Michael C. Ferko

Time-Correlated, Single-Photon Counting Methods in Endothelial Cell Mechanobiology

While mechanical forces are known to guide the development of nearly all biological tissues including bone, cartilage, and many soft tissues, much attention has focused on endothelial cell mechanobiology and the role of blood flow-induced forces in regulating the health of blood vessels. It is now well accepted that modulation of endothelial cell physiology and pathophysiology by fluid mechanical forces is a principal reason why atherosclerotic lesions are located at areas of disturbed flow including at arterial branch points and areas of high arterial curvature. However, the molecular identity of endothelial cell mechanosensors remains elusive largely due to the complexity of cell mechanics and to the difficulty in identifying when and where a candidate mechanosensor has been perturbed. Thus, new methods of cell-specific mechanical modeling along with molecular-scale readouts of perturbation by force are needed to help unravel the magnitude-, time-, and position-dependent responses of endothelial cells to mechanical forces.

Multiscale Stress Analysis of Sheared and Focally-Adhered Endothelial Cells: Role of Subcellular Matrix Moduli in Focal Adhesion-Mediated Signaling

Focal adhesions (FAs) and their associated integrins are thought to act as mechanosensors and transducers of shear stress into intracellular biochemical signals. However, to date there exists no quantification of the magnitude of forces generated at integrin molecules in response to apically-applied fluid shear stress. Thus, we used finite element analysis of fluid dynamics and cellular stresses to compute FA stresses from solid models of focally-adhered endothelial cells. These models were developed from quantitative 3-D microscopy and total internal reflection fluorescence (TIRF) microscopy of calcein-stained endothelial cells. Extrusion coupling variables mapped stresses from the macroscale cell model to individual microscale 3-D models of FAs determined from quantitative TIRF. Included in the microscale FA model were moduli for subcellular matrix (SCM) (e.g. hyaluronan, hyaluronaic acid and other glycocalyx constituents) and extracellular matrix (ECM) (e.g. collagen, fibronectin). Integrin forces were estimated from assumed bonds densities and computed FA stresses. Maximal bond tension obtained from the simulation for a single integrin-extracellular matrix (ECM) bond was .1pN. Thus, it is unlikely that integrin-ECM bonds or chemical activities are appreciably affected by shear stress. The computational model, however, supports an alternative model of activation in and reorganization of FAs in which shear stress-induced forces cause the membrane to bend toward and away from the ECM immediately upstream and downstream of the FA, respectively. The simulation also suggests that the elasticity of the SCM plays an important role in modulating shear-induced FA reorganization. These results support a new model of endothelial cell activation by shear stress in which integrins and FAs participate in the directional biasing of force-induce signaling but do not initiate it.Copyright

Time-resolved fluorescence microscopy of stressed membranes of living endothelial cells

Advances in the understanding of mechanotransduction in endothelial cells require reliable predictive models of stress and readouts of mechano-activation in the same cell. In order to determine correlations between stress and cellular activation, we have coupled rapid 3-D rendering of optical images of stained endothelial cells and computational fluid and mechanics models with photonics-based detection of molecular perturbation. Central to our approach is a multimodal, confocal molecular dynamics microscope with time-correlated, single-photon counting electronics to measure excited-state lifetime dynamics of fluorescent molecules in cell membranes. Preliminary results suggest a correlation of positive shear stress gradients and membrane tension with shear-induced membrane perturbations at sub-micron scales. Such position-dependence of membrane stress concentrations may be used as cues for mechano-chemical signal transduction.