Sanguinarine Rapidly Relaxes Rat Airway Smooth Muscle Cells Dependent on TAS2R Signaling.

Excessive contraction of airway smooth muscle cells (ASMCs) is a hallmark feature of asthma. Intriguing, the activation of bitter taste receptor (TAS2R) in ASMCs can relax ASMCs. However, there is a lack of potent TAS2R agonists that can be used in asthma therapies since those tested agonists cannot relax ASMCs at the dose below a few hundred micromolar. Considering that sanguinarine (SA) is a bitter substance often used in small doses for the treatment of asthma in folk medicine, the present study was to determine the rapid relaxation effect of SA on ASMCs and to reveal the underlying mechanisms associated with TAS2R signaling. Here, cell stiffness, traction force, calcium signaling, cAMP levels, and the mRNA expression were evaluated by using optical magnetic twisting cytometry, traction force microscopy, Fluo-4/AM labeling, enzyme-linked immunosorbent assay (ELISA), and quantitative (q)RT-PCR, respectively. We found that 0.5 µM SA immediately decreased cell stiffness and traction force, which is comparable with the effect of 5 µM isoproterenol. In addition, 0.5 µM SA immediately increased intracellular free calcium concentration ([Ca2+]i) and decreased the mRNA expression of contractile proteins such as calponin and α-smooth muscle actin after the treatment for 24 h. Furthermore, SA-mediated decrease in cell stiffness/traction force and increase in [Ca2+]i were significantly blunted by inhibiting the TAS2Rs signaling. These findings establish the rapid relaxation effect of SA at low concentration (<1 µM) on cultured ASMCs depending on TAS2R signaling, indicating that SA might be developed as a useful bronchodilator in asthma therapy.

[Effect of sanguinarine on biomechanics of rat airway smooth muscle cells].

This study aimed to evaluate the effect of sanguinarine on biomechanical properties of rat airway smooth muscle cells (rASMCs) including stiffness, traction force and cytoskeletal stress fiber organization. To do so, rASMCs cultured in vitro were treated with sanguinarine solution at different concentrations (0.005~5 µmol/L) for 12 h, 24 h, 36 h, and 48 h, respectively. Subsequently, the cells were tested for their viability, stiffness, traction force, migration and microfilament distribution by using methylthiazolyldiphenyl-tetrazolium bromide assay, optical magnetic twisting cytometry, Fourier transform traction microscopy, scratch wound healing method, and immunofluorescence microscopy, respectively. The results showed that at concentration below 0.5 µmol/L sanguinarine had no effect on cell viability, but caused dose and time dependent effect on cell biomechanics. Specifically, rASMCs treated with sanguinarine at 0.05 µmol/L and 0.5 µmol/L for 12 and 24 h exhibited significant reduction in stiffness, traction force and migration speed, together with disorganization of the cytoskeletal stress fibers. Considering the essential role of airway smooth muscle cells (ASMCs) biomechanics in the airway hyperresponsiveness (AHR) of asthma, these findings suggest that sanguinarine may ameliorate AHR via alteration of ASMCs biomechanical properties, thus providing a novel approach for asthma drug development.

Emergent Differential Organization of Airway Smooth Muscle Cells on Concave and Convex Tubular Surface.

Airway smooth muscle cells (ASMCs) exist in a form of helical winding bundles within the bronchial airway wall. Such tubular tissue provides cells with considerable curvature as a physical constraint, which is widely thought as an important determinant of cell behaviors. However, this process is difficult to mimic in the conventional planar cell culture system. Here, we report a method to develop chips with cell-scale tubular (concave and convex) surfaces from fused deposition modeling 3D printing to explore how ASMCs adapt to the cylindrical curvature for morphogenesis and function. Results showed that ASMCs self-organized into two distinctively different patterns of orientation on the concave and convex surfaces, eventually aligning either invariably perpendicular to the cylinder axis on the concave surface or curvature-dependently angled on the convex surface. Such oriented alignments of the ASMCs were maintained even when the cells were in dynamic movement during migration and spreading along the tubular surfaces. Furthermore, the ASMCs underwent a phenotype transition on the tubular (both concave and convex) surfaces, significantly reducing contractility as compared to ASMCs cultured on a flat surface, which was reflected in the changes of proliferation, migration and gene expression of contractile biomarkers. Taken together, our study revealed a curvature-induced pattern formation and functional modulation of ASMCs in vitro, which is not only important to better understanding airway smooth muscle pathophysiology, but may also be useful in the development of new techniques for airway disease diagnosis and therapy such as engineering airway tissues or organoids.