Airway smooth muscle pathophysiology in asthma.

The airway smooth muscle (ASM) cell plays a central role in the pathogenesis of asthma and constitutes an important target for treatment. These cells control muscle tone and thus regulate the opening of the airway lumen and air passage. Evidence indicates that ASM cells participate in the airway hyperresponsiveness as well as the inflammatory and remodeling processes observed in asthmatic subjects. Therapeutic approaches require a comprehensive understanding of the structure and function of the ASM in both the normal and disease states. This review updates current knowledge about ASM and its effects on airway narrowing, remodeling, and inflammation in asthma.

An Official American Thoracic Society Research Statement: Current Challenges Facing Research and Therapeutic Advances in Airway Remodeling.

BACKGROUND: Airway remodeling (AR) is a prominent feature of asthma and other obstructive lung diseases that is minimally affected by current treatments. The goals of this Official American Thoracic Society (ATS) Research Statement are to discuss the scientific, technological, economic, and regulatory issues that deter progress of AR research and development of therapeutics targeting AR and to propose approaches and solutions to these specific problems. This Statement is not intended to provide clinical practice recommendations on any disease in which AR is observed and/or plays a role. METHODS: An international multidisciplinary group from within academia, industry, and the National Institutes of Health, with expertise in multimodal approaches to the study of airway structure and function, pulmonary research and clinical practice in obstructive lung disease, and drug discovery platforms was invited to participate in one internet-based and one face-to-face meeting to address the above-stated goals. Although the majority of the analysis related to AR was in asthma, AR in other diseases was also discussed and considered in the recommendations. A literature search of PubMed was performed to support conclusions. The search was not a systematic review of the evidence. RESULTS: Multiple conceptual, logistical, economic, and regulatory deterrents were identified that limit the performance of AR research and impede accelerated, intensive development of AR-focused therapeutics. Complementary solutions that leverage expertise of academia and industry were proposed to address them. CONCLUSIONS: To date, numerous factors related to the intrinsic difficulty in performing AR research, and economic forces that are disincentives for the pursuit of AR treatments, have thwarted the ability to understand AR pathology and mechanisms and to address it clinically. This ATS Research Statement identifies potential solutions for each of these factors and emphasizes the importance of educating the global research community as to the extent of the problem as a critical first step in developing effective strategies for: (1) increasing the extent and impact of AR research and (2) developing, testing, and ultimately improving drugs targeting AR.

Cyclooxygenase-2 and microRNA-155 expression are elevated in asthmatic airway smooth muscle cells.

Cyclooxygenase-2 (COX-2) expression and PGE2 secretion from human airway smooth muscle cells (hASMCs) may contribute to ß2-adrenoceptor hyporesponsiveness, a clinical feature observed in some patients with asthma. hASMCs from patients with asthma exhibit elevated expression of cytokine-responsive genes, and in some instances this is attributable to an altered histone code and/or microRNA expression. We hypothesized that COX-2 expression and PGE2 secretion might be elevated in asthmatic hASMCs in response to proinflammatory signals in part due to altered histone acetylation and/or microRNA expression. hASMCs obtained from nonasthmatic and asthmatic human subjects were treated with cytomix (IL-1ß, TNF-α, and IFN-γ). A greater elevation of COX-2 mRNA, COX-2 protein, and PGE2 secretion was observed in the asthmatic cells. We investigated histone H3/H4-acetylation, transcription factor binding, mRNA stability, p38 mitogen-activated protein kinase signaling, and microRNA (miR)-155 expression as potential mechanisms responsible for the differential elevation of COX-2 expression. We found that histone H3/H4-acetylation and transcription factor binding to the COX-2 promoter were similar in both groups, and histone H3/H4-acetylation did not increase after cytomix treatment. Cytomix treatment elevated NF-κB and RNA polymerase II binding to similar levels in both groups. COX-2 mRNA stability was increased in asthmatic cells. MiR-155 expression was higher in cytomix-treated asthmatic cells, and we show it enhances COX-2 expression and PGE2 secretion in asthmatic and nonasthmatic hASMCs. Thus, miR-155 expression positively correlates with COX-2 expression in the asthmatic hASMCs and may contribute to the elevated expression observed in these cells. These findings may explain, at least in part, ß2-adrenoceptor hyporesponsiveness in patients with asthma.

MicroRNA-146a and microRNA-146b expression and anti-inflammatory function in human airway smooth muscle.

MicroRNA (miR)-146a and miR-146b are negative regulators of inflammatory gene expression in lung fibroblasts, epithelial cells, monocytes, and endothelial cells. The abundance of cyclooxygenase-2 (COX-2) and IL-1ß is negatively regulated by the miR-146 family, suggesting miR-146a and/or miR-146b might modulate inflammatory mediator expression in airway smooth muscle thereby contributing to pathogenesis of asthma. To test this idea we compared miR-146a and miR-146b expression in human airway smooth muscle cells (hASMCs) from nonasthmatic and asthmatic subjects treated with cytomix (IL-1ß, TNF-α, and IFNγ) and examined the miRNAs' effects on COX-2 and IL-1ß expression. We found that cytomix treatment elevated miR-146a and miR-146b abundance. Induction with cytomix was greater than induction with individual cytokines, and asthmatic cells exhibited higher levels of miR-146a expression following cytomix treatment than nonasthmatic cells. Transfection of miR-146a or miR-146b mimics reduced COX-2 and IL-1ß expression. A miR-146a inhibitor increased COX-2 and IL-1ß expression, but a miR-146b inhibitor was ineffective. Repression of COX-2 and IL-1ß expression by miR-146a correlated with reduced abundance of the RNA-binding protein human antigen R. These results demonstrate that miR-146a and miR-146b expression is inducible in hASMCs by proinflammatory cytokines and that miR-146a expression is greater in asthmatic cells. Both miR-146a and miR-146b can negatively regulate COX-2 and IL-1ß expression at pharmacological levels, but loss-of-function studies showed that only miR-146a is an endogenous negative regulator in hASMCs. The results suggest miR-146 mimics may be an attractive candidate for further preclinical studies as an anti-inflammatory treatment of asthma.

Targeted transgenesis identifies Gαs as the bottleneck in ß2-adrenergic receptor cell signaling and physiological function in airway smooth muscle.

G protein-coupled receptors are the most pervasive signaling superfamily in the body and act as receptors to endogenous agonists and drugs. For ß-agonist-mediated bronchodilation, the receptor-G protein-effector network consists of the ß2-adrenergic receptor (ß2AR), Gs, and adenylyl cyclase, expressed on airway smooth muscle (ASM). Using ASM-targeted transgenesis, we previously explored which of these three early signaling elements represents a limiting factor, or bottleneck, in transmission of the signal from agonist binding to ASM relaxation. Here we overexpressed Gαs in transgenic mice and found that agonist-promoted relaxation of airways was enhanced in direct proportion to the level of Gαs expression. Contraction of ASM from acetylcholine was not affected in Gαs transgenic mice, nor was relaxation by bitter taste receptors. Furthermore, agonist-promoted (but not basal) cAMP production in ASM cells from Gαs-transgenic mice was enhanced compared with ASM from nontransgenic littermates. Agonist-promoted inhibition of platelet-derived growth factor-stimulated ASM proliferation was also enhanced in Gαs mouse ASM. The enhanced maximal ß-agonist response was of similar magnitude for relaxation, cAMP production, and growth inhibition. Taken together, it appears that a limiting factor in ß-agonist responsiveness in ASM is the expression level of Gαs. Gene therapy or pharmacological means of increasing Gαs (or its coupling efficiency to ß2AR) thus represent an interface for development of novel therapeutic agents for improvement of ß-agonist therapy.

Transforming growth factor-ß-induced differentiation of airway smooth muscle cells is inhibited by fibroblast growth factor-2.

In asthma, basic fibroblast growth factor (FGF-2) plays an important (patho)physiological role. This study examines the effects of FGF-2 on the transforming growth factor-ß (TGF-ß)-stimulated differentiation of airway smooth muscle (ASM) cells in vitro. The differentiation of human ASM cells after incubation with TGF-ß (100 pM) and/or FGF-2 (300 pM) for 48 hours was assessed by increases in contractile protein expression, actin-cytoskeleton reorganization, enhancements in cell stiffness, and collagen remodeling. FGF-2 inhibited TGF-ß-stimulated increases in transgelin (SM22) and calponin gene expression (n = 15, P < 0.01) in an extracellular signal-regulated kinase 1/2 (ERK1/2) signal transduction-dependent manner. The abundance of ordered α-smooth muscle actin (α-SMA) filaments formed in the presence of TGF-ß were also reduced by FGF-2, as was the ratio of F-actin to G-actin (n = 8, P < 0.01). Furthermore, FGF-2 attenuated TGF-ß-stimulated increases in ASM cell stiffness and the ASM-mediated contraction of lattices, composed of collagen fibrils (n = 5, P < 0.01). However, the TGF-ß-stimulated production of IL-6 was not influenced by FGF-2 (n = 4, P > 0.05), suggesting that FGF-2 antagonism is selective for the regulation of ASM cell contractile protein expression, organization, and function. Another mitogen, thrombin (0.3 U ml(-1)), exerted no effect on TGF-ß-regulated contractile protein expression (n = 8, P > 0.05), α-SMA organization, or the ratio of F-actin to G-actin (n = 4, P > 0.05), suggesting that the inhibitory effect of FGF-2 is dissociated from its mitogenic actions. The addition of FGF-2, 24 hours after TGF-ß treatment, still reduced contractile protein expression, even when the TGF-ß-receptor kinase inhibitor, SB431542 (10 µM), was added 1 hour before FGF-2. We conclude that the ASM cell differentiation promoted by TGF-ß is antagonized by FGF-2. A better understanding of the mechanism of action for FGF-2 is necessary to develop a strategy for therapeutic exploitation in the treatment of asthma.

Pro-inflammatory and immunomodulatory functions of airway smooth muscle: emerging concepts.

Airway smooth muscle (ASM) is the main regulator of bronchomotor tone. Extensive studies show that in addition to their physical property, human airway smooth muscle (ASM) cells can participate in inflammatory processes modulating the initiation, perpetuation, amplification, and perhaps resolution of airway inflammation. Upon stimulation or interaction with immune cells, ASM cells produce and secrete a variety of inflammatory cytokines and chemokines, cell adhesion molecules, and extracellular matrix (ECM) proteins. These released mediators can, in turn, contribute to the inflammatory state, airway hyperresponsiveness, and airway remodeling present in asthma. As our knowledge of ASM myocyte biology improves, novel bioactive factors are emerging as potentially important regulators of inflammation. This review provides an overview of our understanding of some of these molecules, identifies rising questions, and proposes future studies to better define their role in ASM cell modulation of inflammation and immunity in the lung and respiratory diseases.

Upstream stimulatory factor 1 activates GATA5 expression through an E-box motif.

Silencing of GATA5 gene expression as a result of promoter hypermethylation has been observed in lung, gastrointestinal and ovarian cancers. However, the regulation of GATA5 gene expression has been poorly understood. In the present study, we have demonstrated that an E (enhancer)-box in the GATA5 promoter (bp -118 to -113 in mice; bp -164 to -159 in humans) positively regulates GATA5 transcription by binding USF1 (upstream stimulatory factor 1). Using site-directed mutagenesis, EMSA (electrophoretic mobility-shift analysis) and affinity chromatography, we found that USF1 specifically binds to the E-box sequence (5'-CACGTG-3'), but not to a mutated E-box. CpG methylation of this E-box significantly diminished its binding of transcription factors. Mutation of the E-box within a GATA5 promoter fragment significantly decreased promoter activity in a luciferase reporter assay. Chromatin immunoprecipitation identified that USF1 physiologically interacts with the GATA5 promoter E-box in mouse intestinal mucosa, which has the highest GATA5 gene expression in mouse. Co-transfection with a USF1 expression plasmid significantly increased GATA5 promoter-driven luciferase transcription. Furthermore, real-time and RT (reverse transcription)-PCR analyses confirmed that overexpression of USF1 activates endogenous GATA5 gene expression in human bronchial epithelial cells. The present study provides the first evidence that USF1 activates GATA5 gene expression through the E-box motif and suggests a potential mechanism (disruption of the E-box) by which GATA5 promoter methylation reduces GATA5 expression in cancer.

Building Career Paths for Ph.D., Basic and Translational Scientists in Clinical Departments in the United States: An Official American Thoracic Society Workshop Report.

Rationale: To identify barriers and opportunities for Ph.D., basic and translational scientists to be fully integrated into clinical units. Objectives: In 2022, an ad hoc committee of the American Thoracic Society developed a project proposal and workshop to identify opportunities and barriers for scientists who do not practice medicine to develop successful careers and achieve tenure-track faculty positions in clinical departments and divisions within academic medical centers (AMCs) in the United States. Methods: This document focuses on results from a survey of adult and pediatric pulmonary, critical care, and sleep medicine division chiefs as well as a survey of workshop participants, including faculty in departmental and school leadership roles in both basic science and clinical units within U.S. AMCs. Results: We conclude that full integration of non-clinically practicing basic and translational scientists into the clinical units, in addition to their traditional placements in basic science units, best serves the tripartite mission of AMCs to provide care, perform research, and educate the next generation. Evidence suggests clinical units do employ Ph.D. scientists in large numbers, but these faculty are often hired into non-tenure track positions, which do not provide the salary support, start-up funds, research independence, or space often associated with hiring in basic science units within the same institution. These barriers to success of Ph.D. faculty in clinical units are largely financial. Conclusions: Our recommendation is for AMCs to consider and explore some of our proposed strategies to accomplish the goal of integrating basic and translational scientists into clinical units in a meaningful way.

Nuclear import of serum response factor in airway smooth muscle.

We have previously shown that the transcription-promoting activity of serum response factor (SRF) is partially regulated by its extranuclear redistribution. In this study, we examined the cellular mechanisms that facilitate SRF nuclear entry in canine tracheal smooth muscle cells. We used in vitro pull-down assays to determine which karyopherin proteins bound SRF and found that SRF binds KPNA1 and KPNB1 through its nuclear localization sequence. Immunoprecipitation studies also demonstrated direct SRF-KPNA1 interaction in HEK293 cells. Import assays demonstrated that KPNA1 and KPNB1 together were sufficient to mediate rapid nuclear import of SRF-GFP. Our studies also suggest that SRF is able to gain nuclear entry through an auxiliary, nuclear localization sequence-independent mechanism.

Akt activation induces hypertrophy without contractile phenotypic maturation in airway smooth muscle.

Airway smooth muscle (ASM) hypertrophy is a cardinal feature of severe asthma, but the underlying molecular mechanisms remain uncertain. Forced protein kinase B/Akt 1 activation is known to induce myocyte hypertrophy in other muscle types, and, since a number of mediators present in asthmatic airways can activate Akt signaling, we hypothesized that Akt activation could contribute to ASM hypertrophy in asthma. To test this hypothesis, we evaluated whether Akt activation occurs naturally within airway myocytes in situ, whether Akt1 activation is sufficient to cause hypertrophy of normal airway myocytes, and whether such hypertrophy is accompanied by excessive accumulation of contractile apparatus proteins (contractile phenotype maturation). Immunostains of human airway sections revealed concordant activation of Akt (reflected in Ser(473) phosphorylation) and of its downstream effector p70(S6Kinase) (reflected in Thr(389) phosphorylation) within airway muscle bundles, but there was no phosphorylation of the alternative Akt downstream target glycogen synthase kinase (GSK) 3ß. Artificial overexpression of constitutively active Akt1 (by plasmid transduction or lentiviral infection) caused a progressive increase in size and protein content of cultured canine tracheal myocytes and increased p70(S6Kinase) phosphorylation but not GSK3ß phosphorylation; however, constitutively active Akt1 did not cause disproportionate overaccumulation of smooth muscle (sm) α-actin and SM22. Furthermore, mRNAs encoding sm-α-actin and SM22 were reduced. These results indicate that forced Akt1 signaling causes hypertrophy of cultured airway myocytes without inducing further contractile phenotypic maturation, possibly because of opposing effects on contractile protein gene transcription and translation, and suggest that natural activation of Akt1 plays a similar role in asthmatic ASM.

Fibrillar collagen clamps lung mesenchymal cells in a nonproliferative and noncontractile phenotype.

Pulmonary fibrosis is characterized by phenotypic changes to mesenchymal cells and an increase in the deposition of fibrillar collagen (fCollagen). This study investigated the effect of type I fCollagen on the phenotypic plasticity of human parenchymal fibroblasts (PFbs) in vitro. Cell numbers were 45% lower when cultured on fCollagen as compared with culture on its degradation product, monomeric collagen (mCollagen). DNA profiles indicated that fCollagen is antiproliferative, rather than proapoptotic. fCollagen suppressed basic fibroblast growth factor-stimulated increases in the levels of cyclin E and CDK2 mRNA. fCollagen also suppressed transforming growth factor-beta (100 pM)-stimulated increases in the mRNA and protein levels of alpha-smooth muscle actin (alpha-SMA), a marker of the myofibroblast phenotype. However, in cells exposed to fCollagen, the levels of matrix metalloproteinase (MMP)-1 and -14 mRNA, as well as active MMP-2 protein, were increased by between two- and fivefold. The MMP inhibitors, ilomastat (10 microM) and doxycycline (30 microM), attenuated the dissolution of collagen fibrils by fibroblasts maintained on fCollagen, with a corresponding decrease in cell number. Ilomastat also reduced alpha-SMA expression and the capacity of PFb to contract three-dimensional fCollagen gels. Thus, exposure of fibroblasts to the fibrillar form of type I collagen in vitro reduces cell proliferation, increases MMP production and activation, and attenuates differentiation of PFb into myofibroblasts. fCollagen appears to apply a phenotypic clamp on lung fibroblasts that may be partially released by autocrine MMP activity.

Alternative promoter and GATA5 transcripts in mouse.

GATA5 is a member of the GATA zinc finger transcription factor family involved in tissue-specific transcriptional regulation during cell differentiation and embryogenesis. Previous reports indicate that null mutation of the zebrafish GATA5 gene results in embryonic lethality, whereas deletion of exon 1 from the mouse GATA5 gene causes only derangement of female urogenital development. Here, we have identified an alternate promoter within intron 1 of the mouse GATA5 gene that transcribes a 2.5-kb mRNA that lacks exon 1 entirely but includes 82 bp from intron 1 and all of exons 2-6. The alternative promoter was active during transient transfection in cultured airway myocytes and bronchial epithelial cells, and it drove reporter gene expression in gastric epithelial cells in transgenic mice. The 2.5-kb alternative transcript encodes an NH(2)-terminally truncated "short GATA5" comprising aa 226-404 with a single zinc finger, which retains ability to transactivate the atrial natriuretic factor promoter (albeit less efficiently than full-length GATA5). Another new GATA5 transcript contains all of exons 1-5 and the 5' portion of exon 6 but lacks the terminal 1143 bp of the 3'-untranslated region from exon 6. These findings extend current understanding of the tissue distribution of GATA5 expression and suggests that GATA5 expression and function are more complex than previously appreciated.

Regulation of Smad-mediated gene transcription by RGS3.

Regulator of G protein signaling (RGS) proteins are united into a family by the presence of the homologous RGS domain that binds the alpha subunits of heterotrimeric G proteins and accelerates their GTPase activity. A member of this family, RGS3 regulates the signaling mediated by G(q) and G(i) proteins by binding the corresponding Galpha subunits. Here we show that RGS3 interacts with the novel partners Smad2, Smad3, and Smad4-the transcription factors that are activated through a transforming growth factor-beta (TGF-beta) receptor signaling. This interaction is mediated by the region of RGS3 outside of the RGS domain and by Smad's Mad homology 2 domain. Overexpression of RGS3 results in inhibition of Smad-mediated gene transcription. RGS3 does not affect TGF-beta-induced Smad phosphorylation, but it prevents heteromerization of Smad3 with Smad4, which is required for transcriptional activity of Smads. This translates to functional inhibition of TGF-beta-induced myofibroblast differentiation by RGS3. In conclusion, this study identifies a novel, noncanonical role of RGS3 in regulation of TGF-beta signaling through its interaction with Smads and interfering with Smad heteromerization.

FIZZ1 plays a crucial role in early stage airway remodeling of OVA-induced asthma.

The aim of this study was to elucidate the role of Found in Inflammatory Zone 1 (FIZZ1, also known as RELM-alpha or resistin-like molecule-alpha) in airway remodeling in asthma. We used a rat model of ovalbumin (OVA) sensitization and challenge to induce lung inflammation and remodeling. Expression of alpha -SMA in the lungs of OVA-treated rats was significantly elevated in the peribronchial regions compared with control saline-treated animals. Expression of FIZZ1 mRNA in alveolar epithelial type II cells (AECII) isolated from OVA-treated animals was higher than in control animals. Forced expression of recombinant FIZZ1 in rat-1 lung fibroblast cell line enhanced production of collagen type I and alpha -SMA compared with control transfected cells. These results suggest that FIZZ1 can induce fibroblasts to express markers of myofibroblast differentiation such as alpha -SMA and collagen type I, which are characteristic of early stages of airway remodeling seen in asthma.

Transforming growth factor-beta1 and disorders of the lung.

The transforming growth factor (TGF) superfamily encompasses about 30 members in mammals. The effect of TGF-beta subfamily members is exerted and regulated via selective pathways of synthesis and signaling that involve activation of latent TGF-beta, specific and high-affinity binding to cell membrane serine/threonine kinase receptors, activation of intracellular cascades that include Smad molecules and mitogen-activated protein kinases, and regulated termination of the effect by diverse mechanisms including protein degradation and transcriptional activation. Several comprehensive reviews on TGF-beta biology in general and on the role of this cytokine in other diseases have been published recently. In recent years an unexpected role of TGF-beta on lung homeostasis has been revealed. Here, we discuss the contribution of TGF-beta to the pathogenesis of asthma and chronic obstructive pulmonary disease, two common illnesses of the lung, as well as of lymphangioleiomyomatosis, a rare disease in women. The information we collate and integrate places TGF-beta at a pivotal point within complex networks that control lung physiology as well as the physiopathology of these lung diseases.