Glycocalyx

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The Structure and Functionof the EndothelialGlycocalyx LayerSheldon Weinbaum,1,2 John M. Tarbell,1

and Edward R. Damiano3

1Department of Biomedical Engineering and 2Department of MechanicalEngineering, The City College of New York, New York, NY 10031;email: [email protected], [email protected] of Biomedical Engineering, Boston University, Boston, Massachusetts02215; email: [email protected]

Annu. Rev. Biomed. Eng. 2007. 9:121–67

First published online as a Review in Advance onMarch 20, 2007

The Annual Review of Biomedical Engineering isonline at bioeng.annualreviews.org

This article’s doi:10.1146/annurev.bioeng.9.060906.151959

Copyright c© 2007 by Annual Reviews.All rights reserved

1523-9829/07/0815-0121$20.00

Key Words

mechanical and biochemical properties of glycocalyx, revisedStarling principle, cellular interactions with glycocalyx,inflammatory response, mechanotransduction

AbstractOver the past decade, since it was first observed in vivo, there hasbeen an explosion in interest in the thin (∼500 nm), gel-like endothe-lial glycocalyx layer (EGL) that coats the luminal surface of bloodvessels. In this review, we examine the mechanical and biochemi-cal properties of the EGL and the latest studies on the interactionsof this layer with red and white blood cells. This includes its de-formation owing to fluid shear stress, its penetration by leukocytemicrovilli, and its restorative response after the passage of a whitecell in a tightly fitting capillary. We also examine recently discoveredfunctions of the EGL in modulating the oncotic forces that regulatethe exchange of water in microvessels and the role of the EGL intransducing fluid shear stress into the intracellular cytoskeleton ofendothelial cells, in the initiation of intracellular signaling, and inthe inflammatory response cascade.

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EC: endothelial cell

EGL: endothelialglycocalyx layer

FSS: fluid shear stress

Contents

1. INTRODUCTION AND OVERVIEW. . . . . . . . . . . . . . . . . . . . . . . . . . . . . 122Early Indications of the EGL in Microvessels In Vivo . . . . . . . . . . . . . . . . 123The EGL in Large Vessels and Atherogenesis . . . . . . . . . . . . . . . . . . . . . . . . 123EM Observations Prior to 2000 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 124Direct Intravital Microscopic Evidence for the Full Extent of the EGL

In Vivo . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 124The EGL and the Starling Principle . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 125

2. STRUCTURE AND COMPOSITION OF THE EGL. . . . . . . . . . . . . . 127Biochemical Composition . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 127Recent EM Observations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 130Relation between Biochemical Composition and EM Observations . . . . 131

3. MECHANICAL AND ELECTROCHEMICAL PROPERTIES . . . . . 132EGL Thickness Using Microviscometric Analysis and μ-PIV . . . . . . . . . 132Elastic Properties of EGL Fibers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 134Electrochemical Properties. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 135

4. MODELS FOR STRUCTURAL INTEGRITY ANDRESTORATION . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 137Hydrodynamic Models for Flow in the EGL . . . . . . . . . . . . . . . . . . . . . . . . . 137Models for the Restoring Mechanisms of the EGL . . . . . . . . . . . . . . . . . . . 138

5. CELLULAR INTERACTIONS WITH THE EGL . . . . . . . . . . . . . . . . . 143Red Cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 143White Cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 144

6. PHYSIOLOGICAL FUNCTIONS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 146Permeability and the Revised Starling Principle . . . . . . . . . . . . . . . . . . . . . . 146Mechanotransduction. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 149Inflammatory Response and Ischemia–Reperfusion Injury . . . . . . . . . . . . 155

7. UNRESOLVED ISSUES AND FUTURE DIRECTIONS . . . . . . . . . . 158

1. INTRODUCTION AND OVERVIEW

It is now well recognized that the luminal surface of the endothelial cells (ECs) thatline our vasculature is coated with a glycocalyx of membrane-bound macromoleculescomprised of sulfated proteoglycans, hyaluronan, glycoproteins, and plasma proteinsthat adhere to this surface matrix. A similar coating is observed on the apical surfaceof epithelial cells that line many of our internal organs, but this review focuses onlyon the endothelial glycocalyx layer (EGL). We examine the remarkable propertiesof this hydrated gel-like structure and the three principal functions that it plays asthe interface between the ECs and the flowing blood with its plasma and cellularcomponents. In particular, we explore its function as a modulator of permeabilityin the transcapillary exchange of water; as a mechanotransducer of fluid shear stress(FSS) to the endothelial cytoskeleton, including the resulting biochemical responses;

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WBC: white blood cell

EM: electron microscopy

and as a regulator of red and white blood cell (WBC) interactions, with emphasis onthe inflammatory response. Much of the early literature on the EGL is summarizedin an excellent review by Pries et al. (1). Therefore, we provide only a brief overviewof the literature prior to 2000 in this Introduction.

Early Indications of the EGL in Microvessels In Vivo

The concept that a thin endocapillary layer might cover the entire endothelial sur-face was first proposed in the 1940s by Danielli (2) and Chambers & Zweifach (3),and was subsequently reexamined by Copley (4) who suggested that the layer wasan immobile sheet of plasma and macromolecules. However, this layer evaded ob-servation by light and electron microscopy (EM) until 1966, when Luft (5), usingruthenium red staining, detected a thin layer (∼ 20 nm thick) in rat intestinal mu-cosa. For reasons that were not clear at the time, this layer was far thinner than themuch thicker EGL proposed by Klitzman & Duling (6) to account for the low cap-illary tube hematocrits (i.e., the instantaneous volume fraction of red cells residentin the capillary) that they observed in skeletal-muscle capillaries. Evidence that themacromolecules of the EGL might interfere with flow in a much thicker plasma layernear the capillary wall was first reported by Desjardins & Duling (7). After enzymetreatment targeted at cleaving specific proteoglycan molecules within the EGL, theyobserved up to a twofold increase in capillary tube hematocrit. In addition to theseobservations, evidence that the EGL contributes to microvascular flow resistance wasprovided by Pries et al. (8). Combining network simulations with measurements ofblood flow in large-scale microvascular networks, Pries et al. (8) concluded that theresistance to blood flow in microvessels up to 30 μm in diameter was dramaticallyhigher than in glass tubes of the same diameter. In a subsequent study, Pries et al. (9)found that the resistance to blood flow in microvascular networks decreased markedlyafter enzyme treatment to remove the EGL. These studies and those of Duling andcoworkers suggested that the EGL could serve to retard plasma flow near the vesselwall, which in turn would result in enhanced resistance to blood flow and lower capil-lary tube hematocrits in vivo than in smooth glass tubes. These experimental studieswere accompanied by a series of theoretical models of increasing sophistication thatdescribed the axisymmetric single-file motion of spherical particles and red cells in acylindrical capillary with a porous matrix on its luminal surface (10–13).

The EGL in Large Vessels and Atherogenesis

Although early studies on the EGL were limited largely to microvessels, the associ-ation of altered EGL characteristics with atherosclerosis-prone locations in arterieswas recognized in the early 1980s. Lewis et al. (14) observed the coronary arteriesof White Carneau pigeons and noted that the EGL, as assessed by ruthenium redstaining, was thinnest in areas with high disease predilection, and that upon choles-terol challenge, the EGL thickness was reduced in all arterial zones. This idea ofassociation between EGL abundance and arterial disease has been revisited recentlyby van den Berg et al. (15), who showed that the EGL thickness in the disease-prone

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LDL: low-densitylipoprotein

sinus region of the mouse internal carotid artery is significantly less than in the nearbycommon carotid artery that is spared of disease. They also reported that the EGLis diminished upon systemic atherogenic challenge by a high-fat, high-cholesteroldiet. These observations are consistent with previous studies demonstrating rapidshedding of the EGL from the endothelial surface upon acute stimulation with ele-vated plasma levels of oxidized low-density lipoproteins (LDL) or acute exposure toinflammatory agents and FSS-induced synthesis of EGL components (reviewed inReference 16).

EM Observations Prior to 2000

As noted above, the first visualization of the EGL by EM used the cationic dye ruthe-nium red that binds to acidic mucopolysaccharides and generates electron density inthe presence of osmium tetroxide (5). Subsequent studies by Baldwin & Winlove (17)and Clough & Moffitt (18) used gold colloids and immunoperoxidase labeling. Adam-son & Clough (19) demonstrated, using a large charged marker protein, cationizedferritin, to demarcate the edge of the EGL in frog microvessels, that, in the ab-sence of plasma proteins, the EGL would collapse, presumably owing to eliminationof intramolecular interactions with plasma proteins, and that its undisturbed thick-ness was several times greater than the 20 nm observed with ruthenium red. All ofthese methods suffer from dehydration artifacts associated with aqueous fixatives thatlikely dissolve all but the protein cores of proteoglycans. A method developed to pre-serve water-soluble structures using fluorocarbons as nonaqueous carriers of osmiumtertroxide was applied to microvessels to obviate some of these limitations by Sims &Horne (20). Further elaborations of the fluorocarbon-glutaraldehyde fixation meth-ods by Rostgaard & Qvortrup (21) revealed a filamentous brush-like surface coatingon capillary walls with a layer thickness of <50 nm, suggesting a cleavage of more su-perficial matrix structures. All of the foregoing EM studies suggested an EGL with athickness of less than 100 nm. None of these studies shed light on the possible organi-zation of the EGL and its possible relationship to the F-actin scaffold beneath the api-cal membrane that was first reported in Squire et al. (22), as described later in Figure 2.

Direct Intravital Microscopic Evidence for the Full Extentof the EGL In Vivo

What was missing from in vivo experiments prior to 1996 was a direct in vivo mea-surement of the thickness of either a positively labeled layer of macromolecular con-stituents of the EGL or an exclusion zone to red cells and fluorescently labeledmacromolecules. The first important breakthrough along these lines came with thedye-exclusion technique developed by Vink & Duling (23). Using a 70 kDa FITC-dextran plasma tracer, which they showed was sterically excluded by the EGL, theywere able to provide the first estimate of the in vivo thickness of the layer in capil-laries. Comparing the anatomical diameter of hamster cremaster-muscle capillaries,visualized under brightfield illumination, with measurements of the functional diam-eter available to WBCs, red cells, and fluorescent macromolecules, they concluded


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SOD: superoxide dismutase

μ-PIV: microparticle imagevelocimetry

that the thickness of the EGL was ∼0.4–0.5 μm, which is ∼15%–20% of the radiusof the smallest capillaries in the microcirculation. This estimate of the in vivo thick-ness of the EGL is four to five times greater than previous estimates derived fromEM studies, which likely significantly underestimated the value owing either to thedehydration of the extracellular matrix or the cleavage of its outer matrix componentsthat accompanies tissue fixation. This discrepancy was a catalyst for much of the workthat has followed on the estimation of EGL thickness and its function as a barrier incellular interactions.

Beyond providing the first estimate of the in vivo thickness of the EGL, the Vink& Duling experiments were significant in several other important ways. In particular,they showed that except at velocities <20 μm/s, red blood cells do not invade theregion occupied by the EGL in tightly fitting capillaries, whereas the much stifferand larger WBCs crush the layer. However, when flow is arrested, red cells alsoappear to have this ability because they fill nearly the entire capillary lumen. Fromfundamental fluid mechanical considerations, and based on our understanding of redcell mechanics, these data provided quantitative insight into important mechanicalproperties of the EGL, as discussed in further detail in Sections 2–4. Finally, Vink& Duling showed that exposing FITC-dextran perfused capillaries to epifluorescentillumination for ∼5 min resulted in significant degradation of the EGL and that thisdegradation was effectively blocked by administering superoxide dismutase (SOD)and catalase. (SOD is an enzyme that catalyzes the dismutation of the superoxideanion radical into oxygen and hydrogen peroxide, whereas catalase is an enzyme thatcatalyzes the decomposition of hydrogen peroxide into water and oxygen.) This sup-ports the hypothesis that damage to the EGL owing to light-dye treatment mightbe mediated by oxygen-derived free radicals. Further support for this hypothesiswas demonstrated when Vink et al. (24) showed that oxidized LDL caused a simi-lar degradation of the EGL, whereas native LDL did not. While it is known thatheparan sulfate proteoglycans in the EGL bind SOD (25), these results suggest animportant role for the EGL in scavenging free radicals from the blood. Such a rolecould have important implications for cardiovascular health and disease ranging frominflammation to atherosclerosis.

Although elegant, the dye-exclusion technique developed by Duling and cowork-ers does not provide adequate resolution in vessels larger than ∼12–15 μm in diame-ter (26) because of the increased out-of-focus light and optical difficulties associatedwith the fluorescent dye column near the vessel wall. In an effort to test whether theEGL extended into microvessels beyond the capillary regime, a new technique hasbeen developed that uses high-resolution, near-wall, intravital fluorescent micropar-ticle image velocimetry (μ-PIV) to examine the velocity profile near the vessel wallin postcapillary venules of the mouse cremaster muscle. These studies (27–29) arediscussed in Section 3.

The EGL and the Starling Principle

For more than a century, investigators interested in fluid exchange across microvess-sels have widely assumed that the oncotic forces that drive this flow are determined


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by the difference in plasma protein concentration between the vessel lumen and tis-sue. The key role of the EGL in this exchange was not appreciated. The fact thatthe Starling principle may not have been correctly applied was highlighted at the1996 Starling conference celebrating the one-hundredth anniversary of Starling’s pi-oneering paper outlining his hypothesis for the filtration and absorption of waterin capillaries and the formation of lymph (30). At this meeting, Levick presented aprovocative paper based on his earlier review (31), which showed that when tissueoncotic pressures were carefully measured using the latest experimental methods,the widely accepted classical Starling force balance was violated in all tissues exceptthe kidney and the intestinal mucosa (tissues whose main function is venous reab-sorption), and there was no reabsorption on the venous side of capillaries, contraryto long-accepted views. Without venous reabsorption, one could not properly ac-count for the low whole-body lymph flows measured in vivo. These observationswere further supported by the experiments of Michel & Phillips (32) on single iso-lated perfused frog microvessels. They showed that when capillary pressures weresuddenly dropped, there would initially be a short transient period of reabsorption,which would quickly decay, followed by a very low level of filtration in the new steadystate. These observations clearly did not satisfy the classical application of the Starlingprinciple.

Subsequent to the 1996 Starling conference, Michel (30) and Weinbaum (33)independently proposed a revised Starling hypothesis, now referred to as the Michel-Weinbaum model (34), which proposes that the primary molecular sieve for plasmaproteins is the EGL and that the Starling forces are determined, not by the globaldifference in oncotic pressure between lumen and tissue, but by the local differencein protein concentration across the EGL alone. This revised hypothesis is describedmore fully in Hu & Weinbaum (35), where a detailed three-dimensional model ofthe EGL and the interendothelial cleft is developed. This revised model has sincebeen experimentally confirmed (see Section 6) in frog and mouse microvessels (36,37) and in EC monolayers in culture (38).

As summarized in this introduction, much of the research related to the EGLprior to 2000 has been focused on hydrodynamic problems examining the hemat-ocrit defect and hydrodynamic resistance of microvascular networks, ultrastructuralproblems related to measuring the thickness of the EGL and its uniformity, theo-retical models describing the axisymmetric motion and deformation of red cells incapillaries with an undeformed EGL, and a revisiting of the classical Starling prin-ciple. Since 2000, major new research directions have evolved as the multifacetedfunctions of the EGL have become more apparent. In particular, these new studiesof the EGL have focused on (a) its detailed structure and biochemical composition,including its mechanical and electrochemical properties, (b) its structural integrityand response to deformation, (c) its interactions with red blood cells and WBCs,(d ) its role as the primary mechanotransducer of FSS, (e) its role in the inflam-matory response cascade and ischemia–reperfusion injury, and ( f ) experiments toconfirm the revised Starling principle. These are the major themes of the presentreview.


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SA: sialic acid

GAG: glycosaminoglycan

HS: heparan sulfate

CS: chondroitin sulfate

HA: hyaluronic acid(hyaluronan)

2. STRUCTURE AND COMPOSITION OF THE EGL

Biochemical Composition

The surface of ECs is decorated with a wide variety of membrane-bound macro-molecules, which constitute the EGL. From glycoproteins bearing acidic oligosac-charides and terminal sialic acids (SAs) to proteoglycans along with their associatedglycosaminoglycan (GAG) side chains, the polyanionic nature of its constituents im-parts to it a net negative charge. Under physiological conditions, an extended en-dothelial surface layer arises from the association of components of the EGL withblood-borne molecules (1, 19). Plasma proteins, enzymes, enzyme inhibitors, growthfactors, and cytokines, through cationic sites in their structure, as well as cationicamino acids, cations, and water, all associate with this matrix of biopolyelectrolytes(39, 40). An additional level in the complexity of this biological structure arises fromits dynamic nature. The interactions between GAGs and proteins are highly depen-dent on the conditions of their local microenvironment, such as cation content andconcentration, and pH (41–45). Furthermore, ECs actively regulate the content andphysicochemical properties of GAGs on their surface by having high rates of contin-uous metabolic turnover that allow adaptation to changes in the local environment(46–48).

GAGs are linear polydisperse heteropolysaccharides, characterized by distinct di-saccharide unit repeats (49). Specific combinations of these give rise to different GAGfamilies, such as the heparan sulfate (HS), chondroitin/dermatan sulfate (CS), andhyaluronic acid or hyaluronan (HA) found on ECs (50). Proteoglycans are proteinsthat contain specific sites where sulfated GAGs are covalently attached (49). HS andCS chains vary between 50 and 150 disaccharide units and have an average molecularweight of approximately 30 kDa (51). Sulfated GAGs form extended helical coils,whose conformation depends on the local patterns of sulfation, the flexibility of themonosaccharides involved, and the degree of intramolecular electrostatic interactions(52). Local ionic strength and pH strongly influence the level of extensibility of a GAGchain, and it appears that this is maximal in a NaCl solution having physiologicalconcentration, as demonstrated recently by Seog et al. (53). Under these conditions,GAGs are considered to extend to approximately 80% of their contour length, so achain containing 100 disaccharide units would correspond to 80 nm (1, 53). Recently,it was demonstrated that in small arteries of the rat mesentery, ionic strength is amajor determinant of the overall state of the EGL, which can move from a collapsedto an extended state as ionic strength decreases (54).

The association of GAGs with proteins impacts their structure. Adamson andClough (19) demonstrated how, in the absence of plasma proteins, addition of asolution of a large charged marker protein (cationized ferritin, 440 kDa, 12 nm)reveals a collapsed EGL in frog mesenteric capillaries. Addition of a 2% frog plasmasolution was enough to significantly lift the layer of ferritin above the endothelialsurface, whereas a 5% albumin solution had a less striking effect (19). The preferentialbehavior that the EGL displayed for the less oncotic native plasma over albumin alonemakes it evident that specific interactions are essential to the physiological structureof the EGL.


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GPI:glycosylphosphatidylinositol

The GAGs commonly associated with the vasculature are HS, CS, and HA, withlevels varying among cell types. The most prominent on the surface of ECs are HS,accounting for 50%–90% of the total GAG pool, the rest being comprised of CS andHA (50). Owing to structural analogy with heparin, HS and associated proteoglycanshave been the most extensively studied, with attention recently shifting toward theirability to function as signal transduction molecules (55, 56). The transmembranesyndecans, the membrane-bound glypicans, and the basement matrix-associated per-lecans are the three major protein core families of HS proteoglycans found on ECs(57).

Syndecans-1 (33 kDa), -2 (22 kDa), and -4 (22 kDa), which are expressed on ECs,have three GAG attachment sites, close to their N terminus and distal to the apicalsurface, substituted primarily, but not exclusively, by HS (57, 58). Syndecan-1 con-tains two additional sites that are close to the membrane and reserved for CS (59).On the other side of the plasma membrane, their cytoplasmic tails associate withthe cytoskeleton and assist in its organization, through molecules such as tubulin,dynamin, and α-actinin (60, 61). Active participation in signaling stems from thephosphorylation of certain intracytoplasmic residues, which act as switches control-ling the oligomerization state and altering the binding properties of syndecans (55,56, 60).

Of the glypicans, glypican-1 (64 kDa) is the only one expressed on ECs (57).Close to the membrane, its three to four GAG attachment sites are exclusively sub-stituted with HS (62). Glypican-1 is bound directly to the plasma membrane througha C-terminal glycosylphosphatidylinositol (GPI) anchor (62). Most importantly, theGPI anchor localizes this proteoglycan to lipid rafts, which are cholesterol- andsphingolipid-rich membranous domains involved in vesicular transport and cell sig-naling (62–64). Caveolae can be considered a subset of lipid rafts, which arise from theincorporation of protein caveolin-1, a cholesterol carrier, into the membrane, wherethey may form characteristic cave-like structures (∼100 nm) that are supported bythe cytoskeleton (65).

In contrast with CS and HS, HA is a much longer disaccharide polymer, on theorder of 1000 kDa, which is synthesized on the cell surface and is not covalentlyattached to a core protein (66). It is not sulfated, but obtains its negative charge fromcarboxyl groups that endow it with exceptional hydration properties (66). HA weavesinto the EGL through its interaction with surface HA receptors, such as the trans-membrane CD44, and CS chains (26). CD44 contains two GAGs, either CS or HS,and localizes along with HA in caveolae, where it has various functional interactions(67). Completing the picture, glycoproteins with short, branched oligosaccharidesattached to their core are also found on the surface of ECs. These oligosaccha-rides are capped by SA, the nine-carbon monosaccharides that contribute to thenet negative charge of the EGL, through their ionization at physiological pH (68).Many important receptors on the cell surface, such as selectins, integrins, and mem-bers of the immunoglobulin superfamily, have oligosaccharides attached to them andare classified as glycoproteins (1). An illustration that integrates all of the compo-nents of the EGL described above is shown in Figure 1 (adapted from Reference69).


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ba

c

K+, Na+, Ca++, L-arginine

Albumin, bFGF, LPL, polycationic peptides

Cholesterol and glycosphingolipids

Caveolin-1

K+, Na+, Ca++, L-arginine channels

Hyaluronic acid

Heparan sulfate

Chondroitin sulfate

Glypican

Syndecans

CD44

Shedding

Glycoprotein

Sialic acids

Figure 1Representation of proteoglycans and glycoproteins on the surface of ECs. Caveolin-1associates with regions high in cholesterol and sphingolipids in the membrane (darker circles,left) and forms cave-like structures, caveolae (right). Glypicans, along with their HS chainslocalize in these regions. Transmembrane syndecans are shown to cluster in the outer edge ofcaveolae. Besides HS, syndecans also contain CS, further down the core protein. Aglycoprotein with its short oligosaccharide branched chains and their associated SA “caps” aredisplayed in the middle part of the figure. HA is a very long GAG that weaves into theglycocalyx and binds with CD44. Transmembrane CD44 can have CS, HS, andoligosaccharides attached to it and it localizes in caveolae. Plasma proteins, along with cationsand cationic amino acids (red circles), are known to associate with GAGs. (a) The cytoplasmicdomains of syndecans can associate with linker molecules, which connect them to cytoskeletalelements. (b) Oligomerization of syndecans helps them make direct associations withintracellular signaling effectors. (c) A series of molecules involved in eNOS signaling localizein caveolae. (This illustration is not drawn to scale and a few GAG attachments were omittedfor simplicity.)


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ACC: actin corticalcytoskeleton

Recent EM Observations

Little was known about the ultrastructural organization of the EGL and its relationto the underlying F-actin cytoskeleton until the pioneering study of Squire et al. (22).Using computed autocorrelation functions and Fourier transforms of EM imagesobtained from both new (22) and previous studies (70) of frog mesentery capillaries,these authors were able to identify for the first time the quasi-periodic structureof the EGL and the anchoring foci that appear to emanate from the underlyingactin cortical cytoskeleton (ACC). The computer-enhanced images showed that theEGL is a three-dimensional fibrous network with 10–12-nm-diameter focal scatteringcenters that have a characteristic spacing of 20 nm in all directions. Freeze-fracturereplicas from rare sections where the fracture plane passed parallel and close to theendothelial surface showed that the anchoring foci formed a clearly defined hexagonalarray with a spacing of 100 nm. This spacing is very similar to the spacing of thetruncated bush-like structures that were seen by Rostgaard & Qvortrop (21) usinga fluorocarbon oxygen fixation technique, which preserved the portion of the EGLthat was close to the EC surface. Using these combined studies, Squire et al. (22)proposed an ultrastructural model for the EGL and its linkage to the underlyingACC.

A modified sketch of this ultrastructural model, adapted from Weinbaum et al.(71), is shown in Figure 2a, where the basic organization of the bush-like core pro-tein structures and the underlying ACC are sketched showing the spacing and size ofvarious components. This basic ultrastructural model was then converted into the ide-alized mathematical model with hexagonal symmetry shown in Figure 2b. Weinbaumet al. (71) use this idealized model to predict the flow in the EGL, the drag forcesand bending moments on the core proteins, and the resulting local deformation ofthe ACC. More recently, Zhang et al. (72) have used this same idealized model todevelop a theory for the osmotic flow in the EGL and its reflection coefficient.

Another important EM study of the EGL came with the work of van den Berget al. (73), who used a perfusion fixation technique followed by Alcian blue stainingto stabilize anionic carbohydrate structures on the vessel wall of rat ventricular my-ocardial capillaries. After fixation, capillaries were sectioned and analyzed with EM.The luminal surface of normal capillaries were coated with discrete hairy, bush-likestructures that were uniformly distributed over the luminal surface. The observedcolocalization of certain lectins to these structures is suggestive of the structures hav-ing a saccharine nature rather than merely being crystallization artifacts associatedwith the fixation procedure. The mean thickness of the surface coat in normal cap-illaries measured between ∼0.2 and 0.5 μm, whereas in capillaries exposed to 1 h ofhyaluronidase treatment, the mean thickness measured between ∼0.1 and 0.2 μm. Itis noteworthy that while hyaluronidase appeared to have no effect on EC thickness,the interstitial space between capillaries and their surrounding tissue increased from0.28 μm to 0.46 μm after hyaluronidase treatment. The authors hypothesize that thisincrease in the pericapillary space after degradation of the EGL with hyaluronidaseis suggestive of a protective role for the EGL (and hyaluronan in particular) in pre-venting myocardial edema.


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Integrins

Glycocalyx bushstructure

20 nm

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α-actinin

Corticalcytoskeleton

Junctionalcomplex

Actin stressfibers

Extracellularmatrix

Periodic bushstructure

Actin filaments

Core proteinSpacing: 20 nm

Diameter: 10-12 nm

Cytoskeletal fociSpacing: 100 nm

a

b

Nucleus

Figure 2Structural model for the EGL. (a) Sketch of the arrangement of core proteins in the EGL andits anchorage to the underlying actin cortical cytoskeleton. (b) En face view of the idealizedmathematical model in Weinbaum et al. (71) showing the hexagonal arrangement of coreproteins and cluster foci. Adapted from Squire et al. (22) and figure 1 in Weinbaum et al. (71).

Relation between Biochemical Composition and EM Observations

One of the most important areas of future study is to create a bridge between theconsiderable base of knowledge that has been accumulated on the biochemical com-position of the EGL (shown in Figure 1) and the structural organization observed


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in EM (shown in Figure 2). At present, it appears that the EGL may be organizedinto two layers: an inner region of several tens of nanometers near the apical mem-brane surface, seen, for example, in the EM observations of Rostgaard & Qvortrop(21), and an outer layer up to 0.5 μm thick, which contains the extended core pro-teins. At the interface between these regions there could be a layer of HA becausethe membrane-bound molecules, CD44 and CS, have attachment sites for HA andCS has attachment sites for the various core proteins. The fact that HA lies closeto the apical surface can be inferred from the fact that Henry & Duling (26) haveshown that two hours of hyaluronidase treatment produces only minor changes inpermeability and EGL thickness, suggesting that HA is in a deeper protected regionnear the membrane surface. In contrast, heparinase III, an enzyme that cleaves HSproteoglycans (74), has a dramatic effect on the ECs’ ability to produce NO (74) andtransduce FSS to the actin cytoskeleton (75) (as further discussed in Section 6).

3. MECHANICAL AND ELECTROCHEMICAL PROPERTIES

EGL Thickness Using Microviscometric Analysis and μ-PIV

As mentioned in the Introduction, above, the dye exclusion technique developed byDuling and coworkers (23, 26, 76, 77) was inadequate to study the EGL in vivo inmicrovessels larger than 12–15 μm in diameter (26) and it provided no informationabout the velocity profiles near the vessel wall and within the EGL. Smith et al. (27)addressed these limitations by using intravital near-wall μ-PIV (see Figure 3a–c).

−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−→Figure 3(a) Typical dual-flash bright-field images showing a double exposure of one microsphere(∼ 0.5 μm diameter) near the vessel wall (top image) and near the vessel centerline (bottomimage) of a mouse cremaster-muscle venule in vivo. The dual images of the microsphere(encircled in white upstream and black downstream) are separated in time by the double-flashinterval. (b) Fluorescent intravital μ-PIV data in the plasma-rich region of a mousecremaster-muscle venule (∼33 μm diameter) obtained from images similar to those in (a).Notice that a linear extrapolation of the velocity profile leads to a negative intercept at thevessel wall. A highly nonlinear velocity distribution through the EGL is required to satisfy theno-slip condition at the vessel wall. (c) Hydrodynamically effective EGL thickness (mean andstandard deviation) before (10 vessels) and after light-dye treatment (10 vessels), assumingdifferent values of hydraulic resistivity, K, based on near-wall μ-PIV data similar to (b).(d ) Full-field intravital μ-PIV data fit to the velocity distribution over the cross-section of acontrol vessel using nonlinear regression analysis. (e) Using microviscometric analysis, theEGL thickness is estimated from the minimum in the least-squares error, E, in the fit to μ-PIVdata (similar to the fit shown in d ) before and after light-dye treatment to degrade the EGL.For a particular value of K, an iterative search for the corresponding value of EGL thickness,R–a, that corresponds to the optimal fit to the μ-PIV data in a least-squares sense (i.e. the localminimum for each curve in e) is taken to be the hydrodynamically effective EGL thickness.( f ) Hydrodynamically effective EGL thickness (mean and standard deviation) before(10 vessels) and after light-dye treatment (9 vessels) assuming different values of K based onfull-field μ-PIV data similar to (d ) and least-squares error analyses similar to (e). Adapted fromSmith et al. (27) and Damiano et al. (29).


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** *

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r (µm)

v z (

µm

/s)

Hydraulic resistivity, K (dyn-s/cm4)Estimated layer thickness (µm)

K ∞K = 1010 dyn-s/cm4

Hydraulic resistivity, K (dyn-s/cm4)

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Their technique involves obtaining measurements of the instantaneous transla-tional speeds and radial positions of FITC-labeled microspheres using dual-flashepi-illumination in an optical section through the median plane of the vessel. Thefluid-particle translational speeds are inferred from these measurements and a detailedthree-dimensional analysis of the local fluid dynamics in the vicinity of the vascularendothelium and its EGL (78). Using μ-PIV data in vessels before and after light-dyetreatment to degrade the layer revealed a strongly exponential rather than linear ve-locity distribution very near the vessel wall in control vessels (see Figure 3c). Theseresults show a significant effect of the layer on near-wall microfluidics and providethe first direct estimates of the effective hydrodynamic thickness of the EGL in vivo.This thickness was estimated to be between ∼0.3 and 0.35 μm (assuming a hydraulicresistivity, K, of the EGL that is greater than 1010 dyn-s/cm4; see Figure 3c).

In the initial analysis of Smith et al. (27), the authors assumed a uniform shearfield in the plasma-rich zone (a zone which extends approximately 2 μm from thevessel wall), and only considered particle tracers within the plasma-rich zone. In asubsequent study, Damiano et al. (29) show that this assumption leads to a lower-bound estimate of the effective hydrodynamic thickness of the EGL. The full-fieldanalysis of the fluid dynamics over the entire cross section of the microvessel providedby Damiano et al. (29), which was validated in glass tubes and in microvessels in vivo(28), led to a slightly more accurate estimate of the EGL thickness than could beobtained solely from near-wall μ-PIV data. By taking a full-field approach, and usingμ-PIV data over the entire cross section (see Figure 3d ), Damiano et al. (29) wereable to account for the variation in shear rate throughout the plasma-rich regionnear the vessel wall, which they found increased monotonically in absolute value withincreasing radial position up to the EGL interface. As such, the maximum shear rate,which occurs at the EGL interface, slightly exceeds the mean shear rate within theplasma-rich region used by Smith et al. (27). Extrapolation based on the true velocitygradient at the EGL interface leads to a greater negative intercept at the vessel walland hence a greater (and more accurate) EGL thickness estimate than does a linearextrapolation based on the mean shear rate in the plasma-rich region.

Taking this approach, which they referred to as microviscometry (28), Damianoet al. (29) showed that in mouse cremaster-muscle venules (20–40 μm diameter),the mean hydrodynamically effective EGL thickness was ∼0.5 μm in control vesselsand ∼0.2 μm after light-dye treatment to degrade the EGL (see Figure 3e, f ). Theestimates of Damiano et al. (29) are approximately 40%–50% greater than the esti-mates of the effective EGL thickness that Smith et al. (27) found in the control andlight-dye treated vessels that they analyzed using near-wall μ-PIV applied only to theplasma-rich layer.

Elastic Properties of EGL Fibers

In the elastohydrodynamic theory for the structural integrity of the EGL, the im-portant parameter for determining how the proteoglycan core proteins transmit FSSfrom the free surface of the EGL to the ACC is the flexural rigidity, EI, of thecore proteins of the bush-like structures shown in Figure 2. There are no direct


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measurements of EI for the core proteins of EGL proteoglycans analogous to theexperiments that have been performed for F-actin, collagen fibrils, and microtubules(79–81). Weinbaum et al. (71) determine EI using an indirect approach in which theypredict the measured characteristic time for the time-dependent restoration of theEGL after it has been crushed by the passage of a WBC in a tightly fitting capillary,as first reported in Vink et al. (82).

The model in Weinbaum et al. (71) treats the core proteins as elastic fibers that arefirmly attached by linker molecules to the more rigid actin cortical network beneaththe membrane surface, much like bamboo shoots are interconnected by a network ofroots beneath the surface of the ground. The model uses small deflection theory topredict the long-time final decay of the fiber’s elastic recoil and neglects the initialshort-lived, large deformation of the core protein fibers after the passage of the WBC.The hydrodynamic interaction between the fibers is considered, but the motion ofthe entrained fluid is neglected. This linearized model predicts that EI is 700 pNnm2, about 1/20 of the measured value of EI of an F-actin filament, 1.3 × 103 pNnm2 (80). Because the diameter of a core protein is approximately one-half that of anactin filament, and the moment of inertia, I, of the cross section varies as the fourthpower of the radius, this is a reasonable prediction if values of E are comparable. Morerecently, Han et al. (83) have developed a more sophisticated large deformation modelfor elastica that predicts the time-dependent changes in shape of the core proteinsfrom their initial large deformation to their final restoration after the passage of thewhite cell. This more realistic model predicts that EI is 490 pN nm2, surprisinglyclose to the highly idealized linear elastic model first proposed in Weinbaum et al.(71). This large deformation theory is described in more detail in Section 4.

Electrochemical Properties

Motivated by results reported by Vink & Duling (76) on the clearance rates of chargedand neutral plasma tracers from the EGL, Stace & Damiano (84) developed an elec-trochemical model of the EGL to gain a quantitative understanding of the transportof negatively charged molecules through the layer. This analysis also provides im-portant new insights into the electrochemical composition of the layer. In particular,one is interested in devising a method for quantifying the fixed-charge density of theEGL in vivo. The Stace & Damiano model consists of a quaternary mixture withsolid, water, cationic, and anionic constituents in which fixed negative charges arebound to the solid matrix. Their model is consistent with the triphasic theory devel-oped by Lai et al. (85) in the absence of an elastic-restoring capability by the fibermatrix and in the limit of small solid-volume fractions. The interaction of the fixednegative charges with cations and anions in the blood leads to a charge distribution inequilibrium that achieves spatial charge neutrality globally but with local imbalancesin charge that give rise to a double layer at the EGL–plasma interface. Considerationof the equilibrium configuration in the model of Stace & Damiano (84) shows thatpolyanionic plasma tracers would be partially excluded by the EGL in equilibriumby virtue of their charge. It also quantitatively predicts the degree to which this ex-clusion would occur as a function of tracer valence, EGL fixed-charge density, and


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the ionic strength of blood plasma. This exclusion would result in a reduction influorescence intensity within the EGL relative to luminal intensity levels, and couldtherefore be detected using epifluorescence illumination in vivo. Thus, if the valanceof a polyanionic plasma tracer were known, then a quantitative measure of the diminu-tion of fluorescence intensity in the EGL relative to the luminal intensity level wouldprovide the information necessary to estimate the fixed-charge density of the EGLin vivo.

Vink et al. (86) compared the intensity distributions of neutral dextran plasmatracers (40 kDa, Texas red) with polyanionic fluorescein-labled dextran tracers (40kDa, FITC dextran sulfate) over the cross section of mouse cremaster-muscle venulesin vivo. They showed a 30%–50% attenuation in the anionic tracer intensity relativeto that of the neutral tracer within ∼0.5 microns from the vessel wall. According tothe electrochemical model of Stace & Damiano (84), such an exclusion correspondsto an EGL fixed-charge density of between 0.7 and 1.3 mEq/l, which is approx-imately 1% of the ionic strength of blood plasma. The electrochemical model ofStace & Damiano (84) was later extended by Damiano & Stace (87) to investigatethe effect of the EGL fixed-charge density on charge-mediated mechanical recov-ery of the layer after deformation induced by a passing leukocyte in a capillary (seeSection 4).

In another analysis that investigated the electrochemical transport of chargedmolecules through the microvascular wall, Fu et al. (88) extended their earlier par-tition model of the interendothelial cleft to include a negatively charged EGL atthe cleft entrance. Their analysis examined solute transport under steady-state con-ditions and did not consider transients. From their analysis, they estimated that theEGL fixed-charge density was ∼25–35 mEq/l. To make this estimate, they consideredfour different fixed-charge density distributions across the EGL and used the resultsof Adamson et al. (89), wherein the permeability of frog mesenteric capillaries to apositively charged globular protein was found to be twice that of a negatively chargedglobular protein having nearly the same molecular weight. This estimate is ∼30-foldgreater than the previous fixed-charge density estimate of Stace & Damiano (84) andDamiano & Stace (87). While model assumptions of the Stace & Damiano model areslightly different from those of Fu et al. (88), the main source of this discrepancy maylie in the fact that the estimate of Fu et al. was based on the permeability measure-ments from frog mesenteric capillaries, whereas the estimate of Stace & Damiano wasbased on experiments in hamster cremaster-muscle capillaries. It is certainly possiblethat both estimates are valid in the context of their respective applications. However,it is noteworthy that estimates of EGL fixed-charge density in mammalian microves-sels must be consistent with the observations of Vink & Duling (23) and Han et al.(83). In particular, the EGL must be sufficiently soft so as to become completelycompressed by red cells brought to rest in 5-μm-diameter capillaries when flow isoccluded and yet at the same time be capable of restoring its equilibrium dimen-sions within ∼1 s after compression by a leukocyte (see Section 4). According to themechano-electrochemical model of the EGL of Damiano & Stace (87), who wereable to use the calculations of single-file red-cell motion in EGL-lined capillariespresented by Secomb et al. (13) to place bounds on the EGL fixed-charge density,


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an EGL fixed-charge density greater than 1 mEq/l would not be consistent with theobservations of Vink & Duling (23) and Han et al. (83). This need not necessarily bethe case, however, for the EGL in amphibian capillaries.

4. MODELS FOR STRUCTURAL INTEGRITYAND RESTORATION

It is clear from intravital imaging and immunofluorescence studies that the thicknessof the EGL is nearly uniform and changes little during the passage of a red cell oncethe cell is moving at a fast enough velocity to lift off of the layer. In marked contrast,the experiments in Vink et al. (82) and Han et al. (83) show that the EGL can becrushed to as much as 20% of its undeformed thickness by the passage of a WBCthrough a capillary. As noted above, the structure is both light sensitive and easilycompressible, as evidenced by the fact that a red cell brought to rest will expand to fillnearly the entire lumen of the capillary (23). Because the membrane of the red cell ishighly flexible, it might seem surprising that the fiber structure of the EGL can bothso easily collapse and yet be able to restore itself to its undeformed thickness typicallywithin 0.5 s after the passage of a WBC. Three different biophysical models will bepresented in this section to describe this paradoxical behavior. A second fundamentalparadox is that all three models predict that the FSS at the edge of the EGL is greatlyattenuated by the fiber matrix, with the result that the FSS at the level of the ECmembrane is vanishingly small. Thus, for each model a critical question that needsto be addressed is how FSS is transmitted to the intracellular cytoskeleton. This isone of the central issues in Section 6 on mechanotransduction.

Hydrodynamic Models for Flow in the EGL

Among the earliest analytical attempts to investigate flow through the EGL wasthe work of Barry et al. (90). Their work considered steady and unsteady flow of aNewtonian fluid in a channel lined with a poroelastic wall layer. Wang & Parker (91)applied mixture theory and two-dimensional lubrication theory to the problem of asphere falling through a quiescent fluid in a cylindrical tube lined with a deformableporous wall layer. This was followed by Damiano et al. (10), who obtained analyticalsolutions for axisymmetric pressure-driven flow of rigid close-fitting particles in acylindrical tube with a poroelastic layer lining its luminal surface. In all of thesestudies, flow through the EGL was modeled using the equations of binary mixturetheory where the solid volume fraction was taken to be small. In the limit of a vanishingsolid volume fraction, these equations degenerate to the Brinkman equation for asolenoidal fluid velocity field given by

−μ∇ × ∇ × v − K v = ∇ p

∇ · v = 0, (1)

where v is the fluid velocity vector, p is the pressure field, and the parameters μ and Kdenote the fluid dynamic viscosity and hydraulic resistivity, respectively. K is relatedto the commonly used Darcy permeability, k, by K = μ/k.


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The important dimensionless quantity for characterizing drag through the EGLis δ2 = μ/(h2K ), where h is the mean effective thickness of the layer. This quantitycharacterizes the ratio of viscous drag forces associated with fluid-velocity gradients inthe EGL to permeation-induced viscous drag forces associated with the fluid motionrelative to the solid constituent of the EGL. For small values of δ, permeation-inducedviscous drag forces dominate and balance the pressure gradient throughout the EGL,except very near the plasma interface where fluid velocity gradients become large.Thus, for small values of δ, a Darcy flow prevails away from the plasma interface. Inparticular, the quantity (μ/K )1/2 = δ h is the characteristic exponential decay length(with respect to distance into the layer) of the axial velocity at the plasma–EGLinterface, i.e., it characterizes the depth of penetration of axial flow through the EGL.

Because it is likely that the solid volume fraction φs � 1 (92), it is reasonable totake the dynamic viscosity of the layer to be equal to that of blood plasma (∼0.01 dyn-s/cm2). Estimates of the hydraulic resistivity, K, of the EGL, based on the recoverytime of the layer in capillaries in the wake of a passing WBC, place it between 1010

and 1011 dyn-s/cm4 (71, 87, 93), which puts δ2 between 10−4 and 10−3 for an EGLthickness of ∼0.5 microns. It is noteworthy that a value of K > ∼1010 dyn-s/cm4 soseverely attenuates flow throughout the EGL that FSS on the EC surface is effectivelyeliminated. This has important implications for understanding mechanisms of ECmechanotransduction, which is further discussed in Section 6.

Models for the Restoring Mechanisms of the EGL

Whereas there has been a general consensus as to how to best mathematically describefluid-dynamical drag through the EGL, a variety of hypotheses have emerged as to theorigins of the restoring force mechanism that prevents the deformation of the layerowing to FSS in the physiological range and allows its recovery after compression bya passing WBC. Most prominently these include the oncotic models of Secomb andcoworkers (12, 13, 94), the elastohydrodynamic models of Weinbaum and cowork-ers (71, 83), and the mechano-electrochemical model of Damiano and coworkers(87, 93).

Oncotic model. Pries et al. (9) first introduced the concept that even a minutedifference between the concentration of plasma proteins adsorbed to the EGL andthat of free plasma proteins in the capillary lumen would be sufficient to generate anoncotic (or colloid osmotic) pressure that would be sufficient to exclude red blood cellsfrom the EGL region near the vessel wall under standard flow conditions in capillaries.This concept was then applied in Secomb et al. (12) to develop a model for single-filered blood cell motion in an EGL-lined capillary. They argued that the source of therestoration capability of a compressed EGL was not mechanical (elastic) but chemicaland suggested that, in equilibrium, the excess oncotic pressure within the EGL relativeto the plasma would be balanced by tension in the EGL matrix. Compression of thelayer would result in a further increase in this pressure differential and a relaxationof the tension in the EGL matrix. Upon recovery of the layer back to its equilibriumconfiguration (after the externally applied load is removed), tension would mount


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within this matrix until a balance is reestablished between macromolecular tensionwithin the EGL matrix and the oncotic pressure within the layer.

In their initial model for the single-file axisymmetric motion of deformable redcells in an EGL-lined capillary, Secomb et al. (12) assume that the red cells do notinvade the EGL and that the restoring forces in the red-cell membrane are assumedto arise from membrane shear elasticity and isotropic membrane tension, but thebending elasticity of the membrane is neglected. The flow through the EGL is mod-eled using the momentum equation given by Damiano et al. (10), which is a specialcase of the Brinkman equation described in the previous subsection when flow in thecapillary is axisymmetric and unidirectional. The pressure in the lubrication layerbetween the red cell and the vessel wall combines with the oncotic pressure imposedby the EGL to determine the magnitude of the normal component of the tractionvector acting on the red-cell membrane. Their calculations assumed an oncotic pres-sure in equilibrium of 200 dyn/cm2, which is approximately 8% of the typical oncoticpressure of the plasma (2500 dyn/cm2 or 25 cm H2O). In a subsequent study, Secombet al. (13) refined their previous analysis to include a more realistic model of thered-cell membrane (one which included the effect of bending elasticity) and consid-ered the problem of the red cell invading the EGL as the flow rate in the capillaryvanishes. Results of this analysis showed that an oncotic pressure of only 20 dyn/cm2

in equilibrium was large enough to exclude red blood cells (but not WBCs) fromthe EGL, and yet was small enough to allow the gap between the red cell and thecapillary wall to approach zero thickness when the velocity vanished, as had beenobserved experimentally (23).

At first glance, this model for oncotic tension in the EGL may seem hard to rec-oncile with the models for the oncotic pressure in the EGL that are presented inSection 6 describing the revised Starling principle. In the case of the latter models,the EGL acts as the primary molecular sieve for plasma proteins, which results in asubstantial drop in oncotic pressure across the EGL (the magnitude of which dependson the luminal pressure or filtration rate). At normal filtration rates in capillaries, theoncotic pressure across the EGL drops from approximately 25 cm H2O (2500 dyn/cm2) to 10 cm H2O (1000 dyn/cm2). This oncotic pressure drop far exceeds the20 dyn/cm2 oncotic pressure increase of the adsorbed proteins estimated by Secombet al. (13), thus placing the EGL in a state of compression rather than tension. Thisdifficulty can be explained if the 100-nm-thick layer observed by Squire et al. (22)using EM (where a quasi-periodic structure was observed) functioned as the molec-ular sieve for plasma proteins and this was coated with a significantly thicker and lessorganized structure that functioned as the effective hydrodynamic layer that is mea-sured in Smith et al. (27) and Damiano et al. (29). This outer layer is often referred toas the endothelial surface layer, in contrast to the EGL where the oncotic sieving isassumed to occur. A small oncotic pressure of 20 dyn/cm2 would be sufficient to keepthis outer layer hydrated and provide the lift force for red cells during the pop-outphenomenon modeled by Feng & Weinbaum (95) and Secomb et al. (13). Viewedfrom this perspective, the much larger oncotic pressures in the EGL could be resistedby the elastic properties of the core proteins in the elastohydrodynamic model thatis presented next.


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Elastohydrodynamic model. In the elastohydrodynamic model, the core proteinfibers have a finite flexural rigidity, EI, which provides (a) for their ability to withstandcollapse owing to the decrease in oncotic pressure required for them to function as amolecular sieve for plasma proteins and (b) for their ability to withstand FSS in thephysiological range without substantially deforming. This structural resistance to de-formation in response to FSS is observed in intravital microscopy studies (23, 26, 77)and microviscometric studies (28, 29) of EGL thickness. The linear elastohydrody-namic model of Weinbaum et al. (71), described in Section 3 to predict EI, treats onlythe final portion of the restoration of the EGL after its initial finite compression bythe passage of a WBC. As noted previously, the predicted value of EI was 700 pN nm2.A much more sophisticated nonlinear elastohydrodynamic model is presented in Hanet al. (83) that uses large deformation theory for elastica and a modified Brinkmanequation to describe the local relative motion of the fibers and the fluid. The analysisalso takes into account the fluid that is imbibed as the EGL expands and uses localhydrodynamic resistance coefficients to describe the local time-dependent hydrody-namic forces parallel and perpendicular to the fiber motion. The experimental partof the paper greatly expands on the brief abstract of Vink et al. (82), in which resultswere first reported for the time-dependent restoration of the EGL after the passageof the WBC.

The nonlinear model in Han et al. (see Figure 4a) predicts that there are twophases for the fiber recoil after the passage of a WBC, an initial phase for largecompressions, where the EGL is less than 0.36 ho (for an undeformed thickness, ho)and the ends of the core proteins overlap and are parallel to the endothelial membrane,and a second phase where the fibers assume a shape that is close to the solution for anelastic bar with linearly distributed loading. As seen in Figure 4a, the transition fromphase I to phase II occurs at t = 0.041 s if the EGL is initially compressed to a thicknessof 0.2 ho. Figure 4b compares the predictions of the elastrohydrodynamic model inHan et al. (solid curve) with the experimental data for the time-dependent restorationof the EGL (solid circles) and the predictions of the mechano-electrochemical modelof Damiano & Stace (87) (open triangles), which is discussed in the next subsection.The predictions of Damiano & Stace are rescaled by a factor of 4900 and shiftedto the right by 0.01 because the starting point of their calculation is h/ho = 0.5,where ho = 400 nm. The predictions of the two models are too close to determinewhich model has greater validity. The principal difference in behavior is that theelastohydrodynamic model does not asymptotically approach the undeformed heightfor long time, as is the case for the mechano-electrochemical model, but instead itabruptly reaches h/ho = 1.0 at t/τ = 0.05, where τ = 14 s is a dimensional scaling timerelated to the flexural rigidity EI. The surprisingly close agreement in the predictionsfor EI of the linear and nonlinear elastica models (700 versus 490 pN-nm2) is dueto the fact that the large deformation portion of the recovery is relatively shortlived.

Mechano-electrochemical model. Damiano & Stace (87) proposed a model for thestructural integrity of the EGL that consists of a mixture of electrostatically chargedmacromolecules hydrated in an electrolytic fluid. Their mechano-electrochemical


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Phase II

Phase I

a b

Model predictions (Reference 83)

Model predictions (Reference 87)

Experimental results (Reference 83)

y/ho

x/ho

h/ho

t / τ

1.0

0.8

0.6

0.4

0.2

0 0.2 0.4 0.6 0.8 1.0

1.5

1.0

0.5

00 0.05 0.10 0.15

0 s0.014 s

0.041 s

0.16 s

0.36 s

0.60 s

0.70 s

Figure 4(a) The time-dependent change in shape of the core protein fibers. y/ho and x/ho are scaledvertical and horizontal coordinates of the fiber position, and ho, the undisturbed fiber-layerthickness, is also fiber length. Note the change from phase I to II occurs at 0.041 s. (b)Comparison of model predictions in Han et al. (83) (solid line) and the predictions (opentriangles) derived from figure 2b in Damiano & Stace (87) with the experimental results in Hanet al. (83) (solid dots). The ESL thickness is normalized by 400 nm, the undisturbed ESLthickness, ho (b). Adapted from figures 6 and 7A in Han et al. (83).

model considers one-dimensional finite deformations of the layer in the absence ofan axial flow in the vessel. As in the electrochemical model of Stace & Damiano (84)described in Section 3, Damiano & Stace (87) model the EGL as an isotropic qua-ternary mixture. The constitutive law for the flux of the solid and ionic constituentsis modeled using the Nernst-Planck equation, where it is assumed that these con-stituents have negligible volume fractions, and the viscosity of the fluid constituent isalso negligible. According to their analysis, EGL recovery after deformation is drivenby an electrochemical potential gradient, which consists of electrostatic and chemical-gradient components. The former accounts for the interactions of the fixed-boundcharges on the matrix with the counter ions in solution, whereas the latter arises as aresult of the higher concentration of bound GAGs, glycoproteins, and proteoglycansin the EGL relative to the concentration of corresponding blood-borne constituentsin the lumen. Leukocyte-induced deformations of the EGL result in disturbancesaway from a nearly electroneutral equilibrium environment. In the wake of the leuko-cyte, fluid flux into the compressed layer, driven by gradients in the electrochemicalpotentials of the ions and macromolecular matrix, results in rehydration of the EGL


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and a restoration of its equilibrium dimensions. It is assumed that the matrix has noelastic restoring capability under compression but can support a tensile stress thatbalances the electrochemical potential gradient in the equilibrium configuration.

The two most important parameters in this model for determining the recoverytime of the EGL after compression by a leukocyte are the dimensionless fixed chargedensity, ξ 0, and the dimensionless macromolecular matrix concentration, F (the di-mensional counterparts of ξ 0 and F are nondimensionalized with the concentration ofmobile cations in blood). The relative size of these two quantities determines which isthe more dominant physical mechanism. Asymptotic and numerical analyses of theirmodel reveal that the 90% recovery time, τ , is given in terms of ξ 0, F, and the dimen-sionless equilibrium thickness of the EGL (see Figure 5). As is evident in Figure 5,the model predicts that the response of the EGL falls into two distinct regimes. Whenξ 0

2 � F, the recovery time is essentially independent of ξ 0 and the chemical-potentialgradient is the dominant restoring force. This mechanism is similar to the oncoticmodel of Secomb and coworkers (12, 13, 94), described above, except that the chem-ical potential gradient is derived not from adsorbed plasma proteins but from GAGsbound to the EGL. On the other hand, when ξ 0

2 � F, the electrostatic potentialgradient dominates and the recovery time varies in proportion to ξ 0

−2. Based on the∼1 s recovery time of the EGL, and the value of K on the order of 5 × 1010 dyn-s/cm4,

F = 10F = 10-4-4

F = 10F = 10-5-5

F = 1.22 x 10F = 1.22 x 10-6-6

F = 10F = 10-7-7 Finite difference solution

Asymptotic equation

F = 10-4

F = 10-5

F = 1.22 x 10-6

F = 10-7

τ(s

)R

eco

very

tim

e,

10-110-210-3

0.01

0.1

1

10

100

10-5 10-4

ξ 0

Figure 5The 90% recovery time as predicted by the mechano-electrochemical model of Damiano &Stace (87) as a function of the dimensionless fixed-charge density, ξ0, for different values of thedimensionless macromolecular matrix concentration, F. Results correspond to a hydraulicresistivity, K = 1011 dyn-s/cm4, a cation concentration of 0.14 M (which is equal to theconcentration of mobile cations in blood), a capillary radius of 2.5 μm, and an EGL thicknessof 0.5 μm. Notice that for all values of F < 10−5, a 1 s recovery time corresponds to ξ0 = 0.01,or equivalently, a dimensional fixed-charge density of the EGL of ∼1 mEq/l. Adapted fromfigure 4 in Damiano & Stace (87).


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a fixed-charge density of the EGL of 1 mEq/l would result in the magnitude of thestress traction exerted by the layer on passing red cells of 20–30 dyn/cm2, which isconsistent with the magnitude necessary to account for the exclusion of red cells bythe EGL observed in skeletal muscle capillaries in vivo (13). This corresponds toapproximately one fixed-bound charge on the EGL for every 100 ions in blood. It isnoteworthy that such a charge density would result in a voltage differential betweenthe undeformed EGL and the capillary lumen of ∼0.1 mV. In a subsequent study,Damiano & Stace (93) developed a detailed model of the fluid dynamics and EGLdeformation in the wake region behind a leukocyte moving through a capillary. Theiranalysis details the radial and axial velocity components arising from an axisymmetricpressure-driven flow of a linearly viscous fluid within the wake region and throughoutthe EGL. The analysis establishes the necessary relationships to connect the resultsof the one-dimensional mechano-electrochemical model of Damiano & Stace (87)with the results of a more realistic approximation of the flow regime arising in thewake of a WBC passing through a capillary.

5. CELLULAR INTERACTIONS WITH THE EGL

The early theoretical studies mentioned in the Introduction on the deformation of amoving red cell in a capillary either neglected the presence of the EGL or describedthe simpler case where the red cell did not enter the EGL. The experimental studiesof Vink & Duling (23) clearly show that a red cell brought to rest will expand to fill theentire lumen of the capillary, thus crushing the EGL, and that when motion resumes,the cell will exhibit a pop-out phenomenon in which the cell will rise through theEGL as its velocity is increased until it reaches a critical velocity, at which time the cellwill lift off of the EGL and create a thin lubricating layer between it and the EGL.In the case of the WBC, both its free interaction with the EGL and its tetheredrolling in the presence of the EGL are of interest. To address these interactions, oneneeds to calculate the forces generated on the WBC microvilli tips for both types ofencounters with the EGL as well as the penetration resistance of the layer. Both ofthese problems are examined in this section.

Red Cells

The first realistic attempts to analyze the single-file motion of red blood cellsin EGL-lined capillaries first appeared in Damiano (11) and Secomb et al. (12)(as described in Section 4), who adopted similar approaches to elucidate the effect ofthe EGL on capillary resistance and capillary tube hematocrit. The apparent viscosityand capillary tube hematocrit predicted by these models showed strong sensitivity tothe presence of an EGL, and predicted that a 0.5-μm-thick hydrated macromolecularlayer lining the capillary wall could result in a marked increase in capillary resistanceand a significant reduction in capillary tube hematocrit relative to model predictionsof blood flow through smooth-walled glass tubes. To maintain continuity of mass, theretarded flow through the EGL demands greater red-cell clearances and results inmore elongated red cell shapes in capillaries than in smooth glass tubes of the same


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diameter. Both models were valid only in a high-velocity limit (>1000 μm/s), wherered cell deformation is at a maximum and red cell shapes are nearly independent offlow velocity (96). However, in skeletal muscle capillaries, red cell velocities are oftenin the range of 100 to 200 μm/s in steady flow and can drop to zero during intermit-tent flow conditions. For these situations, restoring forces in the red cell membraneare of the same order as FSS and pressure forces at the cell surface. This results inbroader and shorter cell shapes with decreasing velocity. Secomb et al. (13) addressedthis limitation by refining the red cell model, as in Secomb et al. (96), to includethe effect of bending elasticity in the red cell membrane (see Section 4). With thisgeneralization, they were able to consider red cell velocities down to 1 μm/s.

In addition, Secomb et al. (13) were able to model the so-called pop-out phe-nomenon, first analyzed in Feng & Weinbaum (95), by considering transient changesin red cell shape as a function of velocity for a step increase in driving pressure acrossa red cell that is initially nearly stationary. Feng & Weinbaum (95) identified an im-portant fluid-dynamical lift force that will arise within the porous EGL matrix owingto the axial flow of plasma through the layer. To generate this lift force, a confin-ing boundary or bearing surface (e.g., the red cell membrane) translating at a smallangle relative to the vessel wall, such as might be provided by a passing red bloodcell in a capillary, is required to prevent fluid leakage out of the layer into the freeluminal space of the vessel. Feng & Weinbaum draw an intriguing analogy betweena human skiing on snow powder and a red cell gliding on the EGL. They show thatthe dimensionless parameter, h/

√k (where h is the EGL thickness and k is the Darcy

permeability), describing the fluid motions in both porous layers is of the same orderof magnitude, although a human and red cell differ in mass by 15 orders of magnitude.Such conditions can certainly arise in capillaries, particularly upon the resumption ofred cell motion after a flow-cessation event. In light of this idea, and by virtue of therelatively well-understood mechanical properties of red blood cells, Secomb et al. (13)were able to provide a quantitative description of the flow-dependent exclusion ofred cells from the EGL, which placed approximate bounds on important mechanicalproperties of the EGL.

White Cells

The adhesion of circulating leukocytes to vascular ECs during inflammation andimmune surveillance is initiated by the adhesive tethering of L-, P-, and E-selectinsand α4 integrins to ligands that are located either on the tips of the WBC microvillior the EC surface. This has stimulated an extensive literature on the mechanicsand kinetics of tethered rolling of WBCs on coated glass substrates in which a hostof ligand-receptor interactions have been studied, starting with the early work ofLawrence & Springer (97). Similarly, there is an extensive literature on the tetheredrolling of WBCs on ECs grown in culture media where the state of the EGL iseither unknown or not expected to be preserved. To our knowledge, there are noexperiments that have explored the role of the EGL in regulating adhesive tetheringor as a modulator of free rolling without adhesion. This is surprising because we shallsee in Section 6 that the EGL plays a central role in mechanotransduction and can


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have a profound influence on the cytoskeletal and biochemical responses of culturedEC monolayers to FSS. In contrast, the role of the EGL in leukocyte adhesion is anuncharted area of research.

A critical question in the case of WBC rolling is whether the microvilli of a free-rolling WBC will be able to penetrate the EGL and initiate tethered rolling attach-ments. The quantitative feasibility of this occurring has only been examined theo-retically (98). Tethered rolling is frequently observed in vivo in postcapillary venules,where one would anticipate that an intact EGL exists, but this rolling always seems tobe initiated at the entrance to a postcapillary venule where a tightly fitting WBC hasjust emerged from a capillary (99). At this location, the microvilli have been in closecontact with the EC surface, the WBC having crushed the EGL during its passagethrough the capillary (82, 83). Because the length of WBC microvilli is typically 0.3to 0.7 μm (100), and thus, comparable to the thickness of the EGL (23, 29), the abilityof a WBC to roll freely through microvessels needs to be questioned. Cell rollingwould be quickly curtailed if the microvilli functioned as human legs trying to crossa field of fresh fallen snow whose depth was the same as their length. Because thereis no evidence that WBCs have the equivalent of snowshoes, one wonders whether aWBC can tip-toe across the EGL much like a Jesus Christ lizard runs across water.

For the case of a free-rolling WBC on a cultured endothelial monolayer, oneneeds to consider sedimentation owing to weak gravitational forces. For a WBCwhose specific gravity is 1.1, this sedimentation force is ≈0.3 pN. However, Zhaoet al. (98) show that this weak gravitational force can be amplified 20- to 100-foldat the microvilli tips owing to viscous forces generated in the thin lubricating layerbetween the body of the WBC and the rolling surface. In vitro WBCs appear toroll as if they were displaced by a distance of ≥500 nm from solid boundaries (101,102), suggesting that the microvilli behave as stiff protuberances on the time-scaleof rolling contact, which is typically approximately 0.2 ms at a FSS of 5.0 dyn/cm2.This stiffness is supported by the experiments of Shao et al. (103), who show that theviscoelastic response of a microvillus under stretch is 0.77 s, at least three orders ofmagnitude longer than the very brief rolling contact times. Early studies on the freerolling of WBCs as a precursor to tethered rolling on solid substrates examined therolling interaction of single microvilli (101, 104, 105) but not the response of multiplemicrovilli, which typically exceed 20 in a cross-section (100). The latter was exploredfor the first time in Zhao et al. (98). These authors predicted that for equal lengthmicrovilli, the contact force would increase from 2 pN at a FSS of 1 dyn/cm2 to 5 pN ata FSS of 10 dyn/cm2. For the small population (5%–10%) of long microvilli (>0.5 μm)in a heterogeneous microvilli array, this force would increase from 8 to 30 pN at thesesame FSS. Note that the contact forces do not scale linearly with the FSS owing tothe nonlinear coupling that arises from the sedimentation between contacts.

When the WBC experiences tethered rolling in vivo or in vitro, the weak grav-itational forces are insignificant because Zhao et al. show that the normal forcesgenerated at the microvilli tips are at least an order of magnitude greater than theyare during free rolling. At a FSS of only 1 dyn/cm2, normal contact forces are alreadyof the order of 100 pN and deep penetration of the EGL is easily achieved even atthese relatively low FSSs, as discussed below.


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To determine the depth of penetration of the microvilli, Feng et al. (106) havedeveloped a theory for the motion of a sphere in a Brinkman medium perpendicularto a planar wall as an idealized model for the normal penetration of the microvilli tips.The microvilli tips are treated spheres (typically 0.1 μm in diameter). The diffusionalresistance of 40-nm-diameter gold particles attached to lipids in the membrane wasmeasured by Lee et al. (107). The theory in Feng et al. is used to scale the resistanceof a 0.1-μm-diameter sphere to this measured resistance. Feng et al. estimated that amicrovillus would penetrate the EGL at a velocity of 6 μm/s under the action of a 1 pNforce. Taking account of the nonlinear change in contact time with FSS, Zhao et al.predict that the depth of penetration at a FSS of 10 dyn/cm2 would be ∼1.0 nm forequal-length microvilli and ∼20 nm for individual longer microvilli of heterogeneouslength. The short penetration depth is due to the very short tip contact times, whichare ≤0.1 ms at this shear stress. However, for a FSS of only 1 dyn/cm2, the penetrationdepth could be as much as 200 nm for the longer microvilli owing to the longer contacttime. This suggests that at least on the arterial side of the circulation, where the FSSexceeds 10 dyn/cm2, the WBC may, in fact, behave like a Jesus Christ lizard duringfree rolling, even in the presence of weak gravitational forces. This could explain whytethered rolling is seldom seen on the arterial side of the circulation except in regionsof very low FSS, such as in recirculation zones. In sharp contrast, the long microvillion a WBC experiencing tethered rolling in vivo, where the normal penetration forceis at least an order of magnitude greater at the same FSS, would easily penetrate theentire thickness of the EGL and form new tethering attachments. It is for this reasonthat once tethered rolling is initiated at the entrance to a postcapillary venule, suchrolling can often be sustained for the entire length of the venule.

6. PHYSIOLOGICAL FUNCTIONS

Permeability and the Revised Starling Principle

The classic Starling equation describing the balance of hydrostatic and colloidosmotic (oncotic) forces across the capillary wall includes four forces,

Jv/A = Lp[(Pc − Pt) − σ (πc − πt)],

where Pc and Pt are the hydrostatic pressures in the capillary lumen and tissue,respectively; π c and π t are the corresponding lumen and tissue oncotic pressures;Jv/A is the filtration rate per unit area of capillary wall; σ is the reflection coefficientto the plasma proteins; and Lp is the hydraulic conductivity. This relation has beentested on numerous occasions in both whole organs and isolated microvessels, butnearly always under conditions where the tissue protein concentration was low owingto wash-down after rapid filtration or washout in exposed superfused tissue. Rarely hasthe tissue been backloaded to examine the effect of π t. Furthermore, as noted in theIntroduction, considering the most accurate measurements of π t, most tissues cannotsustain venous reabsorption and one cannot account for measured lymph flows (31).

A resolution to this important paradox has been proposed by Michel (30) andWeinbaum (33). They hypothesized that the effective osmotic barrier is not the whole


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TJ: tight junction

capillary wall, as previously assumed, but just the luminal EGL, which acts as themolecular sieve for plasma proteins. Moreover, if there is a tight junction (TJ) strandin the cleft with infrequent breaks, back diffusion into the luminal side of the cleftowing to the convective flux of water through these breaks can be severely limited,especially at high filtration flows. For this reason, the tissue oncotic pressure π t candiffer greatly from the oncotic pressure π (0) in the protected region between theluminal side of the TJ strand and the back side of the EGL. Similarly, because a largefraction of the hydraulic resistance is due to the TJ strand, the hydrostatic pressurebehind the EGL, P(0), can differ greatly from the hydrostatic pressure Pt in the tissue.

Ac

pc

x

2d

Junctionstrand

L1

L2

2D

Region B

Cleft exit

LB = 5 µm

Region A

Pt

y = 0 y = D

x = -Lf = –150 nm

x = 0x = Lf = 67 nm

x = L = 411 nm

Surface glycocalyx, EGL

Lc = 0.5 pc Ac-1

L1 = x1 – x0 L2 = x2 – x1

L = x2 – x0

x0x1

2h

x2

Lf

2d

2D

y

Pc

a b c

d

Tissue Space

Figure 6Schematic of a microvessel (a) showing cross-section of an interendothelial cleft covered byEGL and (b) three-dimensional view of TJ strand in plane of cleft (c). (d ) Idealizedmathematical model showing four regions: surface glycocalyx (EGL), cleft with TJ strand(region A), near-field tissue space <5μm (region B), and far-field tissue space >5 μm from cleftexit. Adapted from figure 1 in Reference 37.


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This situation is illustrated in Figure 6, which shows a capillary with a cleft and EGLin cross-section (Figure 6b) and a TJ strand with infrequent breaks of length 2d andaverage spacing 2D (Figure 6c). The idealized mathematical model used to describethis geometry is shown in Figure 6d where the dimensions are taken from the detailedmeasurements in Adamson et al. (37) for rat mesenteric microvessels.

In the new Starling equation, first proposed in Weinbaum (33) to describe therevised Starling principle, π t is replaced by π (0) and Pt by P(0) because the Starlingforce balance is applied just across the EGL. This new equation is much more difficultto apply than Equation 2 because π (0) and P(0), which are evaluated at the cleftentrance behind the EGL, can vary significantly along the length of the cleft becausethe streamlines for water flow and solute flux lines follow a curved path through the TJbreaks, as illustrated in Figure 6d. This is particularly significant for fluid streamlinesbecause a large pressure drop can occur in the narrow protected channel in the clefton the luminal side of the TJ strand. Both π (0) and P(0) are unknown and mustbe determined by solving a coupled three-dimensional boundary value problem inwhich boundary and matching conditions are applied across three regions: the EGLwhere the revised Starling equation is applied, region A (the cleft), and region B(the tissue space). The first detailed three-dimensional solutions for this model werepresented in Hu & Weinbaum (35), predicting that at high-filtration rates the localPeclet number, Pe, at the TJ breaks would be greater than 1 and the concentrationof proteins in the protected region behind the EGL would be nearly independent ofthe tissue concentration, π t. As the lumen pressure decreased, Pe would decrease andback diffusion from the tissue would occur, allowing π (0) to rise.

The foregoing theoretical predictions were tested in two sets of experiments, oneperformed on frog mesenteric microvessels (36) and a second on rat mesenteric mi-crovessels (37), where detailed electron microscopic reconstructions of the cleft werealso obtained for input into the theoretical model. In both sets of experiments, thetissue was backloaded with albumin (by damaging the mesothelium at a distance ofapproximately 100 μm from the perfused microvessel) and a superperfusate was intro-duced that was isotonic (50 mg/ml) with respect to the lumen. The time-dependentchanges in tissue concentration were carefully measured using confocal microscopy.Experimental measurements of Jv/A were not performed until the lumen and tis-sue were observed to be isotonic. According to the classical Starling equation, thereshould be no oncotic force across the vessel wall. However, measurements of σδπ infrog microvessels when Jv/A = 0 showed that nearly the full lumen oncotic pressurewas present despite the fact that confocal microscopy indicated that the interstitialalbumin concentrations were isotonic to within a few microns of the vessel wall.Similarly, in rat microvessels, where the fractional length of the breaks in the TJwas larger, the measurements of σδπ were 70% of the lumen oncotic pressure. Bothexperimental results were accurately predicted by the three-dimensional theoreticalmodel in Hu & Weinbaum (35), as sketched in Figure 6d. Even at relatively low lu-men pressures, the model predicts and the experiments confirm that π t and π (0) candiffer substantially. These surprising findings show that there is a large asymmetry inthe effect of the Starling forces at the lumen and tissue fronts owing to the nonlineareffects of convection on the solute transport through the breaks in the TJ strands. In


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view of the highly unusual nature of these findings, several featured perspectives haveappeared in highlights from the physiological literature summarizing the importanceof this new view of the Starling hypothesis (34, 108). This same behavior has beenobserved in cultured bovine aortic EC monolayers (38).

The proposed structure of the EGL depicted in Figure 1 has also been a catalyst fora major extension from the classic study on the osmotic flow across membranes withlong circular cylindrical pores (109) to a fiber matrix layer (72). One of the widelyused results of this classical analysis is the expression for the reflection coefficientof a spherical solute in a porous membrane, σ = (1 − φ)2, where φ is the partitioncoefficient. This analysis, and the expression for σ , have been used extensively in manyinvestigations of porous media that have applied the fiber matrix theory of Curry &Michel (110). Intuitively, investigators have thought that because the flow betweenfibers is Poiseuille-like, the analysis for a fiber matrix layer would be equivalent tothat for circular pores. However, Zhang et al. (72) show that there is a fundamentaldifference because the exclusion layer where the potential energy function describingthe solute distribution is satisfied corresponds to the fluid annulus surrounding thefiber. Thus, the exclusion layer for the solute molecule corresponds to the exteriorof a circular cylinder rather than the interior of a cylinder, as is the case for a circularpore. Each fiber is surrounded by an equivalent fluid annulus in which pressures andoncotic forces must be determined. The theory is developed for a hexagonal fiberarray corresponding to the fiber geometry shown in Figure 1b. A new closed-formexpression for σ is derived, which turns out to be a function of two dimensionlessparameters, a/R and b/R, and not just φ. Here, a and b are the solute and fiber radiiand R is the radius of the effective fluid annulus. The results differ substantially fromthose associated with circular pores because of the large difference in the shape of theboundary along which the osmotic force is generated.

Mechanotransduction

A major recent development is the growing recognition that the EGL serves a crit-ical role in the transmission of FSS to the actin cytoskeleton and in the initiationof intracellular signaling. Although numerous studies have demonstrated that FSSstimulates intracellular biomolecular responses and vascular regulation, it was widelyassumed that this signaling was initiated either at the base of the cell via focal adhe-sion complexes or by the direct action of FSS on proteins in the apical membrane.The theoretical models in Section 4 have led to a fundamental paradox because allthree models for the structural integrity of the EGL predict that the fluid flow withinthe layer is negligible and, consequently, the FSS at the level of the membrane isvanishingly small. Furthermore, recent experiments in which the structural integrityof the EGL is compromised clearly demonstrate that cytoskeletal reorganization andbiochemical responses can be nearly entirely abolished if the EGL is not intact. Thestudies described in this section convincingly show that the EGL plays a vital role inmechanotransduction.

Cytoskeletal reorganization in response to FSS. The basic premise in the elas-tohydrodynamic model for the EGL proposed in Weinbaum et al. (71) is that the


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core proteins in the bush-like structures in Figure 2 have a flexural rigidity, EI, thatis sufficient to withstand significant deformation owing to FSS in the physiologicalrange. If this is the case, the hydrodynamic drag on the tips of the core proteins atthe edge of the EGL will be transmitted as a bending moment to the ACC beneaththe apical membrane of the EC. This flexural rigidity allows the EGL to both serveas a molecular sieve for plasma proteins and as an exquisitely designed transducerof FSS. As noted earlier, Weinbaum et al. (71) and Han et al. (83) predict that EIfalls in the range of 500–700 pN nm2. For the upper value of EI, one finds that themaximum displacement of the tips of the fibers will typically be less than 10 nm for aFSS of 10 dyn/cm2. In marked contrast, this value of EI is inadequate to prevent thebuckling of the core proteins during the arrest of red cell motion or the passage ofa WBC. Weinbaum et al. (71) predict that the compressive elastic resistance of thecore proteins is at least a factor of 20 smaller than the hydrodynamic forces that arerequired to drain the fluid from the EGL.

Likewise, both the oncotic model of Secomb and coworkers (12, 13) and themechano-electrochemical model of Damiano & Stace (87) can account for the con-nection between the extracellular GAGs in the lumen and the EC cytoskeleton. Thiswould likely occur through connections between the GAG layer and an underly-ing macromolecular sublayer, where the sublayer would contain core proteins withtransmembrane and cytoplasmic domains that anchor the entire ESL to the EC cy-toskeleton. Tension in the thick GAG layer, induced by hydrodynamic drag arisingfrom plasma flow through the layer, would provide an effective means of transmittingFSS through the thin sublayer containing core proteins and into the EC cytoskeleton.Thus, the primary distinction between this mechanism of FSS transmission and thatof the elastohydrodynamic model for the EGL proposed by Weinbaum et al. (71)resides in the manner in which the EGL is thought to bear FSS. In contrast to thetheory of flexural rigidity of the core proteins proposed by Weinbaum et al. (71), theoncotic and mechano-electochemical models describe the EGL as being in a stateof axial tension, and through connections to underlying core proteins, the hydro-dynamic drag force on EGL GAGs pulls, to a greater or lesser extent, dependingon the prevailing flow conditions in the vessel, on cytoskeletal elements. In eithercase, these two mechanisms both predict that fluid drag throughout the EGL is con-verted into mechanical stress in core proteins and cytoskeletal elements. This is instark contrast to the conventionally held view that cytoskeletal stress arises from askin-friction mechanism that acts directly on an (EGL-free) EC lipid bilayer exposeddirectly to the flow. Such a view is not consistent with our new hydrodynamic under-standing of the EGL described earlier because fluid drag so severely attenuates flowthroughout the EGL that FSS on the surface of the EC is effectively eliminated.

To demonstrate the quantitative feasibility of the notion that the EGL might serveas a mechanotransducer of FSS, Thi et al. (75) have performed a series of experimentsto explore the role of the EGL in the reorganization of the actin cytoskeleton andjunction and focal-adhesion-associated proteins in response to FSS when the EGL iseither intact or compromised (by enzymatic degradation or by the absence of plasmaproteins) (19). Florian et al. (74) had previously shown that even partial removal of themost abundant proteoglycan, heparin sulfate, by heparinase III would entirely abolish


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DPAB: dense peripheralactin band

the increase in NO owing to FSS, but would have little effect on PGI2, which is be-lieved to be associated with basal adhesions. In particular, the redistribution of F-actin,vinculin, ZO-1, paxillin, and C × 43 in confluent rat fat pad ECs in response to 5 hof FSS in four different bathing media (DMEM, DMEM + 10% FBS, DMEM + 1%BSA, and DMEM + 1%BSA + Heparinase III) were examined. DMEM alone andDMEM with heparinase III were observed to substantially reduce the cell surfaceexpression of heparin sulfate compared to perfusion media with either BSA or FBS.

The most striking observations in these experiments were that with an intact EGLthere was a severe disruption of the dense peripheral actin band (DPAB), a formationof stress fibers, and a migration of vinculin to cell borders after exposure to FSS. Whilethis has been seen in numerous previous studies, this reorganization was completelyabolished when the integrity of the EGL was compromised. Similarly, there weredisruptions of the tight junctions (indicated by ZO-1) and gap junctions (indicatedby C × 43) for the intact EGL and no changes in the distribution of either of thesejunction-associated proteins when the EGL was compromised. In marked contrast,the distribution of paxillin, a marker for focal adhesions and FAK, was unaffected bythe integrity of the EGL. These results, which were obtained for a steady FSS of10 dyn/cm2, were substantially reduced for a FSS of 5 dyn/cm2 applied for the sameduration. This unequivocally demonstrated that the EGL was a transducer of FSSand that there was a threshold FSS for the cytoskeletal reorganization to occur.

The above results are explained in Thi et al. (75) in a “bumper-car” conceptualmodel to describe the role of the EGL in activating cytoskeletal reorganization (seeFigure 7). In Figure 7a, which describes controls without FSS, the DPABs act as rub-ber bumpers, which are held in lateral register (same elevation) by the weak attractiveforces of the VE cadherins at the level of the adherens junctions. This prevents thestiff DPABs from “denting” the more vulnerable parts of the EC, much like bumperson a car. When the cells are exposed to FSS for a substantial duration, the integratedbending moment owing to the drag on all the core proteins at the apical surface pro-duces a torque on the cell (clockwise for the direction of FSS shown in panel b) thatwould act to lower the front (right side) and lower the rear (left side) of the DPAB.The same rotation is occurring on the cells in front of and behind the central cell inthe sketch causing a disjoining force on the cadherin linkages between cells. Whenthis disjoining force exceeds the strength of the cadherin complexes, which has beenmeasured in Baumgartner et al. (111) as 70–120 pN, an unzipping of the adherensjunction occurs. The mathematical model (appendix C of Reference 75) predicts adisjoining force at the level of the DPAB of 70 pN for a FSS of 10 dyn/cm2. Thus,it is striking that when the FSS was reduced to 5 dyn/cm2, and the disjoining forcereduced to 35 pN, there was little or no cytoskeletal reorganization with an intactEGL. This model also appears to explain why cells orient in the direction of flow;by elongating their planform they are able to lengthen the lever arm of the DPAB,decrease the disjoining force on the adherens junction, and produce greater stabil-ity in response to flow. When this occurs a new DPAB will eventually form after asufficiently long duration of change in average FSS.

As shown in Figure 7b, the disruption of the DPAB leads to a redistribution ofF-actin and the formation of stress fibers on the basal aspects of the cell. In the


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Figure 7Sketch of the conceptual“bumper-car” model for thestructural organization ofthe EC in response to fluidshear stress. In its confluentcontrol state (a), ECs displayan intact dense peripheralactin band (DPAB) that islocalized to the adherensjunction, where it serves asthe base for the actincortical web (ACW).(b) Cytoskeletalreorganization in responseto fluid shear stress.Adapted from figures 4A,Bin Thi et al. (75).

transition to a new steady state, there is migration of vinculin to cell borders toestablish temporary focal adhesions at the periphery of the cell so that a confluentmonolayer is maintained. When the EGL is compromised, the FSS acts on the apicalmembrane directly and is transmitted by stress fibers connecting the apical and basalsurfaces to basal adhesion plaques. These stress fibers bypass the underlying ACC.The latter can be thought of as a geodesic dome that is supported in its interior by amore flexible actin network that is clearly seen in Satcher et al. (112). This dome-likestructure rests at its edges on the much stiffer DPAB at its circumference, and forthis reason, when the EGL is intact, the integrated torque serves to try to rotate the


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DPAB. In sharp contrast, the total force acting on the basal integrin attachments isthe same whether the FSS acts on the apical membrane or is transmitted across theapical membrane to the ACC. Thus, the distribution of paxillin, a marker for focaladhesions, is nearly the same whether the EGL is intact or degraded.

Biomolecular response to FSS. The primary evidence that supports a major rolefor the EGL in mechanotransduction comes from experiments in which enzymes wereused to selectively degrade specific components of the EGL, followed by a reassess-ment of function, or, as noted in the previous section, from using bathing solutionswithout plasma proteins where the EGL is compromised (19) and structural organi-zation examined in response to FSS. Florian et al. (74) used the enzyme heparinase IIIto selectively degrade the HS component of bovine aortic endothelial cell GAGs invitro and observed that the substantial production of NO induced over 3 h by steadyor oscillatory FSS (20 or 10 ± 15 dyn/cm2) could be completely inhibited by an en-zyme dose that removed only 46% of the fluorescence intensity associated with a HSantibody. The enzyme did not degrade CS and displayed negligible protease activity.It was also demonstrated that receptor-mediated NO induction by bradykinin andhistamine were not affected by the enzymatic treatment, demonstrating that eNOSactivity was not impaired directly by the enzyme.

In an earlier study, the enzyme neuraminidase was used to remove SA residues fromsaline-perfused rabbit mesenteric arteries, and it was observed that flow-dependentvasodilation was abolished by a 30-min enzymatic pretreatment (113). Because flow-dependent vasodilation is mediated by NO release in many arteries, this study sug-gested that SA also contributes to FSS-induced production of NO. Similarly, Heckeret al. (114) showed that when intact segments of rabbit femoral arteries were pre-treated with neuraminidase, FSS-induced NO production was inhibited. They alsodemonstrated that the same enzyme treatment had no effect on another hallmark re-sponse of ECs, the FSS-induced production of prostacyclin (PGI2) (115). This studyillustrated the fact that there are multiple mechanisms of mechanotransduction andnot a single mechanotransducer. In a more recent study, the enzyme hyaluronidasewas used in isolated canine femoral arteries to degrade HA from the ESL layer, anda significant inhibition of FSS-induced NO production was demonstrated (116).

A new in vitro study using bovine aortic endothelial cells showed that the en-zyme chondroitinase, employed to selectively degrade CS, did not inhibit the char-acteristic FSS-induced NO production, but treatment with either neuraminidase orhyaluronidase did (117). This study, and the earlier one by Florian et al. (74), illus-trated the specificity of the ESL GAG components in mechanotransduction becauseremoval of CS had no effect, whereas removal of HS and SA blocked the FSS-NOresponse. It was also shown that none of the three enzymes used had an inhibitoryeffect on FSS-induced PGI2 production, again suggesting multiple mechanisms ofmechanotransduction.

As described in the previous section, Thi et al. (75) exposed confluent monolayersof rat fat-pad ECs to 10 dyn/cm2 of steady FSS in a parallel plate flow chamber forfive hours and recorded the distribution of key structural proteins, such as F-actin,vinculin, paxillin, and ZO-1 using confocal microscopy. The experiments showed


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a dramatic reorganization of the DPAB and the actin-associated linker moleculevinculin in response to FSS when the EGL was compromised by either removingplasma proteins or enzymatic partial removal of HS. These results imply that HSlinked to transmembrane proteoglycans, such as the syndecans, mediates the FSS-induced reorganization of actin. One might infer from the results of Thi et al. (75) onactin reorganization and from Florian et al. (74) on NO production that experimentsin protein-free media would not display the characteristic FSS-induced NO produc-tion associated with an intact EGL. This, however, is not the case, as several studiesusing cultured cells in protein-free media have shown the FSS-induced productionof NO (118, 119).

One way of interpreting these seemingly contradictory studies is to realize thatwhen experiments are run in protein-free media, the hydraulic permeability of theEGL is increased as has been shown directly in transport experiments by Dull et al.(120) and Tarbell et al. (121) and the thickness of the ESL is decreased. This im-plies that a much greater fraction of the external FSS is transmitted to solid elementscloser to the membrane, and possibly as FSS directly on the plasma membrane. Onepossible scenario that is consistent with this view is that, at least in part, FSS-inducedNO production is mediated by eNOS in caveolae that may be stimulated throughthe solid matrix components of the EGL, such as the HS of glypicans near the mem-brane. While cytoskeletal reorganization appears to depend on transmembrane HSproteoglycans, the same distinction cannot be made for the NO signaling depen-dence on HS. Because NO transduction occurs regardless of where shear is felt, thecaveolae-associated membrane-bound glypicans and the plasma membrane itself arelikely sensors in protein-free media, whereas syndecans would appear to be moreimportant in physiologic media-containing protein.

The Distinction Between Mechanosensing and Mechanotransduction. Focus-ing on NO, we have considered events from the perspective of sensing force by theGAGs and transmission of force to the apical aspects of the cell—the plasma mem-brane or the cortical cytoskeleton. We turn now to mechanisms by which that forceis transduced into a biomolecular response. HS proteoglycans can be linked to boththe decentralized and centralized mechanisms of mechanotransduction put forth byDavies (122). In terms of the former, syndecans have an established association withthe cytoskeleton (55), and through it can decentralize the signal, by spreading it tomultiple sites within the cell (i.e., nucleus, organelles, focal adhesions, intercellularjunctions). In terms of central transduction, both syndecans and glypicans interactwith signaling proteins that can initiate cascades or directly influence eNOS activa-tion. For example, glypicans reside in caveolae along with a multitude of signalingmolecules, including eNOS. Simply by location, glypicans can be involved in anyone of a multitude of signal transduction pathways as elaborated in greater detail inTarbell & Pahakis (69).

HS may be involved in mechanotransduction through effects that are secondary toeNOS activation, but crucial to NO signaling. Nieuwdorp et al. (123) noted that lackof HS on the EC surface would result in loss of EC-SOD, an enzyme that catalyzes thereaction of superoxide to oxygen. This would result in a pro-oxidant state, damaging


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both the EGL and eNOS, which would lead to decreased NO availability (124). Alongthe same lines is the hypothesis that HS plays a role in the availability of arginine closeto the EC surface and its transporters. The supporting evidence for this comes fromseveral different sources. First, HS displays high affinities for polycationic molecules,such as poly-L-arginines, and the binding sites of all their ligands involve arginineresidues (41, 49, 63). Thus, HS proteoglycans may serve as a means of concentratingarginine close to the plasma membrane. In an older study, it was shown that HSproteoglycans adsorbed to a surface undergo a conformational change when exposedto flow, their core proteins unfolding from a random coil to an extended filament,and their HS chains elongating by 35% (125, 126). This was used to illustrate howNa+ ions bound to HS could be delivered by the stretched GAGs to their transporterchannels; an analogous hypothesis can be made for the case of L-arginine.

In sum, when the EGL is intact, FSS is transmitted to the cell through the coreproteins of the EGL, and the specific connections of these proteins to the actin cy-toskeleton (syndecans) and the plasma membrane (glypicans) mediate specific cellsignaling (e.g., NO production, cytoskeletal reorganization). However, this stress isalso distributed to other regions of the cell, most notably the intercellular junctionsand the basal adhesion plaques, where transduction to intracellular biochemical sig-nals also occurs (122, 127). When the EGL is degraded (e.g., by removal of protein) oractually removed (e.g., by the use of an enzyme such as heparanase), FSS is transmit-ted closer to the plasma membrane and apical signaling can proceed by mechanismsthat differ from those associated with the transmembrane core proteins of the EGL.However, mechanical equilibrium principles illustrated in Figure 7b insure that thestresses delivered to the basal adhesion plaques would be the same with or withoutan intact EGL, as indicated by the response to paxillin discussed earlier, suggestingthat signaling through these structures is independent of the integrity of the EGL.In other words, for a given FSS level, a basal adhesion plaque does not know if thecell has an intact EGL or not; it senses the same stress.

In trying to identify the mechanisms by which the EGL mediates mechanotrans-duction, one confronts a multitude of possibilities: (a) cytoskeleton-associated “decen-tralized” signaling, (b) “centralized” direct association of the EGL with intracellularsignaling molecules, and (c) regulation of local concentration gradients and transportof ions, amino acids, and growth factors. Taken together, these support the conceptof the EGL as an orchestrator of mechanotransduction.

Inflammatory Response and Ischemia–Reperfusion Injury

The presence of a 500-nm-thick EGL in postcapillary venules (27, 28, 87) has impor-tant implications for leukocyte recruitment and the inflammatory response cascade.In particular, we must reconsider conventionally held views about the mechanisms ofleukocyte capture from the free stream in postcapillary venules that neglect the EGL.Rolling leukocytes attach to blood vessel walls through the selectin family of adhesionmolecules, as noted in Section 5. These molecules typically extend ∼20–50 nm abovethe plasma membrane (128). The distance between the ligands on the leukocyte mi-crovilli and the adhesion receptor on the endothelial surface is very unlikely to exceed


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100 nm. Based on current estimates of the hydraulic resistivity and restoring forcesof the layer (13, 84, 87, 95) and the predictions of the penetration depth of microvilliin Zhao et al. (98; see Section 5), even long leukocyte microvilli will be unable topenetrate the EGL, unless the WBC is already tethered. Thus, de novo attachmentof leukocytes from the free stream (primary capture) is unlikely in vivo. Nevertheless,primary capture is the prevailing view of how leukocytes initially begin to roll inpostcapillary venules. Indeed, experimental evidence shows that primary capture isexceedingly rare in inflamed microvessels (129). When free-flowing leukocytes attachto the walls of larger venules, which is also a rare event, this occurs mainly throughsecondary capture, i.e., leukocyte-leukocyte interaction (130).

The gravitational amplification mechanism described in Zhao et al. (98; seeSection 5) applies primarily to free-rolling leukocytes flowing across EC monolay-ers in culture. In this mechanism there is a 20–100-fold amplification of the veryweak gravitational contact forces to typically 10 pN. As discussed earlier, forces ofthis magnitude are insufficient in vitro or in vivo to initiate selectin-mediated adhe-sion between the leukocyte and the endothelium except for very long microvilli atextremely low FSS where the contact time is greatly expanded. This is confirmedby the observation in vivo that tethered rolling does not preferentially begin on the“bottom” luminal surface of a postcapillary venule, but rather is uniformly distributedover the entire luminal surface (27, 93). The primary mechanism for tethered rollingpredicted in Reference 98 is the large penetration force that is generated once aninitial tethering event occurs. As noted in Section 5, the penetration force can in-crease to typically 100 pN for a FSS of as little as 1 dyn/cm2—a force that will easilyallow deep EGL penetration. This implies that once tethered rolling is initiated itwill readily continue. This same conclusion is reached in Reference 27.

It has been observed that the vast majority of rolling leukocytes attach at theentrance of postcapillary venules in vivo (99, 131, 132), where deformed leukocytesexit from capillaries with a diameter smaller than the resting leukocyte diameter. Thesignificant mechanical deformation of the EGL that arises as leukocytes squeeze outof the confined lumen of capillaries and enter into postcapillary venules brings theleukocyte membrane in close apposition to the capillary wall so that initial interactionsbetween selectin molecules and their counterligands can occur (27, 71). Previouswork has shown that the EGL is highly compressed by passing leukocytes (23, 83),and in tightly fitting capillaries, Han et al. (83) predict that this compression is closeto 80%.

There is some evidence that the EGL significantly changes its properties underinflammatory conditions (77, 133), which may facilitate sustained rolling and ad-hesion of leukocytes. In particular, cytokine-mediated activation of proteases eitherdwelling in the EGL or secreted by ECs and leukocytes may partially degrade thelayer and thus provide a mechanism for localizing and chemically regulating leukocyterecruitment preferentially to regions of inflamed vascularized tissue. In essence, then,a ubiquitous intact EGL might serve as a mechano-chemical barrier against leuko-cyte recruitment in uninflamed tissue. If such localized degradation is a necessaryprecursor to promote tethered rolling of leukocytes as they emerge from capillaries(primary rolling), then this would represent a critical early step in the inflammatory


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response cascade. Therapeutic interventions targeted at inhibiting EGL sheddingcould then provide effective regulation of inflammation. Thus, the mechanical com-pression of the EGL as leukocytes emerge from capillaries into postcapillary venulescombined with cytokine-mediated localized enzymatic degradation of the layer pro-vide a new paradigm by which leukocyte recruitment may occur in vivo. As statedearlier, the state of the EGL has not been reported in any of the studies of tetheredrolling in parallel-plate flow chambers. Furthermore, many of these studies are notperformed in plasma, where an intact EGL might be expected. Their objective haslargely been to examine the kinetics of selectin attachment to elucidate the specificityof a variety of ligand interactions.

Using the dye-exclusion method developed by Vink & Duling (23), Duling andcoworkers investigated the mechanism by which ischemia–reperfusion injury de-grades the EGL in capillaries and postcapillary venules of the mouse cremaster musclein vivo (134, 135). Platts et al. (134) showed that ischemia–reperfusion injury rapidlymodifies the EGL and results in a 50% decrease in the size of the dye-exclusion zone incapillaries and postcapillary venules, whereas the red blood cell exclusion zone showedlittle change. Pretreatment with the A2A agonist ATL-146e caused a significant atten-uation in the effect of ischemia–reperfusion injury on the EGL. Their results suggestthat adenosine A2A receptor activation may play an important role in protecting theEGL from damage due to ischemia–reperfusion injury and that the modification tothe EGL that they observed in response to this form of injury may represent animportant early step in the inflammatory response to ischemia–reperfusion. On theother hand, adenosine A3 receptor activation was found to cause a dose-dependentreduction in EGL thickness (136). Whereas at low concentrations, adenosine caninhibit EGL degradation and mast cell degranulation by activating the A2A receptor,at high concentrations, it can cause mast cell degranulation and lead to EGL degrada-tion by activating the A3 receptor. In a subsequent study, Rubio-Gayosso et al. (135)show that EGL degradation resulting from ischemia–reperfusion injury is mediatedby reactive-oxygen species. They suggest that the reactive-oxygen species are gener-ated by xanthine-oxidoreductase, an enzyme bound to the EGL, because inhibitingxanthine-oxidoreductase with allopurinol, or competing with it using heparin, essen-tially prevents the reduction of the dye-exclusion zone after ischemia–reperfusioninjury. Their results show that both heparin and hyaluronan have protective effectsagainst ischemia–reperfusion injury when their circulating concentration is increasedbefore the injury. This supports their hypothesis that during ischemia–reperfusioninjury, xanthine-oxidoreductase might bind to the EGL through its heparin-bindingdomain. After reperfusion, EGL-bound xanthine-oxidoreductase would then localizethe damaging effects of reactive-oxygen species production to the apical EGL. Finally,they show that this process may be dependent on the organization of hyaluronan in theEGL because intravenous administration of exogenous hyaluronan ameliorates thedeleterious consequences of ischemia–reperfusion injury. Thus, Rubio-Gayosso et al.hypothesize that reactive-oxygen species, produced as a consequence of ischemia–reperfusion injury, are a signal and not the direct mediator of the decrease in thethickness of the dye-exclusion zone, as other treatments that produce reactive-oxygenspecies, such as UV light, also modified the red blood cell exclusion zone. Taken


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together, the results of Duling and coworkers offer compelling evidence of a multitudeof physiological and pathophysiological roles for the EGL during inflammatoryinsults.

7. UNRESOLVED ISSUES AND FUTURE DIRECTIONS

We complete this review by highlighting some of the central unresolved issues andfuture research directions. A central challenge for future research is to understandthe relationship between the biochemical composition of the EGL and its ultra-structure, currently revealed by EM with its attendant artifacts. One of the puzzlingfeatures is that the EGL is substantially thinner when observed in EM comparedto functional in vivo measurements using intravital microscopy. This has suggestedan inner and outer organization of key proteins with a cleavage plane 50–100 nmfrom the apical membrane. The biochemical composition of the EGL is essential tounderstanding its enzymatic degradation. For example, why does partial removal ofselect components of the EGL (HS, SA, HA) completely block FSS-induced NOproduction? Is FSS-induced NO production mediated by force transmission throughsyndecans or glypicans, or alternatively by changes in the binding of key substrates(e.g., L-arginine) close to the plasma membrane? Why does the integrity of the EGLplay a different role in PGI2 and NO production in response to FSS? What are themechanisms via which reactive-oxygen species mediate damage to the EGL?

From a biophysical standpoint the central unanswered questions relate to themechanisms that provide for the structural integrity of the EGL in response to FSSand its restoration after cellular compression. Is this due to oncotic forces, inherentflexural rigidity of its fibers, electrochemical potential gradients, or some combi-nation of these and possibly other mechanisms? These forces also determine theEGL’s properties as a molecular sieve for plasma proteins. As seen in Zhang et al.(72), the oncotic forces generated in a fiber matrix layer should differ greatly fromthose in a membrane with cylindrical pores. This still needs to be demonstratedexperimentally. Although there is now strong evidence in support of the revised Star-ling principle for water exchange, the role of the EGL in transmitting FSS to thetight junction strands and in turn modulating small-solute permeability has not beenexplored.

It is striking that literally hundreds of papers have been written on tethered rollingon cultured ECs and on substrates with a host of different ligands and receptors, but nostudy has attempted to determine the importance of an intact EGL in either the initia-tion or maintenance of tethered rolling. It is not known whether the EGL is ubiquitousthroughout the microvasculature or if its distribution and relative uniformity are pref-erential to capillaries and postcapillary venules. This has important consequences forinflammation, mechanotransduction, vascular resistance, atherosclerosis, and othervascular diseases. It is safe to say the status of the EGL is still in its infancy and we haveonly scratched the surface in determining its structure and function. However, thefield is beginning to draw considerable attention as the scientific advances and volumeof literature written on this subject since 2000 far surpass cumulative contributionsof the previous 35 years since Luft’s clear demonstration of its existence.


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SUMMARY POINTS

1. The EGL is a complex, multicomponent, biochemical structure that func-tions exquisitely as a molecular sieve, a lubrication layer for red blood cellmotion, and a sensor of flow-induced FSS.

2. When observed in EM, the EGL is <100 nm thick and has a quasi-periodicstructure with focal scattering centers of 10–12 nm in diameter and 20 nmspacing. This extracellular structure appears to be linked to an under-lying actin cortical network that forms a hexagonal lattice with 100 nmspacing.

3. Two methods have been developed for interrogating the EGL using intra-vital microscopy: a fluorescent dye exclusion technique and μ-PIV. Theseapproaches have revealed that the EGL thickness in vivo in capillaries andvenules (5–50 μm in diameter) is ∼0.5 μm thick, which is at least fivefoldthicker than most EM studies indicate.

4. Theoretical models predict that the fluid velocity is greatly attenuated withinthe EGL and hence a negligible FSS acts on the EC membrane. Thus, allof the flow-induced FSS is transferred as a drag to matrix fibers.

5. When the EGL is crushed by the passage of a WBC or during the start-upmotion of a red cell in a tightly fitting capillary, it is restored to full thicknessin <1 s. Three models have been advanced for its restorative mechanismunder these conditions: oncotic forces owing to adsorbed plasma proteins,flexural rigidity of its core proteins, and electrochemical properties owingto its fixed negative charges.

6. The EGL is now believed to be the primary molecular sieve for plasmaproteins and, therefore, the origin of the oncotic forces that controltranscapillary fluid exchange. This has led to a revised Starling princi-ple in which the Starling forces are applied locally across just the EGL,rather than globally across the entire endothelial layer as had been widelyassumed.

7. When the EGL is intact, it serves as the primary sensor of FSS on ECs.The EGL transmits this stress to diverse sites within the cell (e.g., apicalplasma membrane, actin cortical web, basal adhesion plaques, intercellu-lar junctions) where mechanotransduction occurs and intracellular signal-ing is initiated. A “bumper car” model has been proposed for cytoskeletalreorganization.

8. The EGL is likely to play a critical role in the inflammatory response cas-cade, as it can inhibit primary leukocyte capture and systemic leukocyteextravasation and can provide a physical barrier that can regulate and local-ize leukocyte recruitment to inflamed vascularized tissue.


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ACKNOWLEDGMENTS

This work was supported by NIH grants R01-HL44485 (to S.W.), R01-HL57093(to J.M.T.), and R01-HL076499 (to E.R.D.). We thank Danielle Wu for help withcitations.

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Glycocalyx

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