Small airway epithelial cells form a continuous sheet lining the conducting airways, which serves many functions including a physical barrier to protect the underlying tissue. In asthma, injury to epithelial cells can occur during bronchoconstriction, which may exacerbate airway hyperreactivity. To investigate the role of epithelial cell rupture in airway constriction, laser ablation was used to precisely rupture individual airway epithelial cells of small airways (<300-μm diameter) in rat lung slices (∼250-μm thick). Laser ablation of single epithelial cells using a femtosecond laser reproducibly induced airway contraction to ∼70% of the original cross-sectional area within several seconds, and the contraction lasted for up to 40 s. The airway constriction could be mimicked by mechanical rupture of a single epithelial cell using a sharp glass micropipette but not with a blunt glass pipette. These results suggest that soluble mediators released from the wounded epithelial cell induce global airway contraction. To confirm this hypothesis, the lysate of primary human small airway epithelial cells stimulated a similar airway contraction. Laser ablation of single epithelial cells triggered a single instantaneous Ca(2+) wave in the epithelium, and multiple Ca(2+) waves in smooth muscle cells, which were delayed by several seconds. Removal of extracellular Ca(2+) or decreasing intracellular Ca(2+) both blocked laser-induced airway contraction. We conclude that local epithelial cell rupture induces rapid and global airway constriction through release of soluble mediators and subsequent Ca(2+)-dependent smooth muscle shortening.

Epithelial injury and airway hyperresponsiveness are prominent features of asthma. We have previously demonstrated that laser ablation of single epithelial cells immediately induces global airway constriction through Ca(2+)-dependent smooth muscle shortening. The response is mediated by soluble mediators released from wounded single epithelial cells; however, the soluble mediators and signaling mechanisms have not been identified. In this study, we investigated the nature of the epithelial-derived soluble mediators and the associated signaling pathways that lead to the L-type voltage-dependent Ca(2+) channel (VGCC)-mediated Ca(2+) influx. We found that inhibition of adenosine A1 receptors (or removal of adenosine with adenosine deaminase), cyclooxygenase (COX)-2 or prostaglandin E receptor 3 (EP3) receptors, epidermal growth factor receptor (EGFR), or platelet-derived growth factor receptor (PDGFR) all significantly blocked Ca(2+) oscillations in smooth muscle cells and airway contraction induced by local epithelial injury. Using selective agonists to activate the receptors in the presence and absence of selective receptor antagonists, we found that adenosine activated the signaling pathway A1R→EGFR/PDGFR→COX-2→EP3→VGCCs→calcium-induced calcium release, leading to intracellular Ca(2+) oscillations in airway smooth muscle cells and airway constriction.

Methods for tuning extracellular matrix (ECM) mechanics in 3D cell culture that rely on increasing the concentration of either protein or cross-linking molecules fail to control important parameters such as pore size, ligand density, and molecular diffusivity. Alternatively, ECM stiffness can be modulated independently from protein concentration by mechanically loading the ECM. We have developed a novel device for generating stiffness gradients in naturally derived ECMs, where stiffness is tuned by inducing strain, while local mechanical properties are directly determined by laser tweezers based active microrheology (AMR). Hydrogel substrates polymerized within 35 mm diameter Petri dishes are strained non-uniformly by the precise rotation of an embedded cylindrical post, and exhibit a position-dependent stiffness with little to no modulation of local mesh geometry. Here we present the device in the context of fibrin hydrogels. First AMR is used to directly measure local micromechanics in unstrained hydrogels of increasing fibrin concentration. Changes in stiffness are then mapped within our device, where fibrin concentration is held constant. Fluorescence confocal imaging and orbital particle tracking are used to quantify structural changes in fibrin on the micro and nano levels respectively. The micromechanical strain stiffening measured by microrheology is not accompanied by ECM microstructural changes under our applied loads, as measured by confocal microscopy. However, super-resolution orbital tracking reveals nanostructural straightening, lengthening, and reduced movement of fibrin fibers. Furthermore, we show that aortic smooth muscle cells cultured within our device are morphologically sensitive to the induced mechanical gradient. Our results demonstrate a powerful cell culture tool that can be used in the study of mechanical effects on cellular physiology in naturally derived 3D ECM tissues.

Methods for tuning extracellular matrix (ECM) mechanics in 3D cell culture that rely on increasing the concentration of either protein or cross-linking molecules fail to control important parameters such as pore size, ligand density, and molecular diffusivity. Alternatively, ECM stiffness can be modulated independently from protein concentration by mechanically loading the ECM. We have developed a novel device for generating stiffness gradients in naturally derived ECMs, where stiffness is tuned by inducing strain, while local mechanical properties are directly determined by laser tweezers based active microrheology (AMR). Hydrogel substrates polymerized within 35 mm diameter Petri dishes are strained non-uniformly by the precise rotation of an embedded cylindrical post, and exhibit a position-dependent stiffness with little to no modulation of local mesh geometry. Here we present the device in the context of fibrin hydrogels. First AMR is used to directly measure local micromechanics in unstrained hydrogels of increasing fibrin concentration. Changes in stiffness are then mapped within our device, where fibrin concentration is held constant. Fluorescence confocal imaging and orbital particle tracking are used to quantify structural changes in fibrin on the micro and nano levels respectively. The micromechanical strain stiffening measured by microrheology is not accompanied by ECM microstructural changes under our applied loads, as measured by confocal microscopy. However, super-resolution orbital tracking reveals nanostructural straightening, lengthening, and reduced movement of fibrin fibers. Furthermore, we show that aortic smooth muscle cells cultured within our device are morphologically sensitive to the induced mechanical gradient. Our results demonstrate a powerful cell culture tool that can be used in the study of mechanical effects on cellular physiology in naturally derived 3D ECM tissues.

Protein based polymers provide an exciting and complex landscape for tunable natural biomaterials through modulation of molecular level interactions. Here we demonstrate the ability to modify protein polymer structural and mechanical properties at multiple length scales by molecular 'interference' of fibrin's native polymerization mechanism. We have previously reported that engagement of fibrin's polymerization 'hole b', also known as 'b-pockets', through PEGylated complementary 'knob B' mimics can increase fibrin network porosity but also, somewhat paradoxically, increase network stiffness. Here, we explore the possible mechanistic underpinning of this phenomenon through characterization of the effects of knob B-fibrin interaction at multiple length scales from molecular to bulk polymer. Despite its weak monovalent binding affinity for fibrin, addition of both knob B and PEGylated knob B at concentrations near the binding coefficient, Kd, increased fibrin network porosity, consistent with the reported role of knob B-hole b interactions in promoting lateral growth of fibrin fibers. Addition of PEGylated knob B decreases the extensibility of single fibrin fibers at concentrations near its Kd but increases extensibility of fibers at concentrations above its Kd. The data suggest this bimodal behavior is due to the individual contributions knob B, which decreases fiber extensibility, and PEG, which increase fiber extensibility. Taken together with laser trap-based microrheological and bulk rheological analyses of fibrin polymers, our data strongly suggests that hole b engagement increases in single fiber stiffness that translates to higher storage moduli of fibrin polymers despite their increased porosity. These data point to possible strategies for tuning fibrin polymer mechanical properties through modulation of single fiber mechanics.