Targeting the restricted α-subunit repertoire of airway smooth muscle GABAA receptors augments airway smooth muscle relaxation

The prevalence of asthma has taken on pandemic proportions. Since this disease predisposes patients to severe acute airway constriction, novel mechanisms capable of promoting airway smooth muscle relaxation would be clinically valuable. We have recently demonstrated that activation of endogenous airway smooth muscle GABA(A) receptors potentiates β-adrenoceptor-mediated relaxation, and molecular analysis of airway smooth muscle reveals that the α-subunit component of these GABA(A) receptors is limited to the α(4)- and α(5)-subunits. We questioned whether ligands with selective affinity for these GABA(A) receptors could promote relaxation of airway smooth muscle. RT-PCR analysis of GABA(A) receptor subunits was performed on RNA isolated by laser capture microdissection from human and guinea pig airway smooth muscle. Membrane potential and chloride-mediated current were measured in response to GABA(A) subunit-selective agonists in cultured human airway smooth muscle cells. Functional relaxation of precontracted guinea pig tracheal rings was assessed in the absence and presence of the α(4)-subunit-selective GABA(A) receptor agonists: gaboxadol, taurine, and a novel 8-methoxy imidazobenzodiazepine (CM-D-45). Only messenger RNA encoding the α(4)- and α(5)-GABA(A) receptor subunits was identified in RNA isolated by laser capture dissection from guinea pig and human airway smooth muscle tissues. Activation of airway smooth muscle GABA(A) receptors with agonists selective for these subunits resulted in appropriate membrane potential changes and chloride currents and promoted relaxation of airway smooth muscle. In conclusion, selective subunit targeting of endogenous airway smooth muscle-specific GABA(A) receptors may represent a novel therapeutic option for patients in severe bronchospasm.     

Disruption of TGF-β signaling in smooth muscle cell prevents flow-induced vascular remodeling

Transforming growth factor-β (TGF-β) signaling has been prominently implicated in the pathogenesis of vascular remodeling, especially the initiation and progression of flow-induced vascular remodeling. Smooth muscle cells (SMCs) are the principal resident cells in arterial wall and are critical for arterial remodeling. However, the role of TGF-β signaling in SMC for flow-induced vascular remodeling remains unknown. Therefore, the goal of our study was to determine the effect of TGF-β pathway in SMC for vascular remodeling, by using a genetical smooth muscle-specific (SM-specific) TGF-β type II receptor (Tgfbr2) deletion mice model. Mice deficient in the expression of Tgfbr2 (MyhCre.Tgfbr2^f/f) and their corresponding wild-type background mice (MyhCre.Tgfbr2^WT/WT) underwent partial ligation of left common carotid artery for 1, 2, or 4 weeks. Then the carotid arteries were harvested and indicated that the disruption of Tgfbr2 in SMC provided prominent inhibition of vascular remodeling. And the thickening of carotid media, proliferation of SMC, infiltration of macrophage, and expression of matrix metalloproteinase (MMP) were all significantly attenuated in Tgfbr2 disruption mice. Our study demonstrated, for the first time, that the TGF-β signaling in SMC plays an essential role in flow-induced vascular remodeling and disruption can prevent this process.     

Disruption of TGF-β signaling in smooth muscle cell prevents elastase-induced abdominal aortic aneurysm

Transforming growth factor-β (TGF-β) signaling has been significantly implicated in the pathogenesis of aneurysm, prominently the initiation and progression of abdominal aortic aneurysm (AAA). Vascular smooth muscle cell (SMC) is the principal resident cell in aortic wall and is essential for its structure and function. However, the role of TGF-β pathway in SMC for the formation of AAA remains unknown. Therefore, the goal of the present study was to investigate the effect of TGF-β pathway in SMC for AAA pathogenesis, by using a genetical smooth muscle-specific (SM-specific) TGF-βtype II receptor (Tgfbr2) disruption animal model. Mice deficient in the expression of Tgfbr2 (MyhCre.Tgfbr2^f/f and MyhCre.Tgfbr2^WT/f) and their corresponding wild-type background mice (MyhCre.Tgfbr2^WT/WT) underwent AAA induction by infrarenal peri-adventitial application of elastase. Fourteen days after elastase treatment, the aortas were analyzed and indicated that disruption of 1 or 2 alleles of Tgfbr2 in SMC provided markedly step-wise protection from AAA formation. And elastin degradation, medial SMC loss, macrophage infiltration, and matrix metalloproteinases (MMP) expression were all significantly reduced in Tgfbr2 deletion mice. Our study demonstrated, for the first time, that the TGF-β signaling pathway in SMC plays a critical role in AAA and disruption can prevent the aneurysm formation.     

TGF-β regulates Nox4, MnSOD and catalase expression, and IL-6 release in airway smooth muscle cells

Reactive oxygen species (ROS) are generated as a result of normal cellular metabolism, mainly through the mitochondria and peroxisomes, but their release is enhanced by the activation of oxidant enzymes such as NADPH oxidases or downregulation of endogenous antioxidant enzymes such as manganese-superoxide dismutase (MnSOD) and catalase. Transforming growth factor-β (TGF-β), found to be overexpressed in airway smooth muscle (ASM) from asthmatic and chronic obstructive pulmonary disease patients, may be a pivotal regulator of abnormal ASM cell (ASMC) function in these diseases. An important effect of TGF-β on ASMC inflammatory responses is the induction of IL-6 release. TGF-β also triggers intracellular ROS release in ASMCs by upregulation of NADPH oxidase 4 (Nox4). However, the effect of TGF-β on the expression of key antioxidant enzymes and subsequently on oxidant/antioxidant balance is unknown. Moreover, the role of redox-dependent pathways in the mediation of the proinflammatory effects of TGF-β in ASMCs is unclear. In this study, we show that TGF-β induced the expression of Nox4 while at the same time inhibiting the expression of MnSOD and catalase. This change in oxidant/antioxidant enzymes was accompanied by elevated ROS levels and IL-6 release. Further studies revealed a role for Smad3 and phosphatidyl-inositol kinase-mediated pathways in the induction of oxidant/antioxidant imbalance and IL-6 release. The changes in oxidant/antioxidant enzymes and IL-6 release were reversed by the antioxidants N-acetyl-cysteine (NAC) and ebselen through inhibition of Smad3 phosphorylation, indicating redox-dependent activation of Smad3 by TGF-β. Moreover, these findings suggest a potential role for NAC in preventing TGF-β-mediated pro-oxidant and proinflammatory responses in ASMCs. Knockdown of Nox4 using small interfering RNA partially prevented the inhibition of MnSOD but had no effect on catalase and IL-6 expression. These findings provide novel insights into redox regulation of ASM function by TGF-β.     

Could an increase in airway smooth muscle shortening velocity cause airway hyperresponsiveness?

Airway hyperresponsiveness (AHR) is a characteristic feature of asthma. It has been proposed that an increase in the shortening velocity of airway smooth muscle (ASM) could contribute to AHR. To address this possibility, we tested whether an increase in the isotonic shortening velocity of ASM is associated with an increase in the rate and total amount of shortening when ASM is subjected to an oscillating load, as occurs during breathing. Experiments were performed in vitro using 27 rat tracheal ASM strips supramaximally stimulated with methacholine. Isotonic velocity at 20% isometric force (Fiso) was measured, and then the load on the muscle was varied sinusoidally (0.33 ± 0.25 Fiso, 1.2 Hz) for 20 min, while muscle length was measured. A large amplitude oscillation was applied every 4 min to simulate a deep breath. We found that: 1) ASM strips with a higher isotonic velocity shortened more quickly during the force oscillations, both initially (P < 0.001) and after the simulated deep breaths (P = 0.002); 2) ASM strips with a higher isotonic velocity exhibited a greater total shortening during the force oscillation protocol (P < 0.005); and 3) the effect of an increase in isotonic velocity was at least comparable in magnitude to the effect of a proportional increase in ASM force-generating capacity. A cross-bridge model showed that an increase in the total amount of shortening with increased isotonic velocity could be explained by a change in either the cycling rate of phosphorylated cross bridges or the rate of myosin light chain phosphorylation. We conclude that, if asthma involves an increase in ASM velocity, this could be an important factor in the associated AHR.     

IL-17A produced by αβ T cells drives airway hyper-responsiveness in mice and enhances mouse and human airway smooth muscle contraction

Emerging evidence suggests that the T helper 17 (T(H)17) subset of αβ T cells contributes to the development of allergic asthma. In this study, we found that mice lacking the αvβ8 integrin on dendritic cells did not generate T(H)17 cells in the lung and were protected from airway hyper-responsiveness in response to house dust mite and ovalbumin sensitization and challenge. Because loss of T(H)17 cells inhibited airway narrowing without any obvious effects on airway inflammation or epithelial morphology, we examined the direct effects of T(H)17 cytokines on mouse and human airway smooth muscle function. Interleukin-17A (IL-17A), but not IL-17F or IL-22, enhanced contractile force generation of airway smooth muscle through an IL-17 receptor A (IL-17RA)-IL-17RC, nuclear factor κ light-chain enhancer of activated B cells (NF-κB)-ras homolog gene family, member A (RhoA)-Rho-associated coiled-coil containing protein kinase 2 (ROCK2) signaling cascade. Mice lacking integrin αvβ8 on dendritic cells showed impaired activation of this pathway after ovalbumin sensitization and challenge, and the diminished contraction of the tracheal rings in these mice was reversed by IL-17A. These data indicate that the IL-17A produced by T(H)17 cells contributes to allergen-induced airway hyper-responsiveness through direct effects on airway smooth muscle.     

TNF-α and TGF-β synergistically stimulate elongation of human endothelial cells without transdifferentiation to smooth muscle cell phenotype

We earlier reported synergy between tumor necrosis factor-α (TNF-α) and transforming growth factor-β₁ (TGF-β₁) for apoptosis in human umbilical vein endothelium (HUVEC). Here, we study morphological change by circularity measurement of HUVEC surviving this cytokine induced synergistic apoptosis. Contrasting with reports by others studying bovine endothelium, HUVEC did not change morphology in response to TGF-β₁. TNF-α markedly elongated cells (p < 0.001) and this further increased with combination of the two cytokines (p < 0.001), while elongation was accompanied by increased actin stress fibres. Transdifferentiation of HUVEC to a smooth muscle cell phenotype as reported elsewhere was excluded in the current study.     

The mitogenic effect of testosterone and 17β-estradiol on airway smooth muscle cells

Airway disease distribution and/or severity exhibit sex differences suggesting that sex hormones are involved in the respiratory system physiology and pathophysiology. The implication of airway smooth muscle cells (ASMCs) in the physiology of the airways and the pathogenetic mechanism of airway remodeling is of great interest. Therefore, we studied the effect of testosterone and 17β-estradiol on ASMC proliferation and the mechanisms involved.Cell proliferation was estimated using the methyl-[³H]thymidine incorporation and Cell Titer 96^® AQueous One Solution Assay methods. ASMC isolated from adult male or female rabbit trachea were incubated with testosterone (1 pM-1 μM) or 17β-estradiol (1 pM-1 μM), in the presence or absence of the androgen receptor antagonist flutamide (10 nM) or estrogen receptor antagonist ICI182780 (10 nM), as well as of the PI3K inhibitors LY294002 (20 μM) or wortmannin (1 μM), or the MAPK inhibitors PD98059 (100 μM) or U0126 (1 μM).After 24 h of incubation, testosterone and 17β-estradiol increased methyl-[³H]thymidine incorporation and cell number, in ASMC isolated from male or female animals. The induction of ASMC proliferation by testosterone or 17β-estradiol was inhibited by flutamide or ICI182780 respectively, as well as by LY294002, wortmannin, PD98059 or U0126.In conclusion, testosterone and 17β-estradiol have a mitogenic effect on ASMC, which is receptor-mediated and involves the MAPK and PI3K signaling pathways. Moreover, their effect is the same for ASMC from male and female animals. It is possible that gender-related differences in ASMC remodeling, may be influenced by the different patterns of sex steroid hormone secretion in males and females.     

Roxithromycin treatment inhibits TGF-β1-induced activation of ERK and AKT and down-regulation of Caveolin-1 in rat airway smooth muscle cells

Background

Roxithromycin (RXM) has been widely used in asthma treatment; however, the mechanism has not been fully understood. The aim of our study was to investigate the underlying mechanism of RXM treatment in mediating the effect of transforming growth factor (TGF)-β1 on airway smooth muscle cells (ASMCs) proliferation and caveolinn-1 expression.

Methods

Firstly, the rat ovalbumin (OVA) model was built according to the previous papers. Rat ASMCs were prepared and cultured, and then TGF-β1 production in ASMCs was measured by enzyme-linked immunosorbent assay (ELISA). Moreover, the proliferation of ASMCs was determined using cell counting kit (CCK-8) assay. Additionally, the expressions of caveolin-1, phosphorylated-ERK1/2 (p-ERK1/2) and phosphorylated–AKT (p-AKT) in ASMCs treated with or without PD98059 (an ERK1/2 inhibitor), wortannin (a PI3K inhibitor), β-cyclodextrin (β-CD) and RXM were measured by Western blot. Finally, data were evaluated using t–test or one-way ANOVA, and then a P value < 0.05 was set as a threshold.

Results

Compared with normal control, TGF-β1 secretion was significantly increased in asthmatic ASMCs; meanwhile, TGF-β1 promoted ASMCs proliferation (P < 0.05). However, ASMCs proliferation was remarkably inhibited by RXM, β-CD, PD98059 and wortmannin (P < 0.05). Moreover, the expressions of p-ERK1/2 and p-AKT were increased and peaked at 20 min after TGF-β1 stimulation, and then suppressed by RXM. Further, caveolin-1 level was down-regulated by TGF-β1 and up-regulated by inhibitors and RXM.

Conclusion

Our findings demonstrate that RXM treatment inhibits TGF-β1-induced activation of ERK and AKT and down-regulation of caveolin-1, which may be the potential mechanism of RXM protection from chronic inflammatory diseases, including bronchial asthma.     

Salidroside inhibits platelet-derived growth factor–induced proliferation and migration of airway smooth muscle cells

Abnormal proliferation and migration of airway smooth muscle cells (ASMCs) have been found to be important for the airway remodeling during the pathogenesis of asthma. Salidroside a bioactive glucoside that exerts antitumor activity via inhibiting the cell proliferation and migration of cancer cells. The aim of the current study was to evaluate the effects of salidroside on the proliferation and migration of ASMCs. Our results showed that salidroside inhibited the proliferation and migration of ASMCs in response to platelet-derived growth factor (PDGF) stimulation. Salidroside markedly attenuated the PDGF-induced production of matrix metalloproteinase 2 (MMP-2) and MMP-9 in ASMCs. The levels of contractile phenotype markers including smooth muscle α-actin and calponin were reduced in response to PDGF stimulation, which was attenuated by salidroside pretreatment. Salidroside diminished the increase in the expression levels of type I collagen and fibronectin in PDGF-stimulated ASMCs. Furthermore, salidroside blocked the PDGF-induced activation of the nuclear factor-κB (NF-κB) pathway in ASMCs. The results suggested that salidroside functionally regulated the proliferation, migration, phenotype plasticity, and extracellular matrix deposition in PDGF-induced ASMCs and the NF-κB pathway might be implicated in the effects of salidroside on ASMCs induced by PDGF.     

Bronchial inflammation induced PKCζ over-expression: involvement in mechanical properties of airway smooth muscle

Protein kinase C variants (PKCs) have been involved in the control of airway smooth muscle (ASM) tone, and abnormalities in PKC-dependent signaling have been associated with respiratory diseases such as asthma. In this study, the role of atypical PKCζ in airway hyperresponsiveness was investigated, using an in-vitro model of TNFα-treated human bronchi and an in vivo guinea pig model of chronic asthma. Our results demonstrated that PKCζ-specific inhibition produced a significant increase in isoproterenol sensitivity in TNFα-treated bronchi and ovalbumin (OVA)-sensitized guinea pig bronchi. The role of epoxy-eicosanoids, known to exert anti-inflammatory effects in lung, on PKCζ expression and activity in these models was evaluated. An enhanced PKCζ protein expression was delineated in TNFα-treated bronchi when compared with control (untreated) and epoxy-eicosanoid-treated bronchi. Measurements of Ca(2+) sensitivity, performed in TNFα-treated bronchi, demonstrated that treatment with myristoylated (Myr) PKCζ peptide inhibitor resulted in significant reductions of pCa-induced tension. Epoxy-eicosanoid treatments had similar effects on Ca(2+) sensitivity in TNFα-treated bronchi. In control and epoxy-eicosanoid-treated bronchi, the phosphorylated forms of p38MAPK and CPI-17 were significantly decreased compared with the TNFα-treated bronchi. An enhanced expression of PKCζ was ascertained in our in-vivo model of allergic asthma. Hence an increased Ca(2+) sensitivity could be explained by the phosphorylation of p38-MAPK, which in turn leads to phosphorylation and activation of the CPI-17 regulatory protein. This process was reversed upon treatment with the Myr-PKCζ-peptide inhibitor. The present data provide relevant evidence regarding the role of PKCζ in human and rodent models of airways inflammation.     

Conditional inactivation of TGF-β type II receptor in smooth muscle cells and epicardium causes lethal aortic and cardiac defects

To understand the role of TGF-β signaling in cardiovascular development, we generated mice with conditional deletion of the TGF-β type II receptor (TβRII) gene (Tgfbr2) in cells expressing the smooth muscle cell-specific protein SM22α. The SM22α promoter was active in tissues involved in cardiovascular development: vascular smooth muscle cells (VSMCs), epicardium and myocardium. All SM22-Cre^+/−/Tgfbr2 ^flox/flox embryos died during the last third of gestation. About half the mutant embryos exhibited heart defects (ventricular myocardium hypoplasia and septal defects). All mutant embryos displayed profound vascular abnormalities in the descending thoracic aorta (irregular outline and thickness, occasional aneurysms and elastic fiber disarray). Restriction of these defects to the descending thoracic aorta occurred despite similar levels of Tgfbr2 invalidation in the other portions of the aorta, the ductus arteriosus and the pulmonary trunk. Immunocytochemistry identified impairment of VSMC differentiation in the coronary vessels and the descending thoracic aorta as crucial for the defects. Ventricular myocardial hypoplasia, when present, was associated to impaired α-SMA differentiation of the epicardium-derived coronary VSMCs. Tgfbr2 deletion in the VSMCs of the descending thoracic aorta diminished the number of α-SMA-positive VSMC progenitors in the media at E11.5 and drastically decreased tropoelastin (from E11.5) and fibulin-5 (from E.12.5) synthesis and/or deposition. Defective elastogenesis observed in all mutant embryos and the resulting dilatation and probable rupture of the descending thoracic aorta might explain the late embryonic lethality. To conclude, during mouse development, TGF-β plays an irreplaceable role on the differentiation of the VSMCs in the coronary vessels and the descending thoracic aorta.     

Chloride in airway smooth muscle: the ignored anion no longer?

This Perspectives accompanies an Editorial Focus that summarizes new developments concerning the role of chloride in airway smooth muscle physiology. We provide several observations and mechanistic insights to reconcile recent experimental evidence with existing paradigms concerning chloride channel-mediated effects on airway smooth muscle tone. In addition, we highlight the potentially complex and dynamic nature that chloride currents and membrane potential have on calcium handling and airway smooth muscle contractility.     

Inflammation alters regional mitochondrial Ca²⁺ in human airway smooth muscle cells

Regulation of cytosolic Ca(2+) concentration ([Ca(2+)](cyt)) in airway smooth muscle (ASM) is a key aspect of airway contractility and can be modulated by inflammation. Mitochondria have tremendous potential for buffering [Ca(2+)](cyt), helping prevent Ca(2+) overload, and modulating other intracellular events. Here, compartmentalization of mitochondria to different cellular regions may subserve different roles. In the present study, we examined the role of Ca(2+) buffering by mitochondria and mitochondrial Ca(2+) transport mechanisms in the regulation of [Ca(2+)](cyt) in enzymatically dissociated human ASM cells upon exposure to the proinflammatory cytokines TNF-α and IL-13. Cells were loaded simultaneously with fluo-3 AM and rhod-2 AM, and [Ca(2+)](cyt) and mitochondrial Ca(2+) concentration ([Ca(2+)](mito)) were measured, respectively, using real-time two-color fluorescence microscopy in both the perinuclear and distal, perimembranous regions of cells. Histamine induced a rapid increase in both [Ca(2+)](cyt) and [Ca(2+)](mito), with a significant delay in the mitochondrial response. Inhibition of the mitochondrial Na(+)/Ca(2+) exchanger (1 μM CGP-37157) increased [Ca(2+)](mito) responses in perinuclear mitochondria but not distal mitochondria. Inhibition of the mitochondrial uniporter (1 μM Ru360) decreased [Ca(2+)](mito) responses in perinuclear and distal mitochondria. CGP-37157 and Ru360 significantly enhanced histamine-induced [Ca(2+)](cyt). TNF-α and IL-13 both increased [Ca(2+)](cyt), which was associated with decreased [Ca(2+)](mito) in the case of TNF-α but not IL-13. The effects of TNF-α on both [Ca(2+)](cyt) and [Ca(2+)](mito) were affected by CGP-37157 but not by Ru360. Overall, these data demonstrate that in human ASM cells, mitochondria buffer [Ca(2+)](cyt) after agonist stimulation and its enhancement by inflammation. The differential regulation of [Ca(2+)](mito) in different parts of ASM cells may serve to locally regulate Ca(2+) fluxes from intracellular sources versus the plasma membrane as well as respond to differential energy demands at these sites. We propose that such differential mitochondrial regulation, and its disruption, may play a role in airway hyperreactivity in diseases such as asthma, where [Ca(2+)](cyt) is increased.     

TGF-β1 reverses PDGF-stimulated migration of human aortic smooth muscle cells in vitro

Summary Platelet-derived growth factor (PDGF) and transforming growth factor beta-1(TGF-β1) were tested separately or together for the ability to stimulate migration of human aortic vascular smooth muscle cells (VSMC). PDGF (10 ng/ml) stimulated migration of VSMC over a 48-h period. TGF-β1 (10 ng/ml) had no effect on migration during the same period. VSMC exposed simultaneously to both TGF-β1 and PDGF exhibited diminished migration (50%) when compared to cells treated only with PDGF. Cells that migrated in the presence of PDGF possessed short actin cables that extended from cellular processes at the leading edge of migrating cells; focal adhesions containing the α_vβ₃/β₅ integrins localized to the same region. Cells grown in the presence of TGF-β1 exhibited long, intensely stained actin filaments that spanned the entire length of the cell and were similar to untreated control VSMC. Focal adhesions containing α_vβ₃/β₅ distributed evenly on the basal surface in both TGF-β1-treated cells and control cultures. Cellular responses to PDGF were mitigated when TGF-β1 was present in the culture medium. VSMC grown in the presence of both PDGF and TGF-β1 exhibited elongated actin filaments that were similar to nonmotile TGF-β1-treated cultures. Concomitant exposure of VSMC to PDGF and TGF-β1 resulted in focal adhesions that distributed evenly on the lower cell surface. This study suggests that TGF-β1 can partially reverse the stimulatory effect of PDGF on VSMC migration in vitro by modifying the actin cytoskeleton and the distribution of the α _vβ₃/β₅ integrins.     

shRNA targeting β1-integrin suppressed proliferative aspects and migratory properties of airway smooth muscle cells

Dysfunction of airway smooth muscle (ASM) is an essential feature of airway remodeling in chronic asthma. However, the precise mechanisms of this pathological process have not been well studied. In previous study, we found that β1-integrin, which was dramatically upregulated in ASM cells in an asthmatic mouse model, was associated with the cell proliferation. In this study, we employed short hairpin RNA (shRNA) targeting β1-integrin to assess the effect of down-regulation of this receptor on the proliferative aspects and migratory properties of ASM cells in vitro. The cells were treated with shRNA expression vectors directed against β1-integrin, control vectors that included the blank control, empty vector without shRNA, and mismatched shRNA, respectively. The mRNA and protein expressions of β1-integrin were determined by real-time PCR and Western blotting. Cell proliferation was measured by BrdU ELISA and cell cycle by fluorescence-activated cell sorter. Cell apoptosis was detected by Annexin V-PE/7-AAD staining. Cell migration assays were evaluated by transwell assay and expression of IL-6 and IL-8 by ELISA. The results revealed that shRNA targeting β1-integrin significantly decreased the mRNA and protein expressions of β1-integrin, enhanced the proportion of cells in G0/G1 phase, decreased the proportion in S phase, promoted cell apoptosis, inhibited cell proliferation, migration, IL-6 and IL-8 secretion in vitro. In conclusion, the overexpression of β1-integrin in ASM cells is essential for airway dysfunction development because it promotes proliferative aspects and migratory properties of ASM cells. Importantly, shRNA targeting β1-integrin may provide a new approach to preventing airway remodeling in chronic asthma.