Microgravity Effects on Transendothelial Transport

The Endothelial Cell (EC) layer which lines blood vessels from the aorta to the capillaries provides the principal barrier to transport of water and solutes between blood and underlying tissue. Endothelial cells are continuously exposed to the mechanical shearing force (shear stress) and normal force (pressure) imposed by flowing blood on their surface, and they are adapted to this mechanical environment. When the cardiovascular system is exposed to microgravity, the mechanical environmental of endothelial cells is perturbed drastically and the transport properties of EC layers are altered in response. We have shown recently that step changes in shear stress have an acute effect on transport properties of EC layers in a cell culture model, and several recent studies in different vessels of live animals have confirmed the shear-dependent transport properties of the endothelium. We hypothesize that alterations in mechanical forces induced by microgravity and their resultant influence on transendothelial transport of water and solutes are, in large measure, responsible for the characteristic cephalad fluid shift observed in humans experiencing microgravity. To study the effects of altered mechanical forces on transendothelial transport and to test pharmacologic agents as counter measures to microgravity induced fluid shifts we have proposed ground-based studies using well defined cell culture models

The Adaptive Remodeling of Endothelial Glycocalyx in Response to Fluid Shear Stress

The endothelial glycocalyx is vital for mechanotransduction and endothelial barrier integrity. We previously demonstrated the early changes in glycocalyx organization during the initial 30 min of shear exposure. In the present study, we tested the hypothesis that long-term shear stress induces further remodeling of the glycocalyx resulting in a robust layer, and explored the responses of membrane rafts and the actin cytoskeleton. After exposure to shear stress for 24 h, the glycocalyx components heparan sulfate, chondroitin sulfate, glypican-1 and syndecan-1, were enhanced on the apical surface, with nearly uniform spatial distributions close to baseline levels that differed greatly from the 30 min distributions. Heparan sulfate and glypican-1 still clustered near the cell boundaries after 24 h of shear, but caveolin-1/caveolae and actin were enhanced and concentrated across the apical aspects of the cell. Our findings also suggest the GM1-labelled membrane rafts were associated with caveolae and glypican-1/heparan sulfate and varied in concert with these components. We conclude that remodeling of the glycocalyx to long-term shear stress is associated with the changes in membrane rafts and the actin cytoskeleton. This study reveals a space- and time- dependent reorganization of the glycocalyx that may underlie alterations in mechanotransduction mechanisms over the time course of shear exposure

Influence of blood rheology and vessel wall motion on arterial fluid mechanics

Most model studies of arterial fluid mechanics have assumed that blood is a Newtonian fluid and that vessel wall motion driven by the pressure pulse has a small influence on the local velocity and pressure distributions. This paper provides a brief historical review of arterial flow modeling which emphasizes recent developments in non-Newtonian blood analog fluids and studies of the influence of vessel wall motion on local flow fields. It is pointed out that vessel wall motion can have a dominant effect on mean pressure gradient and a significant effect on mean wall shear stress in the aorta

Interaction between the Stress Phase Angle (SPA) and the Oscillatory Shear Index (OSI) Affects Endothelial Cell Gene Expression

Hemodynamic forces play an important role in the non-uniform distribution of atherosclerotic lesions. Endothelial cells are exposed simultaneously to fluid wall shear stress (WSS) and solid circumferential stress (CS). Due to variations in impedance (global factors) and geometric complexities (local factors) in the arterial circulation a time lag arises between these two forces that can be characterized by the temporal phase angle between CS and WSS (stress phase angle±SPA). Asynchronous flows (SPA close to -180Ê) that are most prominent in coronary arteries have been associated with localization of atherosclerosis. Reversing oscillatory flows characterized by an oscillatory shear index (OSI) that is great than zero are also associated with atherosclerosis localization. In this study we examined the relationship between asynchronous flows and reversing flows in altering the expression of 37 genes relevant to atherosclerosis development. In the case of reversing oscillatory flow, we observed that the asynchronous condition upregulated 8 genes compared to synchronous hemodynamics, most of them proatherogenic. Upregulation of the pro-inflammatory transcription factor NFκB p65 was confirmed by western blot, and nuclear translocation of NFκB p65 was confirmed by immunofluorescence staining. A comparative study between nonreversing flow and reversing flow found that in the case of synchronous hemodynamics, reversing flow altered the expression of 11 genes, while in the case of asynchronous hemodynamics, reversing flow altered the expression of 17 genes. Reversing flow significantly upregulated protein expression of NFκB p65 for both synchronous and asynchronous conditions. Nuclear translocation of NFκB p65 was confirmed for synchronous and asynchronous conditions in the presence of flow reversal. These data suggest that asynchronous hemodynamics and reversing flow can elicit proatherogenic responses in endothelial cells compared to synchronous hemodynamics without shear stress reversal, indicating that SPA as well as reversal flow (OSI) are important parameters characterizing arterial susceptibility to disease

Fluid Shear Stress Regulates the Invasive Potential of Glioma Cells via Modulation of Migratory Activity and Matrix Metalloproteinase Expression

Background Glioma cells are exposed to elevated interstitial fluid flow during the onset of angiogenesis, at the tumor periphery while invading normal parenchyma, within white matter tracts, and during vascular normalization therapy. Glioma cell lines that have been exposed to fluid flow forces in vivo have much lower invasive potentials than in vitro cell motility assays without flow would indicate. Methodology/Principal Findings A 3D Modified Boyden chamber (Darcy flow through collagen/cell suspension) model was designed to mimic the fluid dynamic microenvironment to study the effects of fluid shear stress on the migratory activity of glioma cells. Novel methods for gel compaction and isolation of chemotactic migration from flow stimulation were utilized for three glioma cell lines: U87, CNS-1, and U251. All physiologic levels of fluid shear stress suppressed the migratory activity of U87 and CNS-1 cell lines. U251 motility remained unaltered within the 3D interstitial flow model. Matrix Metalloproteinase (MMP) inhibition experiments and assays demonstrated that the glioma cells depended on MMP activity to invade, and suppression in motility correlated with downregulation of MMP-1 and MMP-2 levels. This was confirmed by RT-PCR and with the aid of MMP-1 and MMP-2 shRNA constructs. Conclusions/Significance Fluid shear stress in the tumor microenvironment may explain reduced glioma invasion through modulation of cell motility and MMP levels. The flow-induced migration trends were consistent with reported invasive potentials of implanted gliomas. The models developed for this study imply that flow-modulated motility involves mechanotransduction of fluid shear stress affecting MMP activation and expression. These models should be useful for the continued study of interstitial flow effects on processes that affect tumor progression

High Glucose Attenuates Shear-Induced Changes in Endothelial Hydraulic Conductivity by Degrading the Glycocalyx

Diabetes mellitus is a risk factor for cardiovascular disease; however, the mechanisms through which diabetes impairs homeostasis of the vasculature have not been completely elucidated. The endothelium interacts with circulating blood through the surface glycocalyx layer, which serves as a mechanosensor/transducer of fluid shear forces leading to biomolecular responses. Atherosclerosis localizes typically in regions of low or disturbed shear stress, but in diabetics, the distribution is more diffuse, suggesting that there is a fundamental difference in the way cells sense shear forces. In the present study, we examined the effect of hyperglycemia on mechanotranduction in bovine aortic endothelial cells (BAEC). After six days in high glucose media, we observed a decrease in heparan sulfate content coincident with a significant attenuation of the shear-induced hydraulic conductivity response, lower activation of eNOS after exposure to shear, and reduced cell alignment with shear stress. These studies are consistent with a diabetes-induced change to the glycocalyx altering endothelial response to shear stress that could affect the distribution of atherosclerotic plaques

The Glycocalyx and Its Role in Vascular Physiology and Vascular Related Diseases

Purpose—In 2007 the two senior authors wrote a review on the structure and function of the endothelial glycocalyx layer (Weinbaum in Annu Rev Biomed Eng 9:121–167, 2007). Since then there has been an explosion of interest in this hydrated gel-like structure that coats the luminal surface of endothelial cells that line our vasculature due to its important functions in (A) basic vascular physiology and (B) vascular related diseases. This review will highlight the major advances that have occurred since our 2007 paper. Methods—A literature search mainly focusing on the role of the glycocalyx in the two major areas described above was performed using electronic databases. Results—In part (A) of this review, the new formulation of the century old Starling principle, now referred to as the Michel–Weinbaum glycoclayx model or revised Starling hypothesis, is described including new subtleties and physiological ramifications. New insights into mechanotransduction and release of nitric oxide due to fluid shear stress sensed by the glycocalyx are elaborated. Major advances in understanding the organization and function of glycocalyx components, and new techniques for measuring both its thickness and spatio-chemical organization based on super resolution, stochastic optical reconstruction microscopy (STORM) are presented. As discussed in part (B) of this review, it is now recognized that artery wall stiffness associated with hypertension and aging induces glycocalyx degradation, endothelial dysfunction and vascular disease. In addition to atherosclerosis and cardiovascular diseases, the glycocalyx plays an important role in lifestyle related diseases (e.g., diabetes) and cancer. Infectious diseases including sepsis, Dengue, Zika and Corona viruses, and malaria also involve the glycocalyx. Because of increasing recognition of the role of the glycocalyx in a wide range of diseases, there has been a vigorous search for methods to protect the glycocalyx from degradation or to enhance its synthesis in disease environments. Conclusion—As we have seen in this review, many important developments in our basic understanding of GCX structure, function and role in diseases have been described since the 2007 paper. The future is wide open for continued GCX research

Direct current stimulation of endothelial monolayers induces a transient and reversible increase in transport due to the electroosmotic effect

We investigated the effects of direct current stimulation (DCS) on fluid and solute transport across endothelial cell (EC) monolayers in vitro. Our motivation was transcranial direct current stimulation (tDCS) that has been investigated for treatment of neuropsychiatric disorders, to enhance neurorehabilitation, and to change cognition in healthy subjects. The mechanisms underlying this diversity of applications remain under investigation. To address the possible role of blood-brain barrier (BBB) changes during tDCS, we applied direct current to cultured EC monolayers in a specially designed chamber that generated spatially uniform direct current. DCS induced fluid and solute movement across EC layers that persisted only for the duration of the stimulation suggesting an electroosmosis mechanism. The direction of induced transport reversed with DCS polarity – a hallmark of the electroosmotic effect. The magnitude of DCS-induced flow was linearly correlated to the magnitude of the applied current. A mathematical model based on a two-pore description of the endothelial transport barrier and a Helmholtz model of the electrical double layer describes the experimental data accurately and predicts enhanced significance of this mechanism in less permeable monolayers. This study demonstrates that DCS transiently alters the transport function of the BBB suggesting a new adjunct mechanism of tDCS

The Structural Stability of the Endothelial Glycocalyx after Enzymatic Removal of Glycosaminoglycans

Rationale It is widely believed that glycosaminoglycans (GAGs) and bound plasma proteins form an interconnected gel-like structure on the surface of endothelial cells (the endothelial glycocalyx layer–EGL) that is stabilized by the interaction of its components. However, the structural organization of GAGs and proteins and the contribution of individual components to the stability of the EGL are largely unknown. Objective To evaluate the hypothesis that the interconnected gel-like glycocalyx would collapse when individual GAG components were almost completely removed by a specific enzyme. Methods and Results Using confocal microscopy, we observed that the coverage and thickness of heparan sulfate (HS), chondroitin sulfate (CS), hyaluronic acid (HA), and adsorbed albumin were similar, and that the thicknesses of individual GAGs were spatially nonuniform. The individual GAGs were degraded by specific enzymes in a dose-dependent manner, and decreased much more in coverage than in thickness. Removal of HS or HA did not result in cleavage or collapse of any of the remaining components. Simultaneous removal of CS and HA by chondroitinase did not affect HS, but did reduce adsorbed albumin, although the effect was not large. Conclusion All GAGs and adsorbed proteins are well inter-mixed within the structure of the EGL, but the GAG components do not interact with one another. The GAG components do provide binding sites for albumin. Our results provide a new view of the organization of the endothelial glycocalyx layer and provide the first demonstration of the interaction between individual GAG components