Acid-induced modulation of airway basal tone and contractility: role of acid-sensing ion channels (ASICs) and TRPV1 receptor

The role of extracellular acidosis in inflammatory airway diseases is not well known. One consequence of tissue acidification is the stimulation of sensory nerves via the polymodal H(+)-gated transmembrane channels ASICs and TRPV1 receptor. The present study investigated the effect of acidosis on airway basal tone and responsiveness in the guinea pig. Acidosis (pH 6.8, 10 min, 37 degrees C) significantly decreased the basal tone of tracheal rings (p<0.01 vs. paired control). Moreover, pH fall raised the maximal contraction of tracheal rings to acetylcholine (p<0.05 vs. paired control). The pH-induced relaxation of airway basal tone was inhibited by pretreatments with ASIC1a or ASIC3/ASIC2a inhibitors (0.5 mM ibuprofen, 0.1 mM gadolinium), nitric oxide synthase inhibitor (1 mM L-NAME), and guanylate cyclase inhibitor (1 microM ODQ). In contrast, the pH-induced relaxation of airway basal tone was not modified by epithelium removal or pretreatments with a TRPV1 antagonist (1 microM capsazepine), a combination of NK(1,2,3) receptor antagonists (0.1 microM each), a blocker of voltage-sensitive Na(+) channels (1 microM tetrodotoxin), a cyclooxygenase inhibitor with no activity on ASICs (1 microM indomethacin) or ASIC3 and ASIC3/ASIC2b inhibitors (10 nM diclofenac, 1 microM aspirin). Furthermore, acid-induced hyperresponsiveness to acetylcholine was inhibited by epithelium removal, capsazepine, NK(1,2,3) receptor antagonists, tetrodotoxin, amiloride, ibuprofen and diclofenac. In summary, the initial pH-induced airway relaxation seems to be independent of sensory nerves, suggesting a regulation of airway basal tone mediated by smooth muscle ASICs. Conversely, the pH-induced hyperresponsiveness involves sensory nerves-dependent ASICs and TRPV1, and an unknown epithelial component in response to acidosis.     

Tryptase activates TGFbeta in human airway smooth muscle cells via direct proteolysis

Transforming growth factor β (TGFβ) is a key remodelling factor in asthma. It is produced as a latent complex and the main limiting step in TGFβ bioavailability is its activation. Mast cell tryptase has been shown to stimulate the release of functionally active TGFβ from human airway smooth muscle (ASM) cells [P. Berger, P.O. Girodet, H. Begueret, O. Ousova, D.W. Perng, R. Marthan, A.F. Walls, J.M. Tunon de Lara, Tryptase-stimulated human airway smooth muscle cells induce cytokine synthesis and mast cell chemotaxis, FASEB J. 17 (2003) 2139-2141]. The aim of this study was to determine if tryptase could cause TGFβ activation as well as expression in ASM cells via its receptor, proteinase-activated receptor 2 (PAR2). Tryptase caused TGFβ activation without affecting levels of total TGFβ. This effect was inhibited by the selective tryptase inhibitor FUT175 and leupeptin but not mimicked by the PAR2 activating peptide SLIGKV-NH₂. Furthermore, the ASM cells used in the study did not express PAR2. The results indicate that tryptase activates TGFβ via a PAR2-independent proteolytic mechanism in human ASM cells and may help understanding the role of tryptase in asthma.     

Generation of recombinant monoclonal antibodies to study structure-function of envelope protein VP28 of white spot syndrome virus from shrimp

White spot syndrome virus (WSSV) is a major pathogen in shrimp aquaculture. VP28 is one of the most important envelope proteins of WSSV. In this study, a recombinant antibody library, as single-chain fragment variable (scFv) format, displayed on phage was constructed using mRNA from spleen cells of mice immunized with full-length VP28 expressed in Escherichia coli. After several rounds of panning, six scFv antibodies specifically binding to the epitopes in the N-terminal, middle, and C-terminal regions of VP28, respectively, were isolated from the library. Using these scFv antibodies as tools, the epitopes in VP28 were located on the envelope of the virion by immuno-electron microscopy. Neutralization assay with these antibodies in vitro suggested that these epitopes may not be the attachment site of WSSV to host cell receptor. This study provides a new way to investigate the structure and function of the envelope proteins of WSSV.     

The effect of TGF-beta receptor binding peptides on smooth muscle cells

TGF-beta1 is a potent regulator of vascular smooth muscle cell (VSMC) proliferation, migration, and extracellular matrix (ECM) synthesis. In this study, we selected two peptides, IM-1 and IM-2, that bind to the TGF-beta type II receptor (TGF-beta RII) using phage display. IM-1 and IM-2 bind to the TGF-beta RII, with a K(d) of 1 microM. Like TGF-beta, IM-1 induced VSMC chemotaxis and PAI-1 mRNA expression, as determined using Boyden chambers and real time quantitative PCR. In contrast, IM-2 had no effect on VSMC chemotaxis or PAI-1 induction. Induction of ECM synthesis, involving proteins such as osteopontin and alpha-smooth muscle actin, was determined by ELISA. Osteopontin expression was inhibited by both peptides, but TGF-beta-induced alpha-smooth muscle actin expression could only be inhibited by IM-1. In conclusion, IM-1 activity on VSMC is agonistic with TGF-beta, except for ECM synthesis, whereas the IM-2 peptide is antagonistic for some examined TGF-beta functions.     

Role of Abl in airway hyperresponsiveness and airway remodeling

Background

Asthma is a chronic disease that is characterized by airway hyperresponsiveness and airway remodeling. The underlying mechanisms that mediate the pathological processes are not fully understood. Abl is a non-receptor protein tyrosine kinase that has a role in the regulation of smooth muscle contraction and smooth muscle cell proliferation in vitro. The role of Abl in airway hyperresponsiveness and airway remodeling in vivo is largely unknown.

Methods

To evaluate the role of Abl in asthma pathology, we assessed the expression of Abl in airway tissues from the ovalbumin sensitized and challenged mouse model, and human asthmatic airway smooth muscle cells. In addition, we generated conditional knockout mice in which Abl expression in smooth muscle was disrupted, and then evaluated the effects of Abl conditional knockout on airway resistance, smooth muscle mass, cell proliferation, IL-13 and CCL2 in the mouse model of asthma. Furthermore, we determined the effects of the Abl pharmacological inhibitors imatinib and GNF-5 on these processes in the animal model of asthma.

Results

The expression of Abl was upregulated in airway tissues of the animal model of asthma and in airway smooth muscle cells of patients with severe asthma. Conditional knockout of Abl attenuated airway resistance, smooth muscle mass and staining of proliferating cell nuclear antigen in the airway of mice sensitized and challenged with ovalbumin. Interestingly, conditional knockout of Abl did not affect the levels of IL-13 and CCL2 in bronchoalveolar lavage fluid of animals treated with ovalbumin. However, treatment with imatinib and GNF-5 inhibited the ovalbumin-induced increase in IL-13 and CCL2 as well as airway resistance and smooth muscle growth in animals.

Conclusions

These results suggest that the altered expression of Abl in airway smooth muscle may play a critical role in the development of airway hyperresponsiveness and airway remodeling in asthma. Our findings support the concept that Abl may be a novel target for the development of new therapy to treat asthma.     

Construction of high-density display of CD147 ectodomain on VCSM13 phage via gpVIII: effects of temperature, IPTG, and helper phage infection-period

Production of VCSM13 phage displaying a high density of CD147 ectodomain (CD147Ex) was achieved when culturing conditions were modulated. A phagemid expressing CD147Ex was constructed and used to produce phage display CD147Ex gpVIII fusion protein in TG1 Escherichia coli. Displaying of CD147Ex via gpVIII was successfully increased when growing the transformed TG1 at 25 degrees C with IPTG-stimulation. In addition to temperature and IPTG-stimulation, the VCSM13 helper phage infection-period particularly affected the insertion of CD147Ex into phage progeny. By sandwich ELISA, incorporation of the CD147Ex into phage particle was confirmed. The correct size of the CD147Ex-gpVIII fusion protein at 28kDa was demonstrated by Western immunoblotting. Multivalent display of CD147Ex on phage particles will be valuable in discovering its ligand partner.     

Effects of micropatterned curvature on the motility and mechanical properties of airway smooth muscle cells

Geometric features such as size and shape of the microenvironment are known to alter cell behaviors such as growth, differentiation, apoptosis, and migration. Little is known, however, about the effect of curvature on cell behaviors despite that many cells reside in curved space of tubular organs such as the bronchial airways. To address this question, we fabricated micropatterned strips that mimic airway walls with varying curvature. Then, we cultured airway smooth muscle cells (ASMCs) on these strips and investigated the cells’ motility and mechanical properties using time-lapse imaging microscopy and optical magnetic twisting cytometry (OMTC). We found that both motility and mechanical properties of the ASMCs were influenced by the curvature. In particular, when the curvature increased from 0 to 1/150 μm⁻¹, the velocity of cell migration first decreased (0–1/750 μm⁻¹), and then increased (1/750–1/150 μm⁻¹). In contrast, the cell stiffness increased and then decreased. Thus, at the intermediate curvature (1/750 μm⁻¹) the ASMCs were the least motile, but most stiff. The contractility instead decreased consistently as the curvature increased. The level of F-actin, and vinculin expression within the ASMCs appeared to correlate with the contractility and motility, respectively, in relation to the curvature. These results may provide valuable insights to understanding the heterogeneity of airway constrictions in asthma as well as the developing and functioning of other tubular organs and tissue engineering.     

人源天然ScFv噬菌体抗体库的构建及鉴定

噬菌体抗体库技术是获得治疗性抗体的一条重要途径。以20份健康人外周血为样本,通过提取淋巴细胞、逆转录－PCR（RT PCR）、抗体可变区基因的扩增、重叠PCR获得单链抗体（ScFv）基因,将ScFv克隆入噬粒载体,通过近300次的电转化获得了库容量为1.3×109的全人源天然ScFv噬菌体抗体库。通过随机挑克隆测序和用5种不同抗原筛选对抗体库进行了初步验证。随机测序表明抗体库具有较好的多样性,用5种不同抗原对其进行筛选,均获得了特异性噬菌体抗体的不同富集,表明成功构建了一个多样性良好的人源天然ScFv噬菌体抗体库。     

Age differences in cholinergic airway responsiveness in relation with muscarinic receptor subtypes

Children seem more susceptible to increased airway reactivity than adults. Such an age-dependent discrepancy in airway reactivity may involve different airway smooth muscle functions. Therefore, we compared the in vivo and in vitro responsiveness of airway smooth muscles between two age groups of animals. Rats of 6 and 21 weeks old were challenged in vivo with acetylcholine (ACh) infused intravenously and airway resistance (R(aw)) was measured. Tracheal muscle was also isolated and the isometric force developed to ACh or KCl was measured. Furthermore, the level of genes encoding muscarinic receptor subtypes (M(1-3)) and acetylcholinesterase (AChE) expressed in the tracheal muscle was determined by RT-PCR. In results, the basal R(aw) was similar in the two age groups. The R(aw) at each ACh dose was significantly greater in young rats than older rats (p<0.05, n=22-27). Tracheal muscles from young rats were more sensitive to ACh than older rats (p<0.05, n=20-21), while receptor-independent muscle contraction to KCl was greater in older rats (p<0.05, n=10-19). Genes encoding AChE, M(2) and M(3) muscarinic receptors were more highly expressed in the tracheal muscles from young than older rats (p<0.05, n=4-6). In conclusion, airway smooth muscle in young rat is more sensitive to cholinergic stimulation in vivo and in vitro compared to older rats, which may be due to a higher expression of M(2) and M(3) muscarinic receptors in airway smooth muscle.     

Expression of Fibroblast Growth Factor 9 in Normal Human Lung and Idiopathic Pulmonary Fibrosis

The fibroblast growth factor (FGF) family of signaling ligands contributes significantly to lung development and maintenance in the adult. FGF9 is involved in control of epithelial branching and mesenchymal proliferation and expansion in developing lungs. However, its activity and expression in the normal adult lung and by epithelial and interstitial cells in fibroproliferative diseases like idiopathic pulmonary fibrosis (IPF) are unknown. Tissue samples from normal organ donor human lungs and those of a cohort of patients with mild to severe IPF were sectioned and stained for the immunolocalization of FGF9. In normal lungs, FGF9 was confined to smooth muscle surrounding airways, alveolar ducts and sacs, and blood vessels. In addition to these same sites, lungs of IPF patients expressed FGF9 in a population of myofibroblasts within fibroblastic foci, hypertrophic and hyperplastic epithelium of airways and alveoli, and smooth muscle cells surrounding vessels embedded in thickened interstitium. The results demonstrate that FGF9 protein increased in regions of active cellular hyperplasia, metaplasia, and fibrotic expansion of IPF lungs, and in isolated human lung fibroblasts treated with TGF-β1 and/or overexpressing Wnt7B. The cellular distribution and established biologic activity of FGF9 make it a potentially strong candidate for contributing to the progression of IPF.     

CD117-positive Cells of the Heart: Progenitor Cells or Mast Cells?

Human cardiac stem/progenitor cells and their potential for repair of heart injury are a current hot topic of research. CD117 has been used frequently as a marker for identification of stem/progenitor cells in the heart. However, cardiac mast cells, which are also CD117⁺, have not been excluded by credible means when selecting putative cardiac progenitors by using CD117 as a marker. We evaluated the relationship between CD117⁺ cells and mast cells in the left ventricle of human hearts (n=5 patients, ages 1 week–75 years) with the well-established mast cell markers tryptase, toluidine blue, and thionine. A large number (85–100%) of CD117⁺ cells in the human heart were specifically identified as mast cells. In addition, mast cells showed weak or moderate CD45 immunostaining signals. These results indicate that the majority of CD117⁺ cells in the heart are mast cells and that these cells are distinctly positive for CD45, although staining was weak or moderate. These results strongly suggest that the newly reported CD117⁺/CD45^dim/moderate putative cardiac progenitor cells are mast cells. The significance of this observation in stem cell research of the heart is discussed. (J Histochem Cytochem 58:309–316, 2010)     

血管内皮生长因子及其受体2与哮喘模型大鼠气道平滑肌细胞增殖关系的研究

目的探讨血管内皮生长因子(VEGF)及其受体2(Flk-1)在哮喘大鼠气道平滑肌细胞(ASMC)中表达变化及其对ASMC增殖的影响。方法 SD大鼠18只,随机分为对照组,哮喘模型组和地塞米松干预组各6只,并培养各组气道平滑肌细胞。用免疫组织化学技术检测ASMC增殖细胞核抗原(PCNA)的表达;用RT-PCR及Western blot方法分别检测VEGF和Flk-1mRNA及蛋白质在不同组大鼠ASMC的表达程度。结果(1)哮喘模型组ASMC PCNA表达较对照组和干预组显著增加(P0.05)。(2)哮喘模型组ASMC VEGF164,VEGF188mRNA和VEGF205mRNA的表达较对照组和干预组显著增加(P0.05或P0.01)。(3)哮喘模型组ASMC VEGF及Flk-1蛋白质在大鼠ASMC中的表达较对照组和干预组显著增加(P0.05)。直线相关性分析显示,大鼠ASMC PCNA表达与大鼠ASMC中VEGF205,188,164及Flk-1mRNA表达水平呈正相关(r分别为0.79,0.86,0.83,0.68;P0.05);大鼠ASMC PCNA表达与大鼠ASMC中VEGF及Flk-1蛋白质表达水平也呈正相关(r分别为0.80,0.77;P0.05)。结果 哮喘模型大鼠ASMC中VEGF及其受体Flk-1表达上调,并与气道平滑肌细胞增殖有密切关系。该结果提示VEGF及其受体2可能参与了哮喘气道重建中气道平滑肌细胞增殖的过程。     

The epithelial cholinergic system of the airways

Acetylcholine (ACh), a classical transmitter of parasympathetic nerve fibres in the airways, is also synthesized by a large number of non-neuronal cells, including airway surface epithelial cells. Strongest expression of cholinergic traits is observed in neuroendocrine and brush cells but other epithelial cell types-ciliated, basal and secretory-are cholinergic as well. There is cell type-specific expression of the molecular pathways of ACh release, including both the vesicular storage and exocytotic release known from neurons, and transmembrane release from the cytosol via organic cation transporters. The subcellular distribution of the ACh release machineries suggests luminal release from ciliated and secretory cells, and basolateral release from neuroendocrine cells. The scenario as known so far strongly suggests a local auto-/paracrine role of epithelial ACh in regulating various aspects on the innate mucosal defence mechanisms, including mucociliary clearance, regulation of macrophage function and modulation of sensory nerve fibre activity. The proliferative effects of ACh gain importance in recently identified ACh receptor disorders conferring susceptibility to lung cancer. The cell type-specific molecular diversity of the epithelial ACh synthesis and release machinery implies that it is differently regulated than neuronal ACh release and can be specifically targeted by appropriate drugs.     

Cytoskeleton dynamics: fluctuations within the network

Out-of-equilibrium systems, such as the dynamics of a living cytoskeleton (CSK), are inherently noisy with fluctuations arising from the stochastic nature of the underlying biochemical and molecular events. Recently, such fluctuations within the cell were characterized by observing spontaneous nano-scale motions of an RGD-coated microbead bound to the cell surface [Bursac et al., Nat. Mater. 4 (2005) 557-561]. While these reported anomalous bead motions represent a molecular level reorganization (remodeling) of microstructures in contact with the bead, a precise nature of these cytoskeletal constituents and forces that drive their remodeling dynamics are largely unclear. Here, we focused upon spontaneous motions of an RGD-coated bead and, in particular, assessed to what extent these motions are attributable to (i) bulk cell movement (cell crawling), (ii) dynamics of focal adhesions, (iii) dynamics of lipid membrane, and/or (iv) dynamics of the underlying actin CSK driven by myosin motors.     

Regulation of acetylcholine-induced phosphorylation of PLD1 in porcine tracheal smooth muscle

The muscarinic agonist, acetylcholine (ACh), stimulates phospholipase D (PLD) activity in tracheal smooth muscle cells. Direct activation of protein kinase C (PKC) by phorbol-12-myristate-13-acetate (PMA) also stimulates PLD in this tissue. Activation of ACh-induced PLD was inhibited by the tyrosine kinase inhibitor genistein in a concentration-dependent manner. Presently known isoforms of PLD, PLD1 and PLD2, were identified in tracheal smooth muscle and their activation-induced phosphorylation status studied. Both ACh and PMA increased phosphorylation of PLD1 that was significantly blocked by genistein or the PKC inhibitor calphostin C. PLD2 phosphorylation was not detected in the present experiments. Western blots probed with an anti-phosphotyrosine antibody indicate that PLD1 in this tissue is phosphorylated on tyrosine residues after ACh or PMA stimulation. Tyrosine phosphorylation of PLD1 was blocked by genistein and calphostin C. No tyrosine residues were phosphorylated on PLD2. Taken together, these results demonstrate that porcine tracheal smooth muscle cells express both isoforms PLD1 and PLD2. However, on muscarinic activation only PLD1 in this tissue is phosphorylated by PKC via a tyrosine-kinase-dependent pathway.     

差异显示法对血管平滑肌细胞钙化过程中基因表达变化的研究

目的:本研究运用差异显示技术研究动脉血管平滑肌细胞在钙化过程中基因表达的改变,探讨与动脉钙化相关的基因.方法:体外培养牛主动脉平滑肌细胞,在培养环境中加入10 mmol/L的β-磷酸甘油酯,诱导细胞钙化,作为动脉钙化模型,分别提取对照细胞和钙化细胞的总RNA,用荧光标记的引物进行DD-PCR扩增,电泳显示差异表达的cDNA,再用反向Northern blot对这些差异cDNA进行鉴定确认,并对确认的差异cDNA片段进行克隆测序.结果:DD-PCR显示65个表达差异的片段,经过回收、扩增和反向Northern blot有7个片断确定有持续的差异表达.经过测序和同源性比较,发现有3个片段为新的基因片段.结论:初步确定7个与血管钙化相关的cDNA片段,其中3个片段为新的未知基因片段.     

Modeling of molecular and cellular mechanisms involved in Ca2+ signal encoding in airway myocytes

In airway myocytes signal transduction via cytosolic calcium plays an important role. In relation with experimental results we review models of basic molecular and cellular mechanisms involved in the signal transduction from the myocyte stimulation to the activation of the contractile apparatus. We concentrate on mechanisms for encoding of input signals into Ca²⁺ signals and the mechanisms for their decoding. The mechanisms are arranged into a general scheme of cellular signaling, the so-called bow-tie architecture of signaling, in which calcium plays the role of a common media for cellular signals and links the encoding and decoding part. The encoding of calcium signals in airway myocytes is better known and is presented in more detail. In particular, we focus on three recent models taking into account the intracellular calcium handling and ion fluxes through the plasma membrane. The model of membrane conductances was originally proposed for predicting membrane depolarization and voltage-dependent Ca²⁺ influx triggered by initial cytosolic Ca²⁺ increase as observed on cholinergic stimulation. Cellular models of intracellular Ca²⁺ handling were developed to investigate the role of a mixed population of InsP₃ receptor isoforms and the cellular environment in the occurrence of Ca²⁺ oscillations, and the respective role of the sarcoplasmic reticulum, mitochondria, and cytosolic Ca²⁺-binding proteins in cytosolic Ca²⁺ clearance. Modeling the mechanisms responsible for the decoding of calcium signals is developed in a lesser extent; however, the most recent theoretical studies are briefly presented in relation with the known experimental results.