IgE Regulates the Expression of smMLCK in Human Airway Smooth Muscle Cells

Previous studies have shown that enhanced accumulation of contractile proteins such as smooth muscle myosin light chain kinase (smMLCK) plays a major role in human airway smooth muscle cells (HASM) cell hypercontractility and hypertrophy. Furthermore, serum IgE levels play an important role in smooth muscle hyperreactivity. However, the effect of IgE on smMLCK expression has not been investigated. In this study, we demonstrate that IgE increases the expression of smMLCK at mRNA and protein levels. This effect was inhibited significantly with neutralizing abs directed against FcεRI but not with anti-FcεRII/CD23. Furthermore, Syk knock down and pharmacological inhibition of mitogen activated protein kinases (MAPK) (ERK1/2, p38, and JNK) and phosphatidylinositol 3-kinase (PI3K) significantly diminished the IgE-mediated upregulation of smMLCK expression in HASM cells. Taken together, our data suggest a role of IgE in regulating smMLCK in HASM cells. Therefore, targeting the FcεRI activation on HASM cells may offer a novel approach in controlling the bronchomotor tone in allergic asthma.     

Endobronchial Ultrasound Reliably Quantifies Airway Smooth Muscle Remodeling in an Equine Asthma Model

Endobronchial ultrasonography (EBUS) revealed differences in the thickness of the layer representing subepithelial tissues (L2) between human asthmatics and controls, but whether this measurement correlates with airway smooth muscle (ASM) remodeling in asthma is unknown. In this study, we sought to determine the ability of EBUS to predict histological ASM remodeling in normal and equine asthmatic airways. We studied 109 isolated bronchi from the lungs of 13 horses. They underwent EBUS examination using a 30 MHz radial probe before being processed for histology. ASM remodeling parameters were evaluated in EBUS images (L2 thickness, L2 area, L2 area/internal perimeter [Pi] and L2 area/Pi²) and histological cuts (ASM area/Pi²), and compared. EBUS was then performed ex vivo on the lungs of 4 horses with heaves, an asthma-like condition of horses, and 7 controls to determine whether central bronchial remodeling could be detected with this technique. An optimized approach was developed based on data variability within airways, subjects, and groups, and then validated in 7 horses (3 controls, 4 with heaves) that underwent EBUS in vivo. L2 area was significantly associated to ASM area in isolated lungs (p<0.0001), in the absence of significant bias related to the airway size. Bronchial size significantly affected EBUS ASM-related parameters, except for L2 area/Pi². L2 area/Pi² was increased in the airways of asthmatic horses compared to controls, both ex vivo and in vivo (p<0.05). Bronchial histology confirmed our findings (A_ASM/Pi² was increased in asthmatic horses compared to controls, p<0.05). In both horses with heaves and controls, L2 was composed of ASM for the outer 75% of its thickness and by ECM for the remaining inner 25%. In conclusion, EBUS reliably allows assessment of asthma-associated ASM remodeling of central airways in a non-invasive way.     

Myosin Light Chain Kinase Is Necessary for Tonic Airway Smooth Muscle Contraction

Different interacting signaling modules involving Ca²⁺/calmodulin-dependent myosin light chain kinase, Ca²⁺-independent regulatory light chain phosphorylation, myosin phosphatase inhibition, and actin filament-based proteins are proposed as specific cellular mechanisms involved in the regulation of smooth muscle contraction. However, the relative importance of specific modules is not well defined. By using tamoxifen-activated and smooth muscle-specific knock-out of myosin light chain kinase in mice, we analyzed its role in tonic airway smooth muscle contraction. Knock-out of the kinase in both tracheal and bronchial smooth muscle significantly reduced contraction and myosin phosphorylation responses to K⁺-depolarization and acetylcholine. Kinase-deficient mice lacked bronchial constrictions in normal and asthmatic airways, whereas the asthmatic inflammation response was not affected. These results indicate that myosin light chain kinase acts as a central participant in the contractile signaling module of tonic smooth muscle. Importantly, contractile airway smooth muscles are necessary for physiological and asthmatic airway resistance.     

ERK信号通道调控大鼠气道平滑肌细胞的增殖与凋亡

 为了了解ERK信号通道对正常大鼠气道平滑肌细胞(airway smooth muscle cells, ASMCs)增殖与凋亡的调控. 通过对正常大鼠ASMCs原代培养,4～7代用于实验,以ERK激动剂表皮生长因子（EGF）和抑制剂PD98059干预ASMCs生长,采用RT-PCR和免疫荧光染色观察ASMCs上ERK mRNA和蛋白的表达,MTT法、^３Ｈ-TdR掺入法检测ASMC增殖,Hoechst染色和Annexin-Ⅴ FITC PI双染色法检测细胞凋亡,Western免疫印迹检测ERK1/2、磷酸化ERK1/2和procaspase-3蛋白的表达.结果发现ASMCs上存在ERK mRNA和蛋白的表达,与空白对照组比较,PD98059干预后ASMCs的Ａ_４９０值和细胞DNA合成量均减少(Ｐ<0.05),细胞凋亡指数、早期凋亡细胞百分率均增高(Ｐ<0.05),ERK1/2、磷酸化ERK1/2表达和ERK活化率均降低, procaspase-3蛋白的表达增高.EGF干预后ASMCs的Ａ_４９０值和细胞DNA合成量均增高(Ｐ<0.05),细胞凋亡指数、早期凋亡细胞百分率均下降(Ｐ<0.05),ERK1/2、磷酸化ERK1/2表达和ERK活化率均增高, procaspase-3蛋白的表达降低.P+E组无明显差异(P>0.05).ERK信号通道参与大鼠ASMCs增殖和凋亡的调控,ERK对大鼠ASMCs凋亡的调控与procaspase-3蛋白有关,这一发现将有助于对哮喘ASMCs异常增殖调控机制的深入研究.     

CADM1 Is a Key Receptor Mediating Human Mast Cell Adhesion to Human Lung Fibroblasts and Airway Smooth Muscle Cells

Background

Mast cells (MCs) play a central role in the development of many diseases including asthma and pulmonary fibrosis. Interactions of human lung mast cells (HLMCs) with human airway smooth muscle cells (HASMCs) are partially dependent on adhesion mediated by cell adhesion molecule-1 (CADM1), but the adhesion mechanism through which HLMCs interact with human lung fibroblasts (HLFs) is not known. CADM1 is expressed as several isoforms (SP4, SP1, SP6) in HLMCs, with SP4 dominant. These isoforms differentially regulate HLMC homotypic adhesion and survival.

Objective

In this study we have investigated the role of CADM1 isoforms in the adhesion of HLMCs and HMC-1 cells to primary HASMCs and HLFs.

Methods

CADM1 overexpression or downregulation was achieved using adenoviral delivery of CADM1 short hairpin RNAs or isoform-specific cDNAs respectively.

Results

Downregulation of CADM1 attenuated both HLMC and HMC-1 adhesion to both primary HASMCs and HLFs. Overexpression of either SP1 or SP4 isoforms did not alter MC adhesion to HASMCs, whereas overexpression of SP4, but not SP1, significantly increased both HMC-1 cell and HLMC adhesion to HLFs. The expression level of CADM1 SP4 strongly predicted the extent of MC adhesion; linear regression indicated that CADM1 accounts for up to 67% and 32% of adhesion to HLFs for HMC-1 cells and HLMCs, respectively. HLFs supported HLMC proliferation and survival through a CADM1-dependent mechanism. With respect to CADM1 counter-receptor expression, HLFs expressed both CADM1 and nectin-3, whereas HASMCs expressed only nectin-3.

Conclusion and Clinical Relevance

Collectively these data indicate that the CADM1 SP4 isoform is a key receptor mediating human MC adhesion to HASMCs and HLFs. The differential expression of CADM1 counter-receptors on HLFs compared to HASMCs may allow the specific targeting of either HLMC-HLF or HLMC-HASMC interactions in the lung parenchyma and airways.     

细胞代数和密度影响血管平滑肌细胞表型重塑和收缩反应性的机制

探讨细胞代数和密度对血管平滑肌细胞(vascular smooth muscle cell,VSMC)表型重塑能力的影响及机制,观察血清饥饿诱导的不同代数和密度的VSMC骨架的组构特征及收缩反应性,检测细胞骨架中收缩蛋白的含量和比例变化。结果发现,低代数(3代)、高密度的VSMC经血清饥饿诱导后易于形成束状、极性排列的应力纤维,乙酰胆碱(Ach)刺激可产生明显的收缩反应。Western印迹显示,3代高密度VSMC中,平滑肌22α(SM22α)在F-肌动蛋白中的组成比例及其在F-/G-肌动蛋白的含量之比明显高于8代细胞。结果提示,SM22α在F-肌动蛋白中的分布比例可能决定了应力纤维的排布方式,是细胞获得收缩性的主要调节因素,在VSMC表型重塑过程中具有重要意义。     

Azelnidipine Inhibits Cultured Rat Aortic Smooth Muscle Cell Death Induced by Cyclic Mechanical Stretch

Acute aortic dissection is the most common life-threatening vascular disease, with sudden onset of severe pain and a high fatality rate. Clarifying the detailed mechanism for aortic dissection is of great significance for establishing effective pharmacotherapy for this high mortality disease. In the present study, we evaluated the influence of biomechanical stretch, which mimics an acute rise in blood pressure using an experimental apparatus of stretching loads in vitro, on rat aortic smooth muscle cell (RASMC) death. Then, we examined the effects of azelnidipine and mitogen-activated protein kinase inhibitors on mechanical stretch-induced RASMC death. The major findings of the present study are as follows: (1) cyclic mechanical stretch on RASMC caused cell death in a time-dependent manner up to 4 h; (2) cyclic mechanical stretch on RASMC induced c-Jun N-terminal kinase (JNK) and p38 activation with peaks at 10 min; (3) azelnidipine inhibited RASMC death in a concentration-dependent manner as well as inhibited JNK and p38 activation by mechanical stretch; and (4) SP600125 (a JNK inhibitor) and SB203580 (a p38 inhibitor) protected against stretch-induced RASMC death; (5) Antioxidants, diphenylene iodonium and tempol failed to inhibit stretch-induced RASMC death. On the basis of the above findings, we propose a possible mechanism where an acute rise in blood pressure increases biomechanical stress on the arterial walls, which induces RASMC death, and thus, may lead to aortic dissection. Azelnidipine may be used as a pharmacotherapeutic agent for prevention of aortic dissection independent of its blood pressure lowering effect.     

ADAMTS7 Cleavage and Vascular Smooth Muscle Cell Migration Is Affected by a Coronary-Artery-Disease-Associated Variant

Recent genome-wide association studies have revealed an association between variation at the ADAMTS7 locus and susceptibility to coronary artery disease (CAD). Furthermore, in a population-based study cohort, we observed an inverse association between atherosclerosis prevalence and rs3825807, a nonsynonymous SNP (A to G) leading to a Ser-to-Pro substitution in the prodomain of the protease ADAMTS7. In light of these data, we sought a mechanistic explanation for this association. We found that ADAMTS7 accumulated in smooth muscle cells in coronary and carotid atherosclerotic plaques. Vascular smooth muscle cells (VSMCs) of the G/G genotype for rs3825807 had reduced migratory ability, and conditioned media of VSMCs of the G/G genotype contained less of the cleaved form of thrombospondin-5, an ADAMTS7 substrate that had been shown to be produced by VSMCs and inhibit VSMC migration. Furthermore, we found that there was a reduction in the amount of cleaved ADAMTS7 prodomain in media conditioned by VSMCs of the G/G genotype and that the Ser-to-Pro substitution affected ADAMTS7 prodomain cleavage. The results of our study indicate that rs3825807 has an effect on ADAMTS7 maturation, thrombospondin-5 cleavage, and VSMC migration, with the variant associated with protection from atherosclerosis and CAD rendering a reduction in ADAMTS7 function.     

miRNA Biogenesis Enzyme Drosha Is Required for Vascular Smooth Muscle Cell Survival

miRNA biogenesis enzyme Drosha cleaves double-stranded primary miRNA by interacting with double-stranded RNA binding protein DGCR8 and processes primary miRNA into precursor miRNA to participate in the miRNA biogenesis pathway. The role of Drosha in vascular smooth muscle cells (VSMCs) has not been well addressed. We generated Drosha conditional knockout (cKO) mice by crossing VSMC-specific Cre mice, SM22-Cre, with Drosha ^loxp/loxp mice. Disruption of Drosha in VSMCs resulted in embryonic lethality at E14.5 with severe liver hemorrhage in mutant embryos. No obvious developmental delay was observed in Drosha cKO embryos. The vascular structure was absent in the yolk sac of Drosha homozygotes at E14.5. Loss of Drosha reduced VSMC proliferation in vitro and in vivo. The VSMC differentiation marker genes, including αSMA, SM22, and CNN1, and endothelial cell marker CD31 were significantly downregulated in Drosha cKO mice compared to controls. ERK1/2 mitogen-activated protein kinase and the phosphatidylinositol 3-kinase/AKT were attenuated in VSMCs in vitro and in vivo. Disruption of Drosha in VSMCs of mice leads to the dysregulation of miRNA expression. Using bioinformatics approach, the interactions between dysregulated miRNAs and their target genes were analyzed. Our data demonstrated that Drosha is required for VSMC survival by targeting multiple signaling pathways.     

Prostaglandin Production by Mast Cells and the Regulation of Airway Smooth Muscle Responses

The role of mast cell activation and the generation of mediator release are important factors in determining the reactivity of lung tissue during allergic reactions and asthma. In this article, the advantages of our procedure for isolating mast cells from lung tissue over other methods are discussed. Our studies have demonstrated the importance of cross talk between mast cells and other cell types that also release mediators of bronchoconstriction during activation, thus amplifying the response. In addition, it has been found that inositol phospholipid turnover in response to various mast cell mediators is enhanced in hyperresponsive lung tissue. The consequences to the contractility of the tissue of such alterations in the cellular signaling system in lung tissue are discussed.     

同源异型盒基因对血管平滑肌细胞的调控作用

同源异型盒基因是一类对生物体的生长、发育和分化从时间和空间上进行协调的调控基因。构成血管中膜的血管平滑肌细胞表型具有极大的可塑性。在一些病理性血管重构时,血管平滑肌细胞可发生表型调变,从分化型调变为去分化型,具备增殖和迁移能力。在此过程中,多种同源异型盒基因的表达发挥了重要的调控作用。现就同源异型盒基因与血管平滑肌细胞的表型调变、增殖和迁移的关系等方面的研究进展作一综述。     

A Distribution-Moment Approximation for Coupled Dynamics of the Airway Wall and Airway Smooth Muscle

Asthma is fundamentally a disease of airway constriction. Due to a variety of experimental challenges, the dynamics of airways are poorly understood. Of specific interest is the narrowing of the airway due to forces produced by the airway smooth muscle wrapped around each airway. The interaction between the muscle and the airway wall is crucial for the airway constriction that occurs during an asthma attack. Although cross-bridge theory is a well-studied representation of complex smooth muscle dynamics, and these dynamics can be coupled to the airway wall, this comes at significant computational cost—even for isolated airways. Because many phenomena of interest in pulmonary physiology cannot be adequately understood by studying isolated airways, this presents a significant limitation. We present a distribution-moment approximation of this coupled system and study the validity of the approximation throughout the physiological range. We show that the distribution-moment approximation is valid in most conditions, and we explore the region of breakdown. These results show that in many situations, the distribution-moment approximation is a viable option that provides an orders-of-magnitude reduction in computational complexity; not only is this valuable for isolated airway studies, but it moreover offers the prospect that rich ASM dynamics might be incorporated into interacting airway models where previously this was precluded by computational cost.     

Arsenite-Induced Apoptosis Is Prevented by Selenite in A375 Cell Line

Arsenic trioxide induces apoptosis and clinical remission in patients diagnosed with acute promyelocytic leukemia. The human malignant melanoma A375 cells were treated with NaAsO₂ (0.1–130 μM) and also treated with combined 10 μM NaAsO₂ and 10 μM Na₂SeO₃. NaAsO₂ arrested cell growth in the G1 phase and induced apoptosis in a concentration- and time-dependent manner. In contrast, administration of Na₂SeO₃ antagonized the cell growth inhibition and apoptosis induced by NaAsO₂. The NaAsO₂ treatment resulted in a marked increase in p53 protein as early as 4 h and in Bcl-2 protein level by 12 h. In addition, p53 downregulation accompanied the combined treatment of NaAsO₂ and Na₂SeO₃. Thus, our results indicate upregulation of p53 and Bcl-2 play a crucial role in the NaAsO₂-induced G1 arrest and apoptosis of A375 cells and that downregulation p53 appears to contribute to the inhibition by Na₂SeO₃ of the effects induced by NaAsO₂.