Xuemei Jiang

Substrate stiffness influences TGF-β1-induced differentiation of bronchial fibroblasts into myofibroblasts in airway remodeling

Chronic inflammation and remodeling of the bronchial wall are basic hallmarks of asthma. During the process of bronchial wall remodeling, inflammatory factors, such as transforming growth factor-β1 (TGF-β1), are known to induce the differentiation of fibroblasts into myofibroblasts, which leads to excessive synthesis and secretion of extracellular matrix (ECM) proteins, thus thickening and stiffening the basement membrane. However, it has not been thoroughly studied whether or not substrate stiffening affects the TGF-β1‑induced myofibroblast differentiation. In the present study, the influence of substrate stiffness on the process of bronchial fibroblast differentiation into myofibroblasts in the presence of TGF-β1 was investigated. To address this question, we synthesized polydimethylsiloxane (PDMS) substrates with varying degrees of stiffness (Youngs modulus of 1, 10 and 50 kPa, respectively). We cultured bronchial fibroblasts on the substrates of varying stiffness in media containing TGF-β1 (10 ng/ml) to stimulate the differentiation of fibroblasts into myofibroblasts. Myofibroblast differentiation was examined using semi-quantitative RT-PCR for the expression of α-smooth muscle actin (α-SMA) mRNA and collagen I mRNA, the enzyme-linked immunosorbent assay method was used to assess the expression of collagen I protein and western blotting to assess the expression of α-SMA protein. The optical magnetic twisting cytometry (OMTC) method was used for the changing of cell mechanical properties. Our findings suggest that when fibroblasts were incubated with TGF-β1 (10 ng/ml) on substrate of varying stiffness, the differentiation of fibroblasts into myofibroblasts was enhanced by increasing substrate stiffness. Compared with those cultured on substrate with Youngs modulus of 1 kPa, the mRNA and protein expression of collagen I and α-SMA of fibroblasts cultured on substrates with Youngs modulus of 10 and 50 kPa were increased. Furthermore, with the increase of substrate stiffness, the cell stiffness and contractility were also increased, which also indicated further aggravation of asthma. This finding may help better understand the underlying mechanisms of hyperplasia of myofibroblasts in asthma, which has a marked significance in the therapy of asthma.

Direct UV detection of underivatized amino acids using capillary electrophoresis with online sweeping enrichment

This is an original report proposed a CE method for direct analysis of the underivatized amino acids using UV detection with relatively higher sensitivity, which was based on coordination interactions between amino acids and Cu (II) ions. In addition, an online sweeping preconcentration technique was easily combined to improve the detection sensitivity. Satisfying separations of the amino acids were obtained under optimized conditions: 50 mmol/L CuSO4-0.05% HAc-H2O (pH 4.5), and the separation voltage of 15 kV. The LODs for the analytes ranged from 0.1 to 0.5 micromol/L. The linearity of detection for all analytes was two orders of magnitude with the correlation coefficients greater than 0.99. The repeatability was displayed with an RSD less than 3% for migration time and peak height (n = 5). Moreover, some amino acids in real samples of human saliva and green tea were analyzed by this direct UV detection CE method with acceptable sensitivity.

ADAM33 protein expression and the mechanics of airway smooth muscle cells are highly correlated in ovalbumin-sensitized rats

A disintegrin and metalloproteinase 33 (ADAM33) has been identified as an asthma susceptibility gene; however, the role of ADAM33 in the pathogenesis and progression of asthma remains to be elucidated. As ADAM33 is predominantly expressed in airway smooth muscle cells (ASMCs), it is feasible to investigate whether ADAM33 protein expression is correlated with ASMC mechanics that are ultimately responsible for airway hyperresponsiveness in asthma. To determine this, Sprague Dawley rats were sensitized with ovalbumin (OVA) for up to 12 weeks to simulate asthma symptoms. Subsequently, ASMCs were isolated from the rats and cultured in vitro. The protein expression of ADAM33 and cytoskeletal proteins (including F‑actin and vinculin), cell stiffness and contractility, as well as traction force were measured. The results demonstrated that compared with the non‑sensitized rats, the protein expression of ADAM33 in ASMCs from the OVA‑sensitized rats increased in a time‑dependent manner, reaching a maximum level at 4 weeks of sensitization and gradually subsiding as OVA sensitization continued (P<0.001). The cell stiffness, traction force and expression of vinculin and F‑actin changed similarly, resulting in a positive correlation with ADAM33 protein expression (Pearsons correlation coefficient, 0.864, 0.716, 0.774 and 0.662, respectively; P=0.1‑0.3). The in vivo results of OVA‑induced ADAM33 protein expression and its association with the mechanics of ASMCs suggested that ADAM33 is a mediator of ASMC dysfunction in asthma, and may provide a rationale for the therapeutic targeting of ADAM33 in the treatment of asthma.

Layer-by-layer self-assembly of polydopamine/gold nanoparticle/thiol coating as the stationary phase for open tubular capillary electrochromatography

Much attention has been paid to utilizing polydopamine (PDA) as the stationary phase in open-tubular capillary electrochromatography (OT-CEC) owing to its diverse properties, such as strong adhesion to various surfaces, latent reactivity toward amine and thiol groups and metal ion chelating/redox activities. In this study, a novel open-tubular capillary column coated with polydopamine/gold nanoparticles/thiols (PDA/Au NPs/thiols) has been fabricated based on the multiple properties of PDA for the first time. The capillary inner surface was firstly functionalized with a layer of PDA/Au NPs using the strong adhesive and metal ion redox properties of PDA. Thiols were then introduced and covalently reacted with the hybrid coating based on the Michael addition reaction of PDA and thiols and also Au–S bonds. Moreover, benefitting from the porosity of PDA, layer-by-layer (LBL) self-assembly was further applied to increase the amounts of stationary phase (Au NPs and thiols), which can significantly enhance the separation effectiveness and stability of the coated column. The formation of the PDA/Au NP/thiol coating in the capillary was confirmed and characterized by scanning electron microscopy (SEM), Energy Dispersive Spectrometry (EDS) and AFM (Atomic Force Microscopy). Then the separation effectiveness of the PDA/Au NP/thiol@capillary was verified by the separation of alkylbenzenes, which can achieve baseline separation easily with high column efficiency. In addition, the column showed long lifetime and good stability. The relative standard deviations (RSDs) for intra-day and inter-day repeatability of the PDA/Au NP/thiol@capillary were lower than 5%. Therefore, the layer-by-layer self-assembly of PDA/Au NPs/thiols on the capillary inner-surface could be an effective capillary modification strategy.

A novel peptide ADAM8 inhibitor attenuates bronchial hyperresponsiveness and Th2 cytokine mediated inflammation of murine asthmatic models.

A disintegrin and metalloproteinase 8 (ADAM8) has been identified as a signature gene associated with moderate and severe asthma. Studies in mice have demonstrated that the severity of asthma can be reduced by either transgenic knock-out or by antibodies blocking ADAM8 function, highlighting ADAM8 as potential drug target for asthma therapy. Here, we examined the therapeutic effect of an ADAM8 inhibitor peptide (BK-1361) that specifically blocks cellular ADAM8 activity in ovalbumin-sensitized and challenged Balb/c mice. We found that BK-1361 (25 μg/g body weight) attenuated airway responsiveness to methacholine stimulation by up to 42%, concomitantly reduced tissue remodeling by 50%, and decreased inflammatory cells (e.g. eosinophils down by 54%)/inflammatory factors (e.g. sCD23 down by 50%)/TH2 cytokines (e.g. IL-5 down by 70%)/ADAM8-positive eosinophils (down by 60%) in the lung. We further verified that BK-1361 specifically targets ADAM8 in vivo as the peptide caused significantly reduced levels of soluble CD23 in wild-type but not in ADAM8-deficient mice. These findings suggest that BK-1361 blocks ADAM8-dependent asthma effects in vivo by inhibiting infiltration of eosinophils and TH2 lymphocytes, thus leading to reduction of TH2-mediated inflammation, tissue remodeling and bronchial hyperresponsiveness. Taken together, pharmacological ADAM8 inhibition appears as promising novel therapeutic strategy for the treatment of asthma.

ADAM8 in Asthma. Friend or Foe to Airway Inflammation

Airway inflammation has been suggested as the pathological basis in asthma pathogenesis. Recruitment of leukocytes from the vasculature into airway sites is essential for induction of airway inflammation, a process thought to be mediated by a disintegrin and metalloprotease 8 (ADAM8). However, there is an apparent controversy about whether ADAM8 helps or hampers transmigration of leukocytes through endothelium in airway inflammation of asthma. This review outlines the current contradictory concepts concerning the role of ADAM8 in airway inflammation, particularly focusing on the recruitment of leukocytes during asthma, and attempts to bridge the existing experimental data on the basis of the functional analysis of different domains of ADAM8 and their endogenous processing in vivo. We suggest a possible hypothesis for the specific mechanism by which ADAM8 regulates the transmigration of leukocytes to explain the disparity existing in current studies, and we also raise some questions that require future investigations.

Bisulfite and sulfite as derivatives of sulfur dioxide alters biomechanical behaviors of airway smooth muscle cells in culture

Abstract Sulfur dioxide (SO2) is a common air pollutant that triggers asthmatic symptoms, but its toxicological mechanisms are not fully understood. Specifically, it is unclear how SO2 in vivo affects airway smooth muscle (ASM) cells of which the mechanics is known to ultimately mediate airway hyperresponsiveness (AHR) – a hallmark feature of asthma. To this end, we investigated the effects of bisulfite/sulfite (1:3 M/M in neutral fluid to simulate the in vivo derivatives of inhaled SO2 in the airways), on the viability, migration, stiffness and contractility of ASM cells cultured in vitro. The results showed that bisulfite/sulfite consistently increased viability, migration, F-actin intensity and stiffness of ASM cells in similar fashion as concentration increasing from 10−4 to 10−1 mmol/L. However, bisulfite/sulfite increased the ASM cell contractility induced by KCl only at the concentration between 10−4 and 10−3 mmol/L (p < 0.05), while having no consistent effect on that induced by histamine. At the concentration of 100 mmol/L, bisulfite/sulfite became acutely toxic to the ASM cells. Taken together, the data suggest that SO2 derivatives at low levels in vivo may directly increase the mass, stiffness and contractility of ASM cells, which may help understand the mechanism in which specific air pollutants contribute in vivo to the pathogenesis of asthma.

Overexpression of soluble ADAM33 promotes a hypercontractile phenotype of the airway smooth muscle cell in rat

A disintegrin and metalloproteinase 33 (ADAM33) has been identified as a susceptibility gene for asthma, but details of the causality are not fully understood. We hypothesize that soluble ADAM33 (sADAM33) overexpression can alter the mechanical behaviors of airway smooth muscle cells (ASMCs) via regulation of the cells contractile phenotype, and thus contributes to airway hyperresponsiveness (AHR) in asthma. To test this hypothesis, we either overexpressed or knocked down the sADAM33 in rat ASMCs by transfecting the cells with sADAM33 coding sequence or a small interfering RNA (siRNA) that specifically targets the ADAM33 disintegrin domain, and subsequently assessed the cells for stiffness, contractility and traction force, together with the expression level of contractile and proliferative phenotype markers. We also investigated whether these changes were dependent on Rho/ROCK pathway by culturing the ASMCs either in the absence or presence of ROCK inhibitor (H1152). The results showed that the ASMCs with sADAM33 overexpression were stiffer and more contractile, generated greater traction force, exhibited increased expression levels of contractile phenotype markers and markedly enhanced Rho activation. Furthermore these changes were largely attenuated when the cells were cultured in the presence of H-1152. However, the knock-down of ADAM33 seemed insufficient to influence majority of the mechanical behaviors of the ASMCs. Taken together, we demonstrated that sADAM33 overexpression altered the mechanical behaviors of ASMCs in vitro, which was most likely by promoting a hypercontractile phenotype transition of ASMCs through Rho/ROCK pathway. This revelation may establish the previously missing link between ADAM33 expression and AHR, and also provide useful insight for targeting sADAM33 in asthma prevention and therapy.

Development and Evaluation of Capillary Electrophoresis Based on Coordination Interaction

Abstract In nonaqueous capillary electrophoresis (CE), the electrostatic interactions between additives and analytes can offer additional separation mechanism. Similarly, the dynamic coordination interactions between metal ions and analytes can also be possible of providing separation selectivity. In this work, a new CE mode with applying coordination interaction as separation motivity was developed and it was defined as coordination interaction capillary electrophoresis (CICE). The CICE method was established by using seven arylamides as model compounds and Cu 2+ , Ni 2+ , Zn 2+ as additives of running medium. The effect of operating conditions such as pH value, nature of metal ion, concentration, and separation voltage were investigated. The conditions of running medium for the separation of arylamides were optimized as 3 mM Cu 2+ and 0.1% (v/v) acetic acid in water. The quantitative reliability of CICE, as given by linear range of concentration, correlation coefficient and detection limit, was presented. The potential application of CICE in analysis of real sample was validated by separation of degenerative o -phenylenediamine sample. In addition, good separation results of four sulfonamide compounds were obtained with CICE, demonstrating the great possibilities of CICE as a new, selective, and efficient CE separation mode.

UVA Irradiation Enhances Brusatol-Mediated Inhibition of Melanoma Growth by Downregulation of the Nrf2-Mediated Antioxidant Response

Brusatol (BR) is a potent inhibitor of Nrf2, a transcription factor that is highly expressed in cancer tissues and confers chemoresistance. UVA-generated reactive oxygen species (ROS) can damage both normal and cancer cells and may be of potential use in phototherapy. In order to provide an alternative method to treat the aggressive melanoma, we sought to investigate whether low-dose UVA with BR is more effective in eliminating melanoma cells than the respective single treatments. We found that BR combined with UVA led to inhibition of A375 melanoma cell proliferation by cell cycle arrest in the G1 phase and triggers cell apoptosis. Furthermore, inhibition of Nrf2 expression attenuated colony formation and tumor development from A375 cells in heterotopic mouse models. In addition, cotreatment of UVA and BR partially suppressed Nrf2 and its downstream target genes such as HO-1 along with the PI3K/AKT pathway. We propose that cotreatment increased ROS-induced cell cycle arrest and cellular apoptosis and inhibits melanoma growth by regulating the AKT-Nrf2 pathway in A375 cells which offers a possible therapeutic intervention strategy for the treatment of human melanoma.