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Hypothesis: arterial glycocalyx dysfunction is the first step in the atherothrombotic process

M.I.M. Noble, A.J. Drake-Holland, H. Vink
DOI: http://dx.doi.org/10.1093/qjmed/hcn024 513-518 First published online: 4 March 2008

Abstract

We present evidence that the 0.5 µm thick gel layer, lining the inner wall of healthy blood vessels, the glycocalyx, is the first line of defence against atherothrombotic disease. All blood vessel linings are coated with this gel, a highly negatively charged structure, rich in anionic sites mostly represented by the sialic acid moieties of glycoproteins and the sulphate and carboxyl groups of heparan-sulphate proteoglycans. Blood flow in arteries is associated with a shear stress at the glycocalyx, which signals the underlying endothelial cells to release nitric oxide (NO), an anti-atherogenic factor. Sites of low shear stress in the arterial tree are more susceptible to atheroma due to lack of NO generation through this mechanism, whereas exercise, by increasing blood flow and shear stress, is protective. We postulate that risk factors for atherothrombosis act by impairing glycocalyx function. That luminal hyperglycaemia causes glycocalyx dysfunction has already been shown; we postulate this to be the first step in the atherothrombotic process in patients with diabetes mellitus and metabolic syndrome (insulin resistance). There is also evidence of glycocalyx defects from exposure to oxidized low-density lipoprotein. We postulate that other risk factors will have a similar action on the glycocalyx as the initiating factor in the disease process, e.g. smoking, hyperlipidaemias and hyperhomocystenaemia. These predictions can now be tested in a large animal model of shear-stress-mediated arterial dilatation.

Introduction

In the late 60s and early 70s, Klitzman and Duling1 proposed that the vascular glycocalyx occupies a large volume fraction of capillaries, thereby causing limited capillary blood filling and low capillary haematocrits. Observations on capillary permeability were found not to fit the Starling hypothesis, leading Michel2 and others3,,4 to propose a new modification of the hypothesis, taking into account the presence of vascular gycocalyx. Vascular glycocalyx structure has been characterized, together with its behaviour with respect to handling of macromolecules and cells.5 These findings were derived from micocirculatory studies, but vascular endothelium also separates the vascular endothelial cells from the luminal blood throughout the circulatory system, i.e. not only in the microcirculation, but also in veins, and, of particular relevance to the present article, arteries. Our proposal is that damage to the luminal surface gel covering between the blood and the endothelial cells, allows the start of a progression of changes (e.g. by exposure of the endothelial cell layer to cell adhesion, thrombosis and inflammation) leading to atheroma.

Description of the glycocalyx

All blood vessel linings are coated with a gel, the vascular endothelial glycocalyx, a highly negatively charged structure, rich in anionic sites mostly represented by the sialic acid moieties of glycoproteins and the sulphate and carboxyl groups of heparan-sulphate proteoglycans; this gel is about 0.5 µm thick in capillaries.5 Proteoglycans interact via their trans-membrane core protein with the cell cytoskeleton,6 and electronmicroscopic study has revealed that the gel contains minute proteinaceous ‘hairs’ with such an attachment to the endothelial cell structure (Figure 1).

Figure 1.

Simplified diagram of arterial wall including the glycalyx layer between the blood and the endothelial cells, 0.5 µm thick.

From this standpoint, Weinbaum et al.6 were able to postulate that the glycocalyx acted as a mechanotransducer allowing endothelial cells to ‘sense’ changes in adjacent blood flow and shear stress. Several other groups have contributed to the concept that the glycocalyx may play a role as mechano-transducer of fluid shear stress.7–11

Comment on methods used by other investigators to demonstrate vascular glycocalyx structure and function

The results of visualization of the glycocalyx by electron microscopy are radically dependent on the pretreatment received by the preparation. For instance, a ‘control’ electronmicrograph of a coronary artery from a Krebs-Henseleit perfused heart shows a glycocalyx thickness of only 0.3 µm.11 In contrast, intravital microscopy of cremaster microvessels shows a glycocalyx thickness of 0.5 µm.12 We are of the opinion that conclusions about function should be drawn from intact animals without tissue processing whenever possible, but at least in blood perfused preparations. It has been demonstrated that microvascular resistance of rat mesenteric tissue is reduced by enzymatic damage of the glycocalyx.13 The importance of the volume of glycocalyx gel occupying the lumen of arterial resistance vessels was demonstrated in an open chest coronary perfusion model in which the administration of hyaluronidase reduced minimal microvascular resistance in goat heart: this is consistent with the observations that hyaluronidase reduces glycocalyx volume in myocardial capillaries. The volume of glycocalyx can be measured by using fluorescent dextrans of two molecular weights, one which penetrates the glycocalyx and one which is confined to plasma, the in vivo volume of the glycocalyx can be measured by subtraction of volumes of distribution.14 Such estimates correlate with the estimate of 0.5 µm for glycocalyx thickness.

The consideration concerning the need to use intact animals also applies to the choice of method for assessing shear stress-induced arterial dilatation. This is clear in view of the finding of Jacob et al.,11 that isolated perfused in vitro preparations are to be avoided, as glycocalyx structure and function is dependent on normal albumin concentration. This over-riding consideration has decided the choice of the in vivo iliac artery preparations as used by Kelly et al.10

Kelly et al.10 prepared the pig iliac artery (Figure 2) with two segments in series divided by a snare; there was also a distal snare. Each segment diameter was independently measured by sonomicrometry with pairs of piezo-electric crystals. Blood flow through the artery was measured by transit time ultrasonic flowmeter and shear stress calculated from flow and diameter. A change in shear stress was achieved by closing and opening the distal snare to produce reactive hyperaemia, and the response of artery diameter recorded. It was confirmed that this response was mediated by the glycocalyx by treating the distal (test) segment only with hyaluronidase, which abolished the response of that segment, while the response in the proximal (control) segment to increased shear stress, and the responses of both segments to acetylcholine were preserved. Treatment of the animal with l-NAME [N (G)-nitro-l-arginine methyl ester, an inhibitor of NO production] abolishes all shear stress and acetylcholine responses, confirming the arterial dilatations in these circumstances are mediated by nitric oxide (NO) production by the endothelial cells, and, in the case of shear stress response, transduced by the glycocalyx.15 These authors used a method that allows properly quantified arterial diameter (from sonomicrometry) vs. shear-stress (calculated from blood flow and diameter) relationships.15 This more sophisticated preparation should now be used in studies of the effects of various risk factors for atherothrombosis upon the shear stress-induced arterial dilatation.

Figure 2.

The basic response for studying the function of the glycocalyx in transducing arterial shear stress, using the iliac artery of the anaesthetized pig. At the arrow, acetylcholine was injected downstream into the femoral arterial bed, producing a fall in peripheral resistance so that blood flow (top trace) increases, causing an increase in shear stress (middle trace). Nitric oxide (NO), produced by the endothelial cells is detected by the arterial smooth muscle, which relaxes, causing an increase in arterial diameter (bottom trace), with a lag time required for NO production. As shear stress is inversely related to diameter, this dilatation causes shear stress to fall before the blood flow. This response is abolished (i) by L-NAME, showing that it is NO mediated, (ii) by hyaluronidase, which disrupts the gel structure of the glycocalyx and (iii) by luminal hyperglycaemia, interacting with glycocalyx glycoproteins.

Shear stress-induced arterial dilatation

The mechano-transducer hypothesis applied to arteries leads to the further postulate that an increase in shear stress transduced by the mechanism triggers NO release from the endothelial cells leading to relaxation of arterial smooth muscle, and therefore, arterial dilatation. NO release from endothelial cells is known to be the mediator,16 from the fact that shear stress-induced arterial dilatation is inhibited by l-NAME in animals17 and man.18 Evidence compatible with this idea was shown by the fact that impairment of flow dependent dilation has been shown in rabbit femoral and mesenteric arteries by neuroaminidase degradation of the sialic acid residues of endothelial glycocalyx.8 In addition, NO production by cultured endothelial cells in response to fluid shear stress is inhibited after enzymatic removal of heparan sulphate proteoglycans.19 More substantial evidence comes from the study of Mochizuki et al.9 which showed abolition of shear stress-induced arterial dilatation by hyaluronidase, a finding which has been confirmed by Kelly et al.10 Shear stress-induced arterial dilatation has been fully characterized;15 there is a threshold value of shear stress below which no dilatation occurs, thereafter the stimulus/response curve is approximately linear, backward and forward flow are equally effective, pulsatile flow has little additional effect over and above the effect of mean flow, the dilatation is accompanied by a decrease in arterial compliance. A recent review20 ignores the role of the glycocalyx in mechanotransduction and concentrates only on mechanical changes of the endothelial cell membrane and cytoskeleton. The latter are postulated to be involved by ourselves in that the fine proteinaceous ‘hairs’ that protrude from the endothelial cells into the glycocalyx (as seen by intravital electron microscopy12), are connected to the endothelial cell cytoskeleton and are presumed to trigger the NO production. However, the evidence from hyperglycaemia and hyaluronidase experiments points to a failure of this mechanism if the gel constituents are altered.

Chronic effects of increased shear stress

Continuation of elevated fluid shear stress levels in collateral vessels allows for a continued adaptation and increases in conductance up to 200% of baseline.21 We propose that this long-term effect is dependent on the shear stress response of the glycocalyx, and will be impaired in those pathological conditions, e.g. diabetes mellitus, in which there is glycocalyx damage (see below). Evidence compatible with this idea is the neuroaminidase degradation of the sialic acid residues of endothelial glycocalyx,8 mentioned earlier.

Effect of increased glucose concentration on glycocalyx structure and function

Hyaluronidase is a rather drastic intervention entailing enzymatic digestion of the glycocalyx gel. However, its effect raises the possibility that mechano-transduction is not simply achieved through the proteinaceous ‘hairs’, but involves the mechanical structure of the gel. A more subtle way of influencing this structure was postulated to be hyperglycæmia, because of the glycoproteinaceous nature of the glycocalyx with which glucose interacts. Nieuwdorp et al.18 showed that systemic hyperglycæmia caused generalized glycocalyx thinning, and confirmed that it was accompanied by impaired flow-dependent arterial dilatation in man. Shear stress-induced arterial dilatation is attenuated or abolished when the glucose concentration in the lumen only was increased for 20 min.10

The glycocalyx and risk factors for atherothrombosis

Research results are beginning to emerge from experiments in which risk factors for atherothrombotic disease have been applied to different experimental glycocalyx preparations. These are summarized in Table 1.

View this table:
Table 1

Summary of publications showing the effects of risk factors for atherothrombosis upon the arterial glycocalyx and/or its function

Risk FactorEffectReference
Low shear stress areasBelow thresholdCaro et al.22, Kelly and Snow15, Arisaka et al.23
Hyperglycaemia(i) Abolition of shear responseKelly et al.10
(ii) DamageNieuwdorp et al.24
DiabetesDamageNieuwdorp et al.18
Hyperlipidaemia(i) DamageVink et al.25
(ii) Capillary resistance decreaseConstantinescu et al.26
(iii) Leucocyte adhesionConstantinescu et al.27

Hyperglycæmia, found in diabetes mellitus and non-diabetic insulin resistance (‘metabolic syndrome’), is one of many risk factors for atherothrombosis. Patients at risk from this cause may be normoglycaemic, but can be detected by measurement of the blood glucose × insulin product.28,,29 Another risk factor, which has been studied and shown to impair vascular glycocalyx is the ratio oxidized lipoproteins/LDL.25 This group also showed that the glycocalyx modulates immobilization of leucocytes at the endothelial surface.27 We are also aware of planned studies on hyperinsulinaemia, hyperhomocysteinaemia and smoking, which are awaiting funding. It remains for these studies to be carried out, as well as studies of the effects of lipoprotein(a), hypertension, etc.

Role of glycocalyx in the initiation of atherothrombosis

The vasculoprotective properties of the endothelial glycocalyx have previously been emphasized.30 Atheromatous lesions are more common at sites within the arterial system which have low shear rates.22 Endothelium at these sites are therefore, releasing less NO, particularly if the shear rates are below threshold. The mechanism, via the glycocalyx and various endothelial intracellular pathways, leading to eNOS induction and NO release, have recently been reviewed in Kelly's PhD thesis, 2006 (University College Cork, Ireland). Relatively high shear stress (protective) may suppress atherogenesis by altering the synthesis of glycosaminoglycans.23,,31 NO is an anti-atherothrombotic factor (atheroprotective), so its absence in low shear stress sites of the arterial bed could explain the lesion distribution found by Caro.22 Exercise produces an increase in blood flow and shear stress throughout the arterial tree, particularly in coronary arteries and central arteries supplying the limbs. The increased NO production resulting from this will have an anti-atheromic effect. Glycocalyx degradation allows platelet and leucocyte adhesion to the endothelial cells,27 if unopposed by NO, which will set up an inflammatory reaction. Glycocalyx degradation also leads to sub-endothelial chilomicron accumulation.32 The combination of inflammation and lipid accumulation sets the stage for the eventual formation of the full atherothrombotic lesion. We can therefore, conceive of the normal glycocalyx as an essential barrier to vascular disease.

Diabetes mellitus or metabolic syndrome/insulin resistance may be a cardiovascular disease in which the damage to the vascular glycocalyx could be of particular importance, following from the fact that mere hyperglycaemia causes loss of glycocalyx function followed by glycocalyx thinning and subsequent disease of the underlying vascular tissue. However, in these diseases there is microvascular as well as arterial disease. The association between compromized glycocalyx and microvascular disease in diabetic patients18 might be explained as a consequence of the effect of glucose on glycocalyx throughout the vascular system.

We conclude that arterial glycocalyx dysfunction could be the very first step in the atherothrombotic process. We would point to the importance of testing this hypothesis: (i) in physiologically relevant models that simulate the human situation; (ii) by studying the effect of risk factors and potential treatments and (iii) preventative measures.

It may be necessary to overcome the present prejudice against large animal experiments in order to achieve such objectives.

References

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