-Airway smooth muscle (ASM) cellular and molecular biology is typically studied with single-cell cultures grown on flat 2D substrates. However, cells in vivo exist as part of complex 3D structures, and it is well established in other cell types that altering substrate geometry exerts potent effects on phenotype and function. These factors may be especially relevant to asthma, a disease characterized by structural remodeling of the airway wall, and highlights a need for more physiologically relevant models of ASM function. We utilized a tissue engineering platform known as microfabricated tissue gauges to develop a 3D culture model of ASM featuring arrays of ϳ0.4 mm long, ϳ350 cell "microtissues" capable of simultaneous contractile force measurement and cell-level microscopy. ASM-only microtissues generated baseline tension, exhibited strong cellular organization, and developed actin stress fibers, but lost structural integrity and dissociated from the cantilevers within 3 days. Addition of 3T3-fibroblasts dramatically improved survival times without affecting tension development or morphology. ASM-3T3 microtissues contracted similarly to ex vivo ASM, exhibiting reproducible responses to a range of contractile and relaxant agents. Compared with 2D cultures, microtissues demonstrated identical responses to acetylcholine and KCl, but not histamine, forskolin, or cytochalasin D, suggesting that contractility is regulated by substrate geometry. Microtissues represent a novel model for studying ASM, incorporating a physiological 3D structure, realistic mechanical environment, coculture of multiple cells types, and comparable contractile properties to existing models. This new model allows for rapid screening of biochemical and mechanical factors to provide insight into ASM dysfunction in asthma. 3D culture; tissue engineering; asthma; airway smooth muscle; airway wall remodeling ASTHMA IS AN OBSTRUCTIVE AIRWAY disease characterized by increased airway resistance and hyperresponsiveness (AHR) to certain environmental stimuli. The primary cause of AHR is not clear but ultimately it is airway smooth muscle (ASM) contraction that is responsible for airway narrowing. Alterations in sensitivity, force generation, or shortening velocity of ASM in response to contractile agonists may be possible disease mechanisms. It is now also well established that asthma is characterized by airway wall remodeling, which includes thickening of the airway wall and increased ASM mass (52), altered extracellular matrix (ECM) composition (4, 19), infiltration of inflammatory cells (9), and epithelial dysfunction (33). Since airway wall remodeling may precede clinical symptoms (7), it is possible that a putative defect in ASM contractile function arises as a result of the remodeling process. Structural changes in the airway wall may manifest as altered mechanical loads that alter how ASM force development translates into airway narrowing. Such changes could also modulate ASM contractile phenotype through ECM signaling and mechanotransduction, b...

Objective The underlying causes of abdominal aortic aneurysms (AAAs) remain obscure, although research tools such as the angiotensin II (Ang II) apolipoprotein E-deficient (apoE−/−) mouse model have aided investigations. Longitudinal imaging and determination of biomechanical forces in this small-scale model have been difficult. We hypothesized that high-frequency ultrasound biomicroscopy combined with speckle-tracking analytical strategies can be used to define the role of circumferential mechanical strain in AAA formation in the Ang II/apoE−/− mouse model of AAAs. We simultaneously examined dietary perturbations that might impact the biomechanical properties of the aortic wall, hypothesizing that the generalized inflammatory phenotype associated with diet-induced obesity would be associated with accelerated loss of circumferential strain and aneurysmal aortic degeneration. Methods Receiving either a 60 kcal% fat Western diet or standard 10 kcal% fat normal chow, Ang II-treated apoE−/− mice (n = 34) underwent sequential aortic duplex ultrasound scan imaging (Vevo 2100 System; VisualSonics, Toronto, Ontario, Canada) of their entire aorta. Circumferential strains were calculated using speckle-tracking algorithms and a custom MatLab analysis. Results Decreased strains in all aortic locations after just 3 days of Ang II treatment were observed, and this effect progressed during the 4-week observation period. Anatomic segments along the aorta impacted wall strain (baseline highest in ascending aorta; P < .05), whereas diet did not. At 2 and 4 weeks, there was the largest progressive decrease in strain in the paravisceral/supraceliac aorta (P < .05), which was the segment most likely to be involved in aneurysm formation in this model. Conclusions In the Ang II/apoE−/− aneurysm model, the aorta significantly stiffens (with decreased strain) shortly after Ang II infusion, and this progressively continues through the next 4 weeks. High-fat feeding did not have an impact on wall strain. Delineation of biomechanical factors and AAA morphology via duplex scan and speckle-tracking algorithms in mouse models should accelerate insights into human AAAs.

Mechanical stimulation is recognized as a potent modulator of cellular behaviors such as proliferation, differentiation, and extracellular matrix assembly. However, the study of how cell-generated traction force changes in response to stretch is generally limited to short-term stimulation. The goal of this work is to determine how cells actively alter their traction force in response to long-term physiological cyclic stretch as a function of cell pre-stress. We have developed, to our knowledge, a novel method to assess traction force after long-term (24 h) uniaxial or biaxial cyclic stretch under conditions of high cell pre-stress with culture on stiff (7.5 kPa) polyacrylamide gels (with or without transforming growth factor β1 (TGF-β1)) and low pre-stress by treating with blebbistatin or culture on soft gels (0.6 kPa). In response to equibiaxial stretch, valvular interstitial cells on stiff substrates decreased their traction force (from 300 nN to 100 nN) and spread area (from 3000 to 2100 μm(2)). With uniaxial stretch, the cells had similar decreases in traction force and area and reoriented perpendicular to the stretch. TGF-β1-treated valvular interstitial cells had higher pre-stress (1100 nN) and exhibited a larger drop in traction force with uniaxial stretch, but the percentage changes in force and area with stretch were similar to the non-TGF-β1-treated group. Cells with inhibited myosin II motors increased traction force (from 41 nN to 63 nN) and slightly reoriented toward the stretch direction. In contrast, cells cultured on soft gels increased their traction force significantly, from 15 nN to 45 nN, doubled their spread area, elongated from an initially rounded morphology, and reoriented perpendicular to the uniaxial stretch. Contractile-moment measurements provided results consistent with total traction force measurements. The combined results indicate that the change in traction force in response to external cyclic stretch is dependent upon the initial cell pre-stress. This finding is consistent with depolymerization of initially high-tension actin stress fibers, and reinforcement of an initially low-tension actin cytoskeleton.