Hainsworth Y. Shin

Regulation of endothelial cell proliferation and apoptosis by cyclic pressure.

AbstractThe present study investigated the proliferative and apoptotic responses of human umbilical vein endothelial cells (HUVECs) to well-defined, sinusoidal pressures (60/20, 100/60, and 140/100 mm Hg/mm Hg) at 1 Hz for up to 24 h under Media 199 containing either 1% FBS and 0.04% bovine brain extract (BBE) (low serum/growth factor conditions) or 10% FBS and 0.4% BBE (normal serum/growth factor conditions). Controls were HUVEC maintained under 0.2 mm Hg sustained pressure, but otherwise, similar experimental conditions. Under low serum/growth factor conditions, exposure of HUVEC to 60/20 mm Hg/mm Hg cyclic pressure at 1 Hz for time periods up to 24 h resulted in increases in total cell population density, apoptosis, and DNA synthesis. Under normal serum/growth factor conditions, exposure of HUVEC to either 60/20 or 100/60 mm Hg/mm Hg cyclic pressures resulted in increased DNA synthesis but did not significantly affect cell density or the apoptotic index. A reduced rate of cell death was observed in HUVEC under low serum/growth factor conditions after exposure to 140/100 mm Hg/mm Hg. Under normal serum/growth factor conditions, HUVEC exposed to 140/100 mm Hg/mm Hg cyclic pressure exhibited reduced DNA synthesis. Endothelial cells, therefore, sense and respond to physiologic levels of cyclic pressure by modifying cell proliferation and apoptosis in a mean-pressure-selective manner.

Fluid shear‐induced activation and cleavage of CD18 during pseudopod retraction by human neutrophils

Surface membrane expression and conformational activation of CD18 integrins into an open molecular configuration play critical roles in neutrophil ligand binding, membrane attachment, spreading on the endothelium, and cell migration to sites of inflammation. Previously, we observed pseudopod retraction and concomitant cleavage of CD18 by human neutrophils upon exposure to fluid shear stress. But the underlying cellular mechanism(s) linking these phenomena remains unknown. We hypothesize here that activation of CD18 under the influence of fluid shear stress leads to its increased susceptibility to proteolytic cleavage by lysosomal proteases such as cathepsin B and is a requirement for CD18 cleavage and subsequent pseudopod retraction. Specifically, we report conformational changes in the CD18 extracellular domain on neutrophils exposed to physiological fluid shear stresses. Western blot analysis using a CD18 antibody targeted against the intracellular domain revealed reduced levels of full‐length CD18 after stimulation of neutrophils with either fluid shear stress or with the Ca2+ ionophore phorbol 12‐myristate 13‐acetate (PMA; 100 nM) in the presence of exogenous cathepsin B (0.5 U/ml). Moreover, we identified cathepsin B as one protease that may be released by neutrophils under flow and required for shear‐induced pseudopod retraction. These results suggest that a putative mechanotransduction mechanism involving shear‐induced changes in the conformation of CD18 and its subsequent cleavage from the cell surface serves to regulate pseudopod activity of neutrophils under physiologic shear stress. J. Cell. Physiol. 214: 528–536, 2008.

Membrane cholesterol modulates the fluid shear stress response of polymorphonuclear leukocytes via its effects on membrane fluidity

Continuous exposure of polymorphonuclear leukocytes (PMNLs) to circulatory hemodynamics points to fluid flow as a biophysical regulator of their activity. Specifically, fluid flow-derived shear stresses deactivate leukocytes via actions on the conformational activities of proteins on the cell surface. Because membrane properties affect activities of membrane-bound proteins, we hypothesized that changes in the physical properties of cell membranes influence PMNL sensitivity to fluid shear stress. For this purpose, we modified PMNL membranes and showed that the cellular mechanosensitivity to shear was impaired whether we increased, reduced, or disrupted the organization of cholesterol within the lipid bilayer. Notably, PMNLs with enriched membrane cholesterol exhibited attenuated pseudopod retraction responses to shear that were recovered by select concentrations of benzyl alcohol (a membrane fluidizer). In fact, PMNL responses to shear positively correlated (R(2) = 0.96; P < 0.0001) with cholesterol-related membrane fluidity. Moreover, in low-density lipoprotein receptor-deficient (LDLr(-/-)) mice fed a high-fat diet (a hypercholesterolemia model), PMNL shear-responses correlated (R(2) = 0.5; P < 0.01) with blood concentrations of unesterified (i.e., free) cholesterol. In this regard, the shear-responses of PMNLs gradually diminished and eventually reversed as free cholesterol levels in blood increased during 8 wk of the high-fat diet. Collectively, our results provided evidence that cholesterol is an important component of the PMNL mechanotransducing capacity and elevated membrane cholesterol impairs PMNL shear-responses at least partially through its impact on membrane fluidity. This cholesterol-linked perturbation may contribute to dysregulated PMNL activity (e.g., chronic inflammation) related to hypercholesterolemia and causal for cardiovascular pathologies (e.g., atherosclerosis).

Cyclic pressure modulates endothelial barrier function.

Although numerous studies have documented the importance of mechanical forces in regulating many endothelial cell functions, the effects of these physical stimuli on endothelial barrier function are not well characterized. The present study used a custom-designed, cyclic pressure system to expose human umbilical vein endothelial cells (HUVECs) to physiologically relevant sinusoidal pressures and demonstrated that exposure to 140/100, but not to 60/20, mm Hg cyclic pressure at 1 Hz for 18 h resulted in a significant (p <.05) reduction in transendothelial permeability to albumin. Moreover, these cyclic pressure-selective changes in HUVEC barrier function occurred concomitantly with redistribution of intracellular tight junction protein zona occludens (ZO)-1 and reorientation of the F-actin cytoskeleton. In contrast, exposure of HUVECs to cyclic pressure had no affect on localization of adherens junctions proteins, vascular endothelial (VE)-cadherin, and beta-catenin. These results, therefore, provide the first evidence that select levels of cyclic pressure, a mechanical force pertinent to the hemodynamic vascular milieu, modulates the endothelial barrier function concomitant with an altered distribution of tight junction component, ZO-1.

Receptor-mediated basic fibroblast growth factor signaling regulates cyclic pressure-induced human endothelial cell proliferation.

Vascular endothelial cells sense and respond to pressure by molecular mechanism(s) which, to date, remain poorly understood. The present study investigated basic fibroblast growth factor (bFGF) signaling as a putative mechanotransduction pathway involved in the proliferative responses of human umbilical vein endothelia cells (HUVECs) to 60/20 mm Hg cyclic pressure at 1 Hz for 24 h. Under these conditions, the enhanced proliferative response of these HUVECs was not associated with an increased synthesis/release of bFGF, but involved rapid (within 30 min from the onset of exposure to pressure) tyrosine phosphorylation of the bFGF receptor, FGFR-2. Furthermore, monoclonal antibodies to either bFGF or FGFR-2 attenuated the increased proliferation of HUVECs exposed to 60/20 mm Hg cyclic pressure. HUVECs proliferation under 60/20 mm Hg at 1 Hz cyclic pressure is, therefore, dependent upon bFGF and involves FGFR-2 activation.

Shear-Sensitive Regulation of Neutrophil Flow Behavior and Its Potential Impact on Microvascular Blood Flow Dysregulation in Hypercholesterolemia

Objective— Shear stress–induced pseudopod retraction is an anti-inflammatory measure that minimizes neutrophil activity and is regulated by membrane cholesterol. We tested the hypothesis that a hypercholesterolemic impairment of shear mechanotransduction alters the neutrophil flow behavior leading to microvascular dysfunction. Approach and Results— We examined the shear effects on the flow behavior of human leukocytes. When subjected to shearing during cone-plate viscometry, leukocyte suspensions exhibited parallel time-dependent reductions in viscosity and pseudopod activity. Shear-induced reductions in suspension viscosity were attenuated by membrane cholesterol enrichment. We also showed that enhanced pseudopod activity of leukocyte suspensions in 10% hematocrit significantly (P<0.05) raised the flow resistance of microvascular mimics. These results implicate an impaired neutrophil pseudopod retraction response to shear in hypercholesterolemic microvascular dysfunction. We confirmed this using near-infrared diffuse correlation spectroscopy to assess skeletal muscle blood flow regulation in the hindlimbs of mice subjected to reactive hyperemia. Using a custom protocol for the mouse, we extrapolated an adjusted peak flow and time to adjusted peak flow to quantify the early phase of the blood flow recovery response during reactive hyperemia when shear mechanobiology likely has a maximal impact. Compared with mice on normal diet, hypercholesterolemic mice exhibited significantly (P<0.05) reduced adjusted peak flow and prolonged time to adjusted peak flow which correlated (r=0.4 and r=−0.3, respectively) with neutrophil shear responsiveness and were abrogated by neutropenia. Conclusions— These results provide the first evidence that the neutrophils contribute to tissue blood flow autoregulation. Moreover, a deficit in the neutrophil responsiveness to shear may be a feature of hypercholesterolemia-related microvascular dysfunction.

Linking the Pathobiology of Hypercholesterolemia with the Neutrophil Mechanotransduction

© 2012 Zhang and Shin, licensee InTech. This is an open access chapter distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Linking the Pathobiology of Hypercholesterolemia with the Neutrophil Mechanotransduction

Osteoblast Responses to Steady Shear Stress

Like many tissues in the human body, bone is constantly undergoing changes in its microstructure. The homeostasis of bone can be described as a dynamic equilibrium existing between resorption and deposition of mineral. Although each bone has a genetically determined minimal mass and structure, the normal state of bone in the physiologic environment is a consequence of adaptations to various chemical and physical stimuli (Lanyon 1984). These adaptations are achieved through an active remodeling of the internal and surface characteristics (e.g., structure and mineral content) of bone.