MECHANISMS OF SHEAR-INDUCED ADAPTIVE RHEOLOGY IN ENDOTHELIAL CELLS
Open Access
- Author:
- Dangaria, Jhanvi H.
- Graduate Program:
- Bioengineering
- Degree:
- Doctor of Philosophy
- Document Type:
- Dissertation
- Date of Defense:
- March 06, 2008
- Committee Members:
- Peter J Butler, Committee Chair/Co-Chair
Cheng Dong, Committee Member
William O Hancock, Committee Member
Erin Elizabeth Sheets, Committee Member - Keywords:
- mechanotransduction
particle tracking
viscoelasticity
atherosclerosis
cell mechanics - Abstract:
- Vascular endothelial cells (ECs) constitute the endothelium which lines the entire cardiovascular system and is consequently exposed to mechanical stimuli, such as fluid shear stress due to blood flow and cyclic stretch due to hydrostatic pressure fluctuations, and to chemical stimuli, such as growth factors, inflammatory cytokines, and hormones. Vascular health and pathology is tied to the adaptation of cell mechanics through the regulation of cellular structures that sense and respond to mechanical forces. While long term shear adaptation of endothelial cells has been studied extensively, little is know about the temporal changes in cell mechanics occurring on same time scale as hemodynamic forces. The interplay of force, dynamic cell mechanics, and biochemical signaling is the main focus of the studies summarized in this dissertation. The principal tools used to determine shear-induced temporal changes in cell mechanics were particle tracking microrheology, and analysis of creep functions. To implement these methods, a custom image-correlation based algorithm was designed in LabVIEW to measure nano-scale motions of cellular features and endocytosed beads. Time-lapse images were acquired using high-resolution differential interference contrast digital microscopy of confluent endothelial cells in a flow chamber. These techniques were optimized for use on cells in flow chambers by extensive minimization of motion artifacts. In particular, photolithography was used to microfabricate fiduciary posts on glass coverslips. Position of microposts relative to thermally-induced motion of vesicles in cells was used to subtract low- and high-frequency microscope stage motion artifacts and to facilitate sub-pixel tracking from time-lapse images of adherent cells. This study documents the first reported observations of rapid changes in cell mechanics in response to large temporal fluctuations in shear stress (i.e. step change). Endothelial cells subjected to a step change in shear stress from 0 to 10 dynes/cm2 became significantly more compliant as early as 30 seconds after onset of shear stimulation. Viscoelastic parameters recovered within 4 minutes of shearing even though shear stress was maintained and no further rheological modulation was observed after cessation of shear stress. Using a phenomenological model of a viscoelastic liquid, overall macrorheological parameters were calculated and the time and length scales of viscoelastic deformation were found to be 3 sec and 50 nm, respectively. Furthermore, experiments were designed to explore the role of actomyosin interactions in the dynamic control of EC mechanics. This study grew from knowledge that overall cellular mechanics is strongly dependent on actin and from recent in vitro (model polymer systems) and in vivo (dictyostelium cells and myoblasts) studies suggesting a triple role for myosin II in regulating cytoplasmic rheology through cellular prestress, actin crosslinking, and facilitated polymer diffusion. A novel observation from these studies is that actomyosin regulates macrorheology (i.e. viscoelastic deformation in response to shear stress), microrheology (i.e. constrained thermal motion of small beads) and cellular activation. In fact, the observation of a shear-induced, actin-dependent contraction, the onset time of which depended on myosin II may point the way to a new understanding of the dynamic control of cell mechanics. Together, these studies provide new insight into how early mechanotransduction events may depend on dynamic modulation of mechanical properties and suggest that dynamic adaptive rheology may be one link between hemodynamic force and vascular disease.