin monomers were more abundant ( approximately 1F:2G) in extracts from resting small bronchioles from OVA- or saline-challenged animals. However, while contractile protein levels in bronchioles were reduced by OVA (P < 0.05), the proportion of F:G actin was 1.8-fold greater compared with saline challenge (P < 0.05). Consistent with induction of F-actin after OVA challenge, increases in maximum tension development to carbachol or KCl in small bronchioles from OVA-challenged animals were abrogated (P < 0.01) by actin cytoskeleton disruption with 0.5 microM latrunculin A. Cytoskeletal stabilization of F-actin with 0.1 microM jasplakinolide potentiated maximum contractions to carbachol or KCl (P < 0.05) in bronchioles from OVA- but not saline-treated rats. We conclude that alterations in the composition and/or arrangement of the contractile apparatus after OVA exposure confer enhanced contractile responses, possibly as a result of increased F-actin content. Such a mechanism may have relevance for AHR found in allergic asthma.

Repeated allergen inhalation induces phenotypic modulation of smooth muscle in bronchioles of sensitized rats.

Repeated ovalbumin (OA) or saline exposure of sensitized Brown Norway rats was examined on agonist reactivity, airway smooth muscle (ASM) content, and contractile protein expression in small bronchioles at 24 h, 7 days, and 35 days after challenge. OA increased ASM content (P < 0.05 vs. saline) at 24 h, which resolved by 7 days. Maximum developed tension (T(max)) to carbachol, KCl, and 4-beta-phorbol 12,13-dibutyrate was increased (P < 0.05) by OA in bronchioles at 24 h but was abrogated after correction for ASM. Differences in T(max) were not present at 7 days. In contrast, at 35 days, T(max) was increased (P < 0.05) after correction for ASM. Smooth muscle (sm)-alpha-actin, sm-myosin heavy chain (MHC) isoform 1, calponin, smoothelin-A, and sm-myosin light chain kinase expression were reduced (P < 0.05) by OA at 24 h in bronchioles but not in trachealis. Consistent with contraction findings, no difference in expression of these proteins was detected at 7 days. At 35 days, however, with the exception of sm-alpha-actin, their abundance was again reduced (P < 0.05) by OA. Nonmuscle MHC and beta-actin were unchanged throughout by OA. These findings indicate persistent changes in contractile protein content, consistent with ASM phenotypic modulation in vivo, which occur in response to repeated OA inhalation. Thus, OA exposure induces structural changes in bronchiole ASM content and in agonist responsiveness ex vivo that resemble remodeling in asthma.

Proliferative aspects of airway smooth muscle.

Increased airway smooth muscle (ASM) mass is perhaps the most important component of the airway wall remodeling process in asthma. Known mediators of ASM proliferation in cell culture models fall into 2 categories: those that activate receptors with intrinsic receptor tyrosine kinase activity and those that have their effects through receptors linked to heterotrimeric guanosine triphosphate-binding proteins. The major candidate signaling pathways activated by ASM mitogens are those dependent on extracellular signal-regulated kinase and phosphoinositide 3'-kinase. Increases in ASM mass may also involve ASM migration, and in culture, the key signaling mechanisms have been identified as the p38 mitogen-activated protein kinase and the p21-activated kinase 1 pathways. New evidence from an in vivo rat model indicates that primed CD4(+) T cells are sufficient to trigger ASM and epithelial remodeling after allergen challenge. Hyperplasia has been observed in an equine model of asthma and may account for the increase in ASM mass. Reduction in the rate of apoptosis may also play a role. beta(2)-Adrenergic receptor agonists and glucocorticoids have antiproliferative activity against a broad spectrum of mitogens, although it has become apparent that mitogens are differentially sensitive. Culture of ASM on collagen type I has been shown to enhance proliferative activity and prevent the inhibitory effect of glucocorticoids, whereas beta(2)-agonists are minimally affected. There is no evidence that long-acting beta(2)-agonists are more effective than short-acting agonists, but persistent stimulation of the beta(2)-adrenergic receptor probably helps suppress growth responses. The maximum response of fluticasone propionate against thrombin-induced proliferation is increased when it is combined with salmeterol.