Mechanobiology of the abluminal glycocalyx

Abstract

BACKGROUND:

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.

OBJECTIVES:

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.

RESULTS:

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.

CONCLUSIONS:

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.

Keywords

Glycocalyx endothelium mechanotransduction

1. Introduction

The endothelium lines blood vessels and thus is the very first layer of cells that is exposed to shear stress from flowing blood. Shear stress, in turn, affects the way in which endothelial cells (ECs) regulate their immediate environment [1,2]. For example, in response to shear stress ECs produce vasodilators [3–5], constrictors [6], and anticoagulants [7,8]. Thus, they are the primary regulators of blood flow, blood viscosity, and blood vessel permeability to blood-borne solutes. Since these responses are dynamic, the origins of pathologies such as hypertension and atherosclerosis have been traced to dysfunctional responses to altered fluid flow and other time-dependent mechanical stimuli [9]. For example, laminar flow and high shear stress have been found to be atheroprotective whereas disturbed flow, exhibiting low and oscillatory shear stress such as that experienced by ECs near bifurcations and curved regions of arteries, make those areas prone to development of atherosclerotic lesions [10]. Atherosclerotic lesion formation is due in part to the leaky junctions between ECs and the resultant accumulation of low-density lipoprotein in the intima [11,12]. Understanding the fundamental mechanisms of EC responses to shear stress may uncover a fundamental origin of atherosclerosis. Similarly, since many cells are mechanosensitive, understanding EC mechanotransduction may elucidate the origin of other pathologies related to mechanobiology.

Two complementary leading hypotheses, describing the mechanisms by which ECs sense fluid shear stress, involve the glycocalyx on the luminal side of the cell [13,14] and focal adhesions (FAs) on the abluminal side [9,15,16]. The glycocalyx is a carbohydrate-rich surface coating that extends into the flow stream and thus deforms with blood flow, and transmits forces to membrane lipids and proteins, which, in turn, transmit forces to intracellular organelles (reviewed in [17,18]). Resisting these forces are FAs on the abluminal side of the cell [19]. These adhesion plaques form primarily via integrin adhesion to specific extracellular matrix (ECM) proteins [20]. FAs provide resistance to forces induced by shear stress and are thus considered primary candidates for mechanotransducers [21]. Forces are also likely transmitted from cell to cell through cell-cell junctional proteins such as PECAM-1 and VE-Cadherin, known mechanotransducers [22]. Force-induced changes in protein interaction inside the FA may be a mechanism by which forces are sensed and transduced into different signaling pathways [23]. FAs are considered to be areas where the membrane is within 10–15 nm from the substrate whereas close contacts are within 40–50 nm [24].

A theory is gaining traction in which abluminal glycocalyx and integrins are not independent mechanotransducers, but, rather, coordinate the formation of FAs and thus mechanotransduction [25]. Under this theory, the glycocalyx is required for the formation of FAs. This perspective paper explores this theory and further proposes the hypothesis that after FA development, the abluminal glycocalyx itself participates in mechanotransduction processes when forces are applied on the cell’s luminal surface. We first discuss the mechanisms by which glycocalyx mechanical properties assist in the initial formation of close contacts and FAs. We then present estimates of peri-focal adhesion deformation using a finite element model of a sheared and focally adhered endothelial cell in which focal adhesion topography was determined using quantitative total internal reflection fluorescence microscopy. Such abluminal deformation from luminally applied shear is taken as evidence that forces can be transmitted from the luminal cell surface to the area immediately surrounding focal adhesions. From this model, we propose a new hypothesis that the glycocalyx acts as a regulator of mechanotransduction through two mechanisms, one in which forces are transmitted from the ECM, through the glycocalyx and to the abluminal cell surface upon cell deformation, and another in which the glycocalyx can inhibit, or permit, new integrin ligation of ECM proteins, which could initiate new intracellular signaling [26].

2. The glycocalyx interacts with the cell surface at locations of liquid-ordered domains

The glycocalyx is a multitiered structure with selectins and glycosylated lipids and proteins rising from 10–1000 nm from the cell surface (reviewed in [17,27,28]). For example, emanating from molecules such as CD44, syndecans, and glypicans, are chondroitin sulfate (CS) and heparan sulfate (HS), molecules that extend 50–100 nm from the surface (reviewed in [29]). Entangled in these molecules is hyaluronan, a megadalton linear polysaccharide that is secreted by the cell through hyaluronan synthase at the cell surface. Hyaluronan can both initiate cell adhesion to a surface [30] and provide a significant barrier to cell adhesion, thus preventing red and white cell interaction with the EC surface [31]. Hyaluronan can extend into the blood stream to a distance ranging from a few nm to microns [32]. Consistent with this concept of cell repulsion, hyaluronan was found to be the principal component of the pericellular free layer of fibroblasts in culture when in the presence of red blood cells [33].

The attachment of the glycocalyx to the membrane is facilitated, in part, by glycosylphosphatidylinositol- (GPI-) anchored proteins such as glypican-1. These GPI anchors are known to be associated with cholesterol-rich liquid-ordered domains [34] that include rafts and caveolae. Caveolae, in turn, are closely associated with syndecans which may be bound to the actin cytoskeleton [35]. Therefore, a leading hypothesis for mechanical interaction between blood flow and internal stress transmissions is that flow distends heparan sulfate, acts through glypican-1 and caveoli, working against the anchoring role of actin [13,36]. This force balance may lead to activation of EC nitric oxide synthase, (eNOS) a molecule that catalyzes the conversion of L-arginine into nitric oxide (NO) and L-citrullin [37]. NO is the principal regulator of vasodilation, platelet aggregation, and quiescence of atheroprotective ECs [7]. Loss of this mechanotransduction pathway leads to dysfunction in flow regulation, platelet aggregation, white cell adhesion, and loss of blood fluidity; all atherogenic processes. Therefore, understanding the precise mechanisms of glycocalyx-mediated mechanotransduction, and the deleterious arteriolar effects of loss of this mechanotransduction pathway, could lead to the development of drugs or therapies aimed at ameliorating these effects.

3. The cell adheres to the basement membrane using molecules associated with liquid-ordered domains

On the abluminal side, cells link to the basement membrane via integrins, heterodimeric molecules whose activation increases their affinity for ECM proteins. Primarily through 𝛽₁ integrins, this adhesion leads to integrin clustering to create an integrin signaling layer, followed by association of vinculin and talin to create a force transduction layer, and finally the association of actin stress fibers to create an actin regulatory layer [38]. We have shown that when the ECM is brought into contact with a cell, cholesterol-rich liquid-ordered domains rapidly assemble there within the first few seconds [39]. Talin association with integrins, a sign of integrin activation, followed within 10 seconds after this event. Consistent with results from this study, Schwartz and colleagues found that FAs were, themselves, liquid-ordered domains, implicating these domains as participants in mechanotransduction [40]. Further evidence that liquid-ordered domains participate in mechanotransduction came from our recent studies using optical trap force spectroscopy in which RGD-coated beads were introduced to the cell surface using a calibrated optical trap [41]. Within less than a second, adhesions were formed. These adhesions depended on the integrity of liquid-ordered domains as adhesion was enhanced by benzyl alcohol, which causes domain coalescence [42] and inhibited by vitamin E, which disperses domains. Thus, ordered domains are some of the very first participants in the formation of nascent FAs, making the membrane a necessary participant in mechanotransduction.

4. Glycocalyx is necessary for FA formation

The necessary role of competing repulsive and attractive forces has been well known for decades. The concept of force being necessary to sustain adhesion was proposed by Bell in 1978 [43] and is consistent with the concept that integrin-ECM adhere through catch bonds [44] in which force in the 10–30 pN range increased bond lifetime. In addition, Chrzanowska-Wodnicka and Burridge found that Rho-mediated contractility is necessary for the growth of FAs [45]. Further, the glycocalyx has been implicated as providing competitive force (reviewed in [46]). Under this paradigm, short range attractive forces in the catch bonds between integrins and the ECM molecules, are supported by long range repeller forces from the glycocalyx (Fig. 1). Consistent with this interpretation, recent computational models suggest that integrin-ligand interaction arises from the competitive interaction of integrin-ECM attraction and glycocalyx repulsion [25,47]. In that model, integrin-ECM bonds were modelled as Hookean springs with force-dependent affinities described by a Bell model. Close contacts were governed by the glycocalyx interaction with the ECM. These are converted to focal contacts by integrin adhesion. Results showed that when one integrin-ECM bond occurred (after overcoming the deformation of the abluminal glycocalyx), other bonds formed since the integrins were now in close proximity to the ECM ligands. Additional clustering was counteracted by bond rearrangement and detachment. Without repulsion of the glycocalyx, bonds could form everywhere and there would be no clustering. Further, without the glycocalyx, repulsive forces on the integrins would be reduced and bond lifetimes shortened. Therefore, the glycocalyx mechanical properties directly governed integrin clustering and FA growth. Additionally, integrin clustering was seen to depend on ECM stiffness.

Experimental support for this model is provided by Paszek, et al. [48]. In this study, the authors generated synthetic mucin glycoproteins each with increasing lengths intercalated in the plasma membrane and emanating from the cell surface. Results demonstrated that glycoproteins with a length of 80 nm, which are significantly greater in length than integrins (20 nm), were excluded from FAs, whereas shorter ones were not excluded. Importantly they found that these mucin molecules exerted force on integrin molecules, thus increasing bond lifetime consistent with catch-bond behavior as noted in [44] wherein the glycoproteins were compressed from their maximum length when integrins were locally adhered. Using FRET-based strain gauges with CFP/YFP encoded in the mucin proteins, they showed that these molecules were indeed compressed compared with their normally expressed lengths. Further, this compression was sufficient to apply forces on integrin molecules that was not dependent on actin-myosin contractility, thus implicating the glycocalyx as the source of the force that balanced integrin adhesion.

Fig. 1.

Schematic contour of abluminal membrane taken from electron micrograph of focal plaque of a fibroblast grown on epoxy [49]. Springs correspond to abluminal glycocalyx. Blue rods (center) indicate integrin adhesions. Elastic springs provided by the glycocalyx may provide tension on integrin molecules at the focal adhesion site. Such force could provide the necessary force for integrin bond stabilization.

Further experimental evidence was provided on the single molecule level by Son et al. [41].

In this recent study, we showed that, after the initial adhesion between the RGD peptide and 𝛽₁ integrins, heparan sulfate was necessary for subsequent adhesions. In this study, benzyl alcohol (BA), which increases line tension between liquid-ordered and liquid-disordered domains in the membrane, thus favoring coalescence into larger liquid-ordered domains [42], caused two integrin molecules to adhere to the RGD coated surface within 1.5 seconds, rather than one. Such integrin avidity regulation by liquid-ordered domains was supported in further studies using fluorescence correlation spectroscopy which showed that this procedure also led to integrins diffusing in pairs rather than as single molecules. Supporting the role of domains, it was found that Lyn, a Src family of protein tyrosine kinase enriched in cholesterol-rich membrane microdomains [50] that diffuses with liquid-ordered domains, also dimerized in the presence of BA. The two bonds were demonstrated in bond rupture experiments in which the force (∼50 pN) was almost exactly double that arising from a single bond (∼25 pN). This increase in integrin avidity was blocked by treatment with heparanase, thus implicating heparan sulfate as a participant in the increase of integrin avidity, consistent with the role of glycocalyx in fostering clustering of integrins into FAs.

5. The dynamic interplay between the glycocalyx and integrin adhesion near FAs may point to a new mechanotransduction mechanism

A main contribution of our studies identifying the requirement of glycocalyx repulsion to foster integrin clustering and maintain integrin adhesion, is that it suggests that attention should be focused on the area of the membrane immediately adjacent to the FA (peri-FA) in addition to the traditional attention focused on the FA plaque itself. Because of the juxtaposition of constrained (e.g. integrin ECM) and free (e.g. glycocalyx) areas, it is likely that most deformation will occur at this interface. This prediction is consistent with new data implicating the glycocalyx in shear-enhanced cell migration [51]. Recently, Thi et al. demonstrated that the glycocalyx was required for EC remodeling due to stress fiber reorientation [13]. This remodeling was also demonstrated in vinculin-positive FAs. Such remodeling depended on intact heparan sulfate, as heparanase treatment blocked it. While the authors attributed remodeling to the stresses transmitted through the luminal glycocalyx through stress fibers to the FAs, it is possible that an additional component may be in the abluminal glycocalyx, which may participate in the formation of FAs in the first place.

The results of this analysis point to an alternative mechanism for shear-induced growth and remodeling of the FAs. In short, shear stress induces the bending of membrane toward ECM ligands and facilitates new integrin binding in the downstream side of FAs [26]. Nanometer-order bending would be sufficient to bring new integrin molecules into the vicinity of extra-cellular ligands in the ECM. As dynamic interactions of integrins with ECM ligands is an essential step for mechanotransduction [52,53], demonstrating that shear stress on the luminal surface is sufficient to force the abluminal membrane to close interaction with the abluminal glycocalyx and ECM in the basement membrane, would provide support for the concept that the abluminal glycocalyx may play a role in shear-induced signaling.

6. Finite element modeling of focally adhered ECs reveals a role for glycocalyx in abluminal deformation

Thus, for the purpose of this perspective paper, we updated a finite element model previously reported [54] by including the glycocalyx in the peri-FA space, here called the abluminal glycocalyx (AG). Solid modeling, image analysis, model construction, and finite element analysis was performed as described in our other studies [54,55]. Briefly, solid models were generated from stacks of images obtained by high-resolution microscopy [55]. To generate EC solid models with in vivo topography we used fluorescence imaging with wide field microscopy, deconvolution, and computational corrections for spherical aberrations. Multiscale modeling was performed by integrating cell-scale models and FA scale models using extrusion coupling variables available in COMSOL multiphysics software. Models were constructed from images of bovine aortic ECs (BAECs), grown to confluence on chambered-coverslips (Labtek, Campbell, CA) in Dulbecco’s modified eagle medium (DMEM) supplemented with 10% fetal calf serum (FCS), 2 mM L-glutamine, 100 units/ml penicillin and streptomycin (BioSource, Camarillo, CA). Imaging was performed on live cells in phosphate buffered saline (PBS) and 1% albumin at room temperature. To stain the EC cytoplasm, cells were incubated for 5 minutes in Calcein-AM (Molecular Probes). Calcein stain was prepared from a 1 mM stock solution in dimethylsulfoxide (DMSO) and diluted to 1 μM in warm (37 °C) phosphate buffered saline (PBS). Detailed description of cell staining and imaging is given in [55]. The nucleus was stained using 20 μM DNA fluorochrome Hoechst 33258. To image the membrane, 1 μM DiIC₁₆(3) (1,1’-Dihexadecyl-3,3,3’,3’-Tetramethylindocarbocyanine Perchlorate) in DMSO was introduced to the media and the cell was allowed to incubate for 5 minutes. After incubation, the cells were washed three times in PBS with 1% albumin and then returned to PBS with 1% albumin at 37 °C. Cells were imaged live while confluent. All imaging was completed within 10 minutes after staining.

Imaging was performed on an IX71 microscope (Olympus, Lehigh, PA) equipped with a piezoelectric z-stage (Mad City Labs, Madison WI), a Xenon arc lamp light source, a fiber-coupled laser (Melles Griot, California, US), an objective-based total internal reflection florescence attachment (Olympus, Japan) and a sensicam CCD camera (Cooke Corp, Romulus, MI). 40–50 optical slices spaced at 0.2 μm of adherent cells were obtained from florescence imaging at 532, 488 and 400 nm excitation for DiI, calcein and Hoechst, respectively, and deblurred using blind deconvolution software (Autoquant, Renesselaer, NY). A PlanApo 60x/1.45 NA TIRFM oil-immersion objective (Olympus, Japan), was used for both epi-illumination and total internal reflection florescence microscopy (TIRFM). A 3-D data set of cells with unknown axial dimensions was corrected using a non-linear axial distortion correction function (ADCF) as described previously [55]. FAs of the cultured DiI-stained BAECs were viewed with objective-based total internal reflection fluorescence microscopy (TIRFM). TIRFM images were taken on the same cell and region of interest as the 3-D image sets. Cell-distance from the cover glass was determined using the methods of [54,57]. After background subtraction, a smoothing algorithm was applied to the image to reduce occurrence of sharp edges and kinks in the solid model (Fig. 3). Basal FA topography was obtained from the equation: $\begin{eqnarray}\displaystyle {\rm\Delta}(x,y)={\rm\Delta}_{0}+d_{p}\ln \frac{F_{0}}{F(x,y)} & & \displaystyle\end{eqnarray}$ 1 where Δ(x, y) is the separation distance between the membrane and glass, Δ₀ is the minimum separation distance (Δ₀ = 10 nm), F₀ is the maximum intensity of the image at the minimum separation, and F (x, y) is the pixel intensity [58], d_p = 95 nm. This parameter depends on the penetration depth and depends upon wavelength, refractive index of glass, and refractive index of the cell. Membranes at focal contacts lie between 10–15 nm from the glass substrate and membranes at close contacts are 30–50 nm from the glass [24].

Fig. 2.

Results of finite element analysis of sheared and focally adhered ECs. Nominal shear stress is 10 dynes∕cm². Black arrows indicate analyzed FAs. Blue arrow (above cell) indicates direction of luminal shear stress. Overall cell deformation is estimated in [54] and measured in [56].

Finite element solution for the flow field was accomplished using incompressible Navier Stokes for a nominal shear of 1 Pa (Fig. 2). A linearly elastic model was used for all cellular components (cytoplasm, nucleus, ECM and AG). This 3D cell model was solved for a cytoplasm modulus of 775 Pa [59]. With the help of extrusion coupling variables, these stresses were mapped from the cell scale model to the FA scale model by applying resultant forces at an elliptical region 110 nm above the 2D FA (the cell scale model) to the top surface of 3D model of FAs (Fig. 3). 3D FAs were solved for an ECM modulus of 5000 Pa and two different moduli for abluminal glycocalyx: 40 Pa and 80 Pa [60]. Recent experimental evidence suggests that upstream FAs (those on the side of the cell facing the flow) react differently to shear stress than do centrally located and downstream FAs [53]. Thus upstream, downstream and middle FAs were chosen for further analysis (shown in Fig. 2).

Fig. 3.

A. Image from total internal reflection fluorescence microscopy of a DiI stained EC, with background subtraction and smoothing. B. Contour of abluminal focal contact (FA) and close contact as calculated from equation 1 (note: for clarity, z and x, y scaling is not equal) C. Predictions of distributions of restoring forces of abluminal glycocalyx upon application of luminal shear stress on average of 1 Pa. Simulations predict tensile forces on the upstream side of a focal adhesion (left of center) and compressive forces on the downstream side. Forces arise from tension and compression, respectively, of the abluminal glycocalyx.

Fig. 4.

z-displacement toward (negative) or away from (positive) the basement membrane of abluminal FAs resulting from luminal application of shear stress. Lines indicate deformation to (negative z) or away from (positive z) the underlying substrate. Zero deformation corresponds to the location of integrin adhesion to the ECM.

The simulations predict a z-displacement on the order of 1–10 nm at the downstream side of a FA irrespective of the position of the FA (Fig. 4). Such shear-induced bending in the area immediately adjacent to the adhesion plaque has been measured in amoeboid cells [61] with a magnitude on the same order as that predicted by our simulation. An important observation is that this deformation is greater for upstream FAs compared to downstream FAs with the middle FA having the least deformation, suggesting some shielding by the nucleus. This study supports the hypothesis that forces from shear flow could shift the formation of new FAs by altering the force balance in the peri-FA area, in essence superimposing a polarized (by virtue of shear flow) force distribution on top of the forces already exerted by the abluminal glycocalyx and integrins.

7. Discussion

We present a survey of the literature that provides evidence of an abluminal glycocalyx and its influence on the formation of focal adhesions. We further provide results from an elastic finite element model of a sheared and focally adhered endothelial cells grown in a confluent monolayer. From this analysis, this perspective paper suggests a hypothesis that the abluminal glycocalyx may participate in mechanotransduction events. The experimental evidence for this occurrence is beyond the scope of this study, but could be an intriguing avenue of investigation. Limitations to the model include the fact that the endothelial cell is treated as a continuum elastic body. Nevertheless, the sophistication of the model lies in the fact that it is inclusive of four components (cytoplasm, nucleus, ECM and abluminal glycocalyx), and is constructed from experimental images with the abluminal surface topography provided by quantitative total internal reflection microscopy. An assumption of an elastic abluminal glycocalyx providing repelling forces against the attractive forces between integrins and extracellular matrix proteins is provided by us [41] and others [46]. Other possibilities for the repulsion between the abluminal EC membrane and the basement membrane between focal adhesions could be due to electrostatic and entropic repulsion. However, Sabri and colleagues showed that for some cells, the main components of repulsion between adhesion points arose from the relative length of adhesion molecules and surface repeller elements [62].

8. Conclusion

We have recently published on the earliest events of cell adhesion and found that the membrane and glycocalyx are important players in this dynamic process [41]. Cellular focal adhesions to extracellular matrix proteins enable cells to carry out their most basic functions, including the organization of tissues, cell signaling, and force generation. Integrin-dependent adhesions provide the forces necessary for cell migration, contraction of tissue, and provide a force balance during cell division. What is also known about FAs is that the very occurrence of adhesion can initiate downstream biochemical signaling in cells [52]. While it is well known that FAs are dynamic, little is known about the dynamics of assembly when a membrane containing integrins is presented to a substrate. Thus, an unexplored mechanotransduction pathway includes the presentation of membrane to ECM and the detailed mechanical interaction of the abluminal glycocalyx and integrin adhesion. We know that diffusion of integrins to the place of close approximation of membrane and substrate, binding of integrins to ECM, and associated activation of talin, lead to important downstream signaling events long associated with mechanotransduction. The membrane, which contains integrins, must be close enough to the substrate for adhesion and the integrins need to be in the right place at the right time. Since FAs induce membrane curvature, it is likely that membrane bending must occur to enable FA formation. If this hypothesis is correct, properties that affect membrane bending, such as alterations in composition of the abluminal glycocalyx, will modulate FA initiation. Such analysis of the abluminal glycocalyx in mechanotransduction may yield new insight into the integrated mechanosensing network comprised of the glycocalyx, membrane, and cytoskeleton.

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