Engineering approaches to study lung mechanobiology

Engineering tools and techniques greatly expand the scope of biological studies, permitting investigation of the interplay between mechanical forces, and cell and tissue biology, and also enabling in vitro replication of the physiological environment. This dissertation describes the development of various engineering approaches to investigate the regulation of the pulmonary blood-gas barrier, and endothelial barrier function in particular. The pulmonary blood-gas barrier consists of airway epithelial cells, which are exposed to the inhaled air, and vascular endothelial cells, which line the blood vessels. In Chapter 2 of this thesis, I describe the development of a cyclic cell stretching device to mimic the mechanical environment of the pulmonary endothelium during physiological and pathological levels of cyclic stretch. I further varied the cell substrate stiffness in order to replicate the mechanics of soft and healthy tissue, or stiff, fibrotic tissue. This device also enables dynamic imaging of cells subject to mechanical stretch. Studies conducted with this device (Chapter 2) demonstrated that physiological cyclic stretch and substrate stiffness coordinately protect the pulmonary endothelium against disruption by the inflammatory mediator, thrombin. Further, quantitative immunofluorescence imaging established that cyclic stretch conditioning led to remodeling of endothelial junctions, and changes in the dynamics of cell membrane protrusions called lamellipodia (Chapter 3). These findings provided valuable insights into potential subcellular mechanisms underlying the cyclic stretch-induced protection of lung endothelial monolayers against proinflammatory signals. The integrity of the pulmonary endothelium also depends on the force balance in the monolayer, which results from the interplay between intracellular contractile forces and cell-cell and cell-matrix tethering (adhesion) forces. In Chapter 4, I describe studies using a microfabricated platform to quantify the impact of biochemical, genetic, and matrix-based perturbations to cell-generated mechanical forces. Results from these studies provided important insights into the distribution of forces between cell-cell and cell-matrix adhesions, and conditions under which this distribution was perturbed. Findings directly assessed the impact of genetic and biochemical perturbations on aspects of lung injury conducted in collaboration with the Komarova and Malik research teams at the University of Illinois at Chicago. Finally, to better replicate pulmonary tissue, I developed an in vitro model of the pulmonary blood-gas barrier, consisting of primary airway epithelial cells exposed to air and primary pulmonary endothelial cells cultured in contact with medium on the opposite side of a porous membrane (Chapter 5). This close proximity enabled the two cell types to exchange soluble factors. I devised differentiation conditions under which epithelial cultures exhibited a polarized phenotype with rich mucociliary differentiation, as observed in vivo. This model constitutes a first step towards a lung-on-a-chip device being developed in collaboration with the Kenis and Murphy research teams at University of Illinois at Urbana-Champaign, in order to investigate the effects induced by exposure of the airway epithelium and pulmonary endothelium to aerosolized nanoparticles