Inflammatory edema is mediated by cytoskeletal-based mechanical forces, which induce endothelial-cell retraction. Agents that increase intracellular cAMP protect the endothelium from inflammatory mediators. Understanding the mechanisms by which edemagenic agents and cAMP agonists remodel the endothelial cytoskeleton, which, in turn, regulates barrier function, is critical to precisely develop treatments. We previously reported that edemagenic agents disrupt barrier function independent of expression of actin-myosin contraction. Expression of actin-myosin contraction increases inflammatory edema by impacting the restoration of barrier function. Increased intracellular cAMP does not protect barrier function through inhibition of actin-myosin tension development, but does so by uncoupling this contractile load from disrupting cell adhesion. The PI, a new investigator, will test the hypothesis in human endothelial cells that microtubules modulate endothelial-cell adhesion through a counterbalance force with actin-myosin filaments. By modulating microtubule assembly, the expression of centripetal actin-myosin tension can have differential effects on cell adhesion. To test this hypothesis we have taken an interdisciplinary approach that uses cell biology and engineering to address this fundamental question.
In Aim 1, we will test whether changes in microtubule assembly remodel mechanical forces through shifts in load-bearing forces between microtubules and actin-myosin elements.
In Aim 2, we will localize whether microtubules and actin-myosin forces are directed at cell-cell or cell-matrix sites.
In Aim 3, we will then test whether changes in microtubule assembly alter cell adhesion based on shifts in load-bearing forces, and whether physiological stimuli utilize this basic mechanism.
In Aim 4, we will test an alternative hypothesis that changes in microtubule assembly alter cell adhesion through crosstalk communication through integrin-ligand interactions. We have assembled an interdisciplinary group of biologists, a physician, a physicist and engineers. We will use a mathematical model of transendothelial impedance to localize changes in cell adhesion. We will directly measure centripetal tension and cell stiffness. We will measure biochemical changes in myosin, actin and microtubules. We will utilize molecular approaches and quantitative dynamic imaging approaches in living cells to further validate our hypothesis. We believe that the results of these studies will greatly advance vascular and cell biology.
