2D timelapse and 3D fluorescence microscopy with applications to vascular tissue engineering
Stamatas, Georgios Nikolaou
McIntire, Larry V.
Doctor of Philosophy thesis
Part of the definition given to the new emerging science discipline of tissue engineering is the understanding of the structure-function relationships at the cellular level. In this context it is important for vascular tissue engineering to understand the mechanisms involved in the vascular cell responses to their mechanically active environment. This work has elucidated some aspects of the complicated puzzle of mechanotransduction in vascular smooth muscle cells (SMC) and endothelial cells (EC). Two dimensional timelapse fluorescence microscopy revealed rapid alkalinization occurring in cultured human aortic SMC exposed to well defined fluid flow profiles. The response was reversible and persisted for at least 20 min after flow initiation. The magnitude of the alkalinization (0.14 pH units) was enough to increase the nitric oxide synthase activity and account at least in part for the flow-induced increases in NO production by SMC. Use of specific inhibitors demonstrated the involvement of the Na$\sp+$/H$\sp+$ exchanger in the flow-induced response, whereas the Cl$\sp-$/HCO$\sb3\sp-$ exchanger was active even under stationary conditions. The involvement of calcium as a second messenger in the EC flow-induced mechanotransduction and the localization of possible signals within the cell was addressed by a three dimensional fluorescence microscopy technique. After 5 min of flow initiation there was a significant calcium increase in the nuclear region. The response was cytoskeleton independent. The same technique revealed early flow-induced changes in the three dimensional EC architecture. Nuclear and whole cell heights were reduced by about 1 $\mu$m with a corresponding increase in the cross-sectional area at lower optical sections. Using specific cytoskeleton disrupters we demonstrated that the whole cell height response was cytoskeleton independent and the nuclear height response was microtubule dependent. Thus, it has been shown that force imposed on the EC membrane is rapidly transmitted by microtubules to the endothelial nucleus. A mechanical equivalent model is presented to explain the cytoskeleton involvement in flow-induced structural changes based on tensegrity arguments. The early responses in the nuclear calcium and structure demonstrated in this study may be important for the shear-induced gene regulation.
Biology, Cell; Engineering, Biomedical; Engineering, Chemical