Biorheology

In order to play different roles in vascular functions as a mechanosensor to blood flows and as a barrier to transvascular exchange, the endothelial surface glycocalyx (ESG) should have an organized structure. Due to the limitations of optical and electron microscopy, the ultra-structure of ESG has not been revealed until the recent development of super-resolution optical microscopy, STORM.

To investigate the ESG components and their organization on bEnd3 (mouse brain microvascular endothelial cells) monolayer.

ESG was immunolabeled with anti-heparan sulfate (HS), followed by an ATTO488 conjugated goat anti-mouse IgG, and with biotinylated hyaluronic acid (HA) binding protein, followed by an AF647 conjugated anti-biotin. The ESG was then imaged by the STORM.

HA is a long molecule weaving into a network which covers the endothelial luminal surface. In contrast, HS is a shorter molecule, perpendicular to the cell surface. HA and HS are partially overlapped with each other at the endothelial luminal surface. We also quantified the length, diameter, orientation, and density of HS at the top, middle and bottom regions of the endothelial surface.

Our results suggest that HS plays a major role in mechanosensing and HA plays a major role in the molecular sieve.

The endothelial glycocalyx plays a pivotal role in regulating blood flow, filtering blood components, sensing and transducing mechanical signals. These functions are intimately related to its dynamics at the molecular level.

The objective of this research is to establish the relationship between the functions of the endothelial glycocalyx and its dynamics at the molecular level.

To establish such a relationship, large-scale molecular dynamics simulations were undertaken to mimic the dynamics of the glycocalyx and its components in the presence of flow shear stresses.

First, motions of the glycocalyx core protein and the pertinent subdomains were scrutinised. Three-directional movements of the glycocalyx core protein were observed, although the flow was imposed only in the x direction. Such an observation contributes to understanding the glycocalyx redistribution as reported in experiments. Unsynchronised motion of the core protein subdomains was also spotted, which provides an alternative explanation of macroscopic phenomena. Moreover, the dynamics, root-mean-square-deviations and conformational changes of the sugar chains were investigated. Based on the findings, an alternative force transmission pathway, the role of sugar chains, and potential influence on signalling transduction pathways were proposed and discussed.

This study relates the functions of the glycocalyx with its microscopic dynamics, which fills a knowledge gap about the links between different scales.

Endothelial cells (ECs) sense the forces from blood flow through the glycocalyx, a carbohydrate rich luminal surface layer decorating most cells, and through forces transmitted through focal adhesions (FAs) on the abluminal side of the cell.

This perspective paper explores a complementary hypothesis, that glycocalyx molecules on the abluminal side of the EC between the basement membrane and the EC membrane, occupying the space outside of FAs, work in concert with FAs to sense blood flow-induced shear stress applied to the luminal surface.

First, we summarize recent studies suggesting that the glycocalyx repels the plasma membrane away from the basement membrane, while integrin molecules attach to extracellular matrix (ECM) ligands. This coordinated attraction and repulsion results in the focal nature of integrin-mediated adhesion making the abluminal glycocalyx a participant in mechanotransduction. Further, the glycocalyx mechanically links the plasma membrane to the basement membrane providing a mechanism of force transduction when the cell deforms in the peri-FA space. To determine if the membrane might deform against a restoring force of an elastic abluminal glycocalyx in the peri-FA space we present some analysis from a multicomponent elastic finite element model of a sheared and focally adhered endothelial cell whose abluminal topography was assessed using quantitative total internal reflection fluorescence microscopy with an assumption that glycocalyx fills the space between the membrane and extracellular matrix.

While requiring experimental verification, this analysis supports the hypothesis that shear on the luminal surface can be transmitted to the abluminal surface and deform the cell in the vicinity of the focal adhesions, with the magnitude of deformation depending on the abluminal glycocalyx modulus.

Ultrastructural investigations of the endothelial glycocalyx reveal a layer adjacent to the cell surface with a structure consistent with the primary  ultrafilter of vascular walls. Theory predicts this layer can be no greater than 200–300 nm thick, a result  to be reconciled with observations that red cells and large macromolecules are excluded  from a region 1 micrometer or more from the cell membrane.

To determine whether this apparent inconsistency might be accounted for by a model of steady state water and protein transport through a glycocalyx bi-layer formed by a porous outer layer in series with a more selective inner layer.

Expressions for coupled water and albumin fluxes through the two layers were used to describe steady state ultra-filtration though the bi-layer model.

Albumin accumulates at the interface between the porous layer and the selective inner layer. The osmotic pressure of accumulated albumin significantly modifies the observed permeability properties of the microvessel wall by an effective unstirred layer effect.

 The model places significant constraints on the outer layer permeability properties . The only outer layer properties that are consistent with measured steady state filtration rates and models of red cell flux through microvessels are an albumin permeability coefficient and hydraulic conductivity more than an order of magnitude larger than the those of the inner layer.

The onset of many disease processes depends on the function of the endothelial cell (EC) glycocalyx (GCX) which acts as a flow-dependent barrier to cellular infiltration and molecular transport across the blood vessel wall.

This review aims to examine these processes with the potential end goal of implementing GCX repair to restore EC barrier function and slow the progression of disease.

Cell and mouse studies were employed to examine the state of EC GCX in healthy versus disruptive flow conditions. Correlations of observations of the GCX with a number of EC functions were sought with an emphasis on studies of trans-endothelial barrier integrity against vessel wall infiltration of cells and molecules from the circulation. To demonstrate the importance of GCX as a regulator of trans-endothelial infiltration, assays were performed using ECs with an intact GCX and compared to assays of ECs with an experimentally degraded GCX. Studies were also conducted of ECs in which a degraded GCX was repaired.

In healthy flow conditions, the EC GCX was found to be thick and substantially covered the endothelial surface. GCX expression dropped significantly in complex flow conditions and coincided with a disease-like cellular and molecular accumulation in the endothelium or within the blood vessel wall. Therapeutic repair of the GCX abolished this accumulation.

Regenerating the degraded GCX reverses EC barrier dysfunction and may attenuate the progression of vascular disease.

Previous studies have demonstrated that the glycosaminoglycans (GAGs) heparan sulfate (HS) and hyaluronic acid (HA) are mechanosensors for interstitial flow on cancer cells. The proteins that link the GAGs to the cancer cell for mechanotransduction, however, are not known.

To assess whether the HS proteoglycan core proteins, Glypican-1 and Syndecan-1, or the HA receptor, CD44, provides the mechanical linkage to the cell.

The highly metastatic renal carcinoma cell line (SN12L1) and its companion low metastatic cell line (SN12C) were analyzed by Western blot, siRNA, and a 3-dimensional interstitial flow migration assay.

There was significant elevation of Glypican-1 protein expression in the SN12L1 cells relative to the SN12C cells while there were no significant differences in Syndecan-1 or CD44. Knock down of Glypican-1 by siRNA completely blocked flow induced migration in SN12L1 cells. MAPK inhibitors also blocked flow induced migration in SN12L1 cells.

Glypican-1 provides the mechanical linkage from HS (the flow sensor) to the SN12L1 cell where mechanotransduction leading to the enhancement of migration (metastasis) occurs. MAPKs downstream of Glypican-1 propagate the signal. The HS, Glypican-1, MAPK signaling axis suggests opportunities for pharmaceutical intervention.

The endothelial (EC) surface layer (glycocalyx) has been shown to act as a barrier to transvascular exchange of solutes, and adhesion of leukocytes (WBCs) during the inflammatory process. It is a labile structure whose components are readily shed by the action of proteases and endoglycosidases. Details of shedding of specific constituents of the glycocalyx remain to be determined.

To review the contributions of the primary glycosaminoglycans that comprise the glycocalyx, heparan sulfate (HS), chondroitin sulfate (CS) and hyaluronan (HA), as barrier to WBC-EC adhesion, and elucidate the rates of shedding of each component in response to an inflammatory stimulus. Assess the potential role that stiffness of the glycocalyx plays in resisting infiltration by WBCs during the adhesion process.

Quantitate shedding of the glycocalyx in post-capillary venules of rat mesentery in response to superfusion of the tissue with 10⁻⁶ M fMLP. The presence and loss of HS, CS and HA was assessed by labeling all components with fluorescently labelled lectin (BS-1) or HS antibodies, and HA with fluorescently labelled hyaluronan binding protein (HBP).

Following a 30 min exposure of the mesentery to fMLP about 50% of HBP was lost in contrast to a previously shown loss of 20% of lectin labelled GAGs, and 25% loss of Mab labelled HS. The time constant for HBP shedding (5.8 min) was one-third that for BS-1 labelled GAGs (14.3 min). An attempt was made to assess stiffness of the glycocalyx by observing the motion of adhered lectin coated fluorescently labelled microspheres (FLM) under oscillatory flow conditions. Estimates of the elastic modulus of the glycocalyx revealed a value of 26 mPa, which was orders of magnitude below published data obtained by atomic force microscopy.

The relatively rapid shedding of HA compared to HS was consistent with the hypothesis that HA may form the dominant barrier to WBC-EC adhesion. Prior observations that HA lies closer to and parallel to the endothelial membrane, compared to HS suggests that the compact layer of HA near the EC membrane surrounds WBC adhesion receptors that are much shorter in length than the total thickness of the glycocalyx. The relatively low elastic modulus of the glycocalyx under shear is consistent with the hypothesis that the FLMs adhered to strands of HS normal to the EC surface that extended above the relatively more compact and stiffer HA layer below. Gradients of stiffness within the glycocalyx may not be detected by compressive indentation tests published to date.