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Degradation of the endothelial glycocalyx by atherogenic factors. Microvascular
functional implications
Constantinescu, A.A.
Publication date
2002
Link to publication
Citation for published version (APA):
Constantinescu, A. A. (2002). Degradation of the endothelial glycocalyx by atherogenic
factors. Microvascular functional implications.
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ChapterChapter 7
Generall Discussion
7.11 Endothelial dysfunction in atherogenesis
Althoughh atherosclerotic cardiovascular disease represents the leading cause off morbidity and mortality in the Western societies, the understanding of itss pathogenesis remains incomplete, as well as the modalities to prevent its complicationss 11,2]. From today's perspective, atherosclerosis represents an inflammatory-proliferativee response of the artery wall to various forms of injuryy induced by risk factors such as hypercholesterolemia, hypertension, diabetess mellitus, or tobacco use [3,4]. The response-to-injury hypothesis off atherosclerosis implicates endothelial dysfunction as the first step in de-velopmentt of atherosclerosis [3,4].
AA large body of evidence indicates that atherogenic risk factors induce endotheliall cell activation, which is characterized by increased expression off adhesion molecules, increased formation of oxygen radicals, and de-creasedd production of nitric oxide [3-8]. The result is endothelial dysfunc-tion,, which entails adhesion of leukocytes and platelets, disruption of the endotheliall permeability barrier, alteration of vascular tone and induction off procoagulant instead of anticoagulant properties of vascular endothe-liumm [3-8].
Thee purpose of this thesis was to test the hypothesis that alteration of endotheliall function by atherogenic risk factors involves degradation of thee endothelial glycocalyx. This hypothesis was formulated considering thee implication of the glycocalyx in several physiological aspects of blood-endotheliumm interaction, which represent in turn the aspects that are al-teredd during development of endothelial dysfunction. Our studies provide evidencee that the endothelial glycocalyx is indeed modified by atherogenic riskk factors. In this chapter, our novel findings will be discussed in relation-shipp with the established mechanisms of endothelial dysfunction.
7.22 Degradation of the endothelial glycocalyx by
Ox-LDL L
Elevatedd levels of low density lipoprotein (LDL) cholesterol represent a ma-jorr risk factor for atherosclerosis. Furthermore, it is generally accepted that atherogenicc properties of LDL are enhanced by oxidative modification. Ox-LDLL has been found in atherosclerotic lesions [9] and in plasma of patients withh coronary artery disease [10,11]. The effect of LDL oxidation on en-dotheliall cells has been investigated in many in vitro and in vivo studies, whichh have indicated that OX-LDL can aggressively induce endothelial dys-function.. OX-LDL can stimulate leukocyte-endothelial adhesion and ex-pressionn of adhesion molecules, alter the endothelial permeability barrier,
j.2j.2 Degradation of the endothelial glycocalyx by Ox-LDL 135 inhibitt endothelium-dependent dilation, and induce procoagulant
proper-tiess and apoptosis in endothelial cells [12-23].
Inn agreement with the evidence that OX-LDL induces endothelial dys-function,, we found that OX-LDL in a clinically relevant dose decreased the thicknesss of the endothelial glycocalyx. Our approach consisted of systemic injectionn of OX-LDL in the hamster circulation, which resulted at 20-30 min inn a decrease by approximately 50 % of the luminal domain of the endothe-liall glycocalyx in cremaster muscle capillaries. Concomitantly, we observed increasedd adhesion of platelets to the endothelial surface. Both effects were abolishedd after administration of SOD and catalase, and this was in agree-mentt with other reports showing that OX-LDL effect is mediated by oxygen free-radicalss [12,15].
Wee pursued the effect of OX-LDL by investigating whether the decreased glycocalyxx thickness was the result of glycocalyx compression by poten-tiallyy increased hemodynamic shear stress forces, or represented a struc-turall alteration of glycocalyx constituents by OX-LDL. Capillary red cell ve-locityy did not change after OX-LDL administration, however capillary tube hematocritt increased by two-fold in parallel with the decrease in glycoca-lyxx thickness. Furthermore, we increased the state of oxidation of OX-LDL, butt we found no difference between the effects of severely oxidized as com-paredd to moderately oxidized molecules. These results led us to conclude thatt early stages of modification of LDL molecules are sufficient to induce glycocalyxx alteration, which appears to be structural, because it occurs in-dependentlyy of hemodynamic changes.
Severall consequences of glycocalyx alteration for OX-LDL induced en-dotheliall dysfunction will be discussed further. In the first place, given thee rapid time course in which glycocalyx thickness decreased and recov-ered,, we proposed that the adsorption of plasma proteins to the endothe-liall surface was transiently impaired in the presence of OX-LDL. Given the rolee of plasma proteins in the consolidation of the glycocalyx matrix as aa molecular filter [24], an impaired protein adsorption may contribute to OX-LDLL induced alteration of the endothelial permeability barrier. Further-more,, the deleterious effect of OX-LDL on glycocalyx matrix may also extend overr the heparan sulfate proteoglycans (HSPGs), as reported in cell culture studiess [25,26]. Degradation of HSPGs, which represent an abundant con-stituentt of the endothelial glycocalyx, may determine a considerable reduc-tionn of glycocalyx thickness [27].
Inn the second place, a relevant aspect for endothelial dysfunction is thee relationship between glycocalyx degradation and production of oxy-genn free-radicals. Administration of SOD and catalase completely prevented glycocalyxx degradation, indicating that the OX-LDL effect was mediated by
oxygenn free radicals. However, as we hypothesized in chapter 3, a pos-itivee feedback between glycocalyx degradation and formation of oxygen radicalss may occur in response to OX-LDL. In support of this hypothesis it mustt be noted that HSPGs of the glycocalyx are responsible for the localiza-tionn of endogenous SOD at the endothelial surface [28]. Thus, degradation off HSPGs may facilitate production of oxygen radicals due to a decreased availabilityy of scavengers at the endothelial surface. Furthermore, the par-allell increase in capillary tube hematocrit and red cell flux may determine aa proportional increase in capillary oxygen content, which may become an additionall source of free radicals during exposure to OX-LDL.
Inn the third place, platelet adhesion to the endothelial surface increased inn the presence of OX-LDL in parallel with degradation of the endothelial glycocalyx.. Although platelet activation by OX-LDL cannot be excluded, itt is probable that the non-thrombogenic nature of the endothelial surface wass altered by glycocalyx degradation.
7.33 Degradation of the endothelial glycocalyx
stimulatess leukocyte-endothelial adhesion
Ann important early feature of endothelial dysfunction in atherogenesis is adhesionn of leukocytes to the endothelial surface. Leukocyte - endothelial adhesionn is a process driven by adhesion molecules. In this respect, many reportss have shown that OX-LDL and early stages of hypercholesterolemia increasee expression and activation of adhesion molecules on both leuko-cytess and endothelial cells [13,16,17,29,30].
Thee role of the endothelial glycocalyx in the leukocyte adhesion process iss less well understood, although it is generally accepted that the natural non-adhesivenesss of the endothelial surface is maintained by the negatively-chargedd glycocalyx coat. One of the aims of our study was to test the hy-pothesiss that degradation of the endothelial glycocalyx contributes to en-hancedd leukocyte adhesion. We found that local microperfusion with hep-aritinase,, an enzyme that specifically degrades HSPGs of the glycocalyx, didd not affect leukocyte rolling, but induced firm adhesion of leukocytes to endotheliall cells to a similar extent as a clinically relevant dose of OX-LDL. Thiss finding indicates that primary degradation of the endothelial glycoca-lyxx may be in itself a trigger for the activation of endothelial cell adhesion moleculess or may facilitate leukocyte - endothelium interaction by reduc-ingg electrostatic repulsion between cells. On the other hand, the endothe-liall glycocalyx appears to be neutral with respect to leukocyte rolling, and thiss finding is in agreement with the positioning of L-selectins on the tips off microvilli, which are approximately 0.5 um long and appear to be able to
7.4.7.4. Glycocalyx degradation during diet-induced hyperlipidemia 137
penetratee the glycocalyx at shear rates existent in venules [31].
Withh respect to the clinical relevance of glycocalyx degradation, its con-tributionn to OX-LDL induced l e u k o c y t e - e n d o t h e l i a l cell adhesion is of in-terest.. OX-LDL is k n o w n to induce expression and activation of P-selectin, L-selectin,, I C A M - I , ICAM-2, and C D i i / 1 8 [13,16,17]. Therefore it is difficult too differentiate between a direct effect of OX-LDL on adhesion molecules, a n dd an effect mediated by glycocalyx degradation. We attempted to locally counteractt the effect of OX-LDL by consolidating the endothelial glycocalyx byy intraluminal microinjections of heparan-sulfate (HS). The n u m b e r of ad-herentt leukocytes elicited by OX-LDL w a s significantly lower in the venules microperfusedd with HS. However, HS has binding sites for leukocyte L-selectins,, a n d injected HS may inhibit leukocyte rolling by competing with L-selectinn binding to endothelial ligands [32]. In our experiments, injected HSS inhibited leukocyte rolling before OX-LDL administration, b u t not after exposuree to OX-LDL. We concluded that the effect of HS on leukocyte rolling, whichh was probably compensated by up-regulation of rolling molecules af-terr administration of OX-LDL, could not solely account for the decrease in thee n u m b e r of firm adhesion events. We p r o p o s e d therefore that binding off HS to the endothelial cells m a y partially re-constitute the glycocalyx, and therebyy m a y decrease firm leukocyte adhesion. However, additional exper-imentss are needed to assess by visualization the binding of injected HS at thee endothelial surface.
AA role of endothelial glycocalyx degradation in leukocyte adhesion in hypercholesterolemiaa is supported by the finding that capillaries with lipid depositss and decreased glycocalyx thickness of hyperhpidemic mice pre-sentedd spontaneous leukocyte adhesion. Thus, although leukocyte - endo-theliall cell adhesion is the result of interaction between adhesion molecules, thee endothelial glycocalyx has the potential to m o d u l a t e this process.
7.44 Degradation of the endothelial glycocalyx during
diet-inducedd hyperlipidemia
7.4.11 Subendothelial accumulation of chylomicrons
Previouss electron microscopy reports on endothelial surface changes dur-ingg diet-induced hypercholesterolemia showed decreased thickness of the endotheliall glycocalyx a n d altered binding of lectins in vascular areas sus-ceptiblee to turbulent h e m o d y n a m i c conditions [33-35]- Consistently, in the presentt study intravital microscopy examination of the luminal domai n off the glycocalyx in cremaster muscle capillaries indicated a considerably reducedd thickness of the endothelial glycocalyx after administration of a
high-cholesteroll diet. Furthermore, intravital microscopy allowed visual-izationn of extravasated lipoproteins in the subendothelial space of capillar-iess with reduced glycocalyx thickness.
Thee endothelial glycocalyx is considered to play a major role in the molecularr sieving properties of vascular endothelium [24]. Therefore, it iss probable that glycocalyx degradation is associated with alteration of the endotheliall permeability barrier. An altered endothelial barrier function is ann early feature of atherosclerosis, leading to lipid accumulation and for-mationn of atherosclerotic plaques in large vessels [3,4]. Alteration of the endotheliall barrier has been reported to occur after exposure of endothelial cellss to OX-LDL, hypercholesterolemia or lipolysis products [18,36-38].
InIn the present study, degradation of the endothelial glycocalyx during diet-inducedd hypercholesterolemia was found to be associated with suben-dotheliall accumulation of chylomicrons in capillaries. Subendothelial re-tentionn of chylomicrons was observed also in larger microvessels, i.e. arte-rioless and venules. The endothelial glycocalyx was not measured in these vessels,, because the intravital microscopy assessment of glycocalyx thick-nesss is less accurate in larger vessels [39]. We aimed to differentiate be-tweenn endothelial glycocalyx changes during mild hypercholesterolemia in C57BL/66 mice as compared to severe hypercholesterolemia and extensive atherosclerosiss in ApoE3-Leiden mice [40,41]. However, we found that in bothh groups of mice the decrease in glycocalyx thickness was similar in capillariess with subendothelial chylomicrons, irrespective of the severity of hypercholesterolemia.. Nevertheless, the proportion of affected capillaries increasedd with the duration of high cholesterol diet.
Inn chapter 5, we proposed that a positive feedback may have occurred betweenn glycocalyx degradation and subendothelial retention of chylomi-crons.. Subendothelial presence of chylomicrons was associated with an inflammatoryy reaction indicated by the presence of phagocytotic cells and increasedd leukocyte-endothelial cell adhesion. Thus, degradation of the endotheliall glycocalyx, which is initiated probably during early stages of hypercholesterolemiaa [29,33-35], m ay t*e amplified by cytokines produced duringg the inflammatory reaction associated with accumulation of chylomi-cronss [42]. The resulting ongoing alteration of the endothelial permeability barrierr associated with degradation of the endothelial glycocalyx may be involvedd in early as well as in advanced phases of atherogenic endothelial dysfunction. .
Itt remains to be established whether subendothelial accumulation of li-poproteinss at the capillary level is limited to striated muscle or pertains alsoo to areas of major interest during atherogenesis, such as myocardial microvessels.. Furthermore, it is of interest to investigate whether
chylomi-J4J4 Glycocalyx degradation during diet-induced hyperlipemia 139 cronss contribute to the formation of atherosclerotic plaques in large arteries.
Thee concept of postprandial hyperlipidemia as being atherogenic has been postulatedd decades ago [43]. In support of this concept, it has been shown thatt fluorescently-labelled chylomicron remnants penetrate and accumu-latee in the vessel wall of isolated arteries [44,45]. On the other hand, it has beenn shown that small lipoproteins are more atherogenic than larger ones. Accordingly,, at a given cholesterol level, large numbers of small apoBioo-containingg lipoproteins are more atherogenic than lower numbers of large apoBiooo lipoprotein particles [46]. Thus, whether subendothelial accumu-lationn of large chylomicrons during hypercholesterolemia is a feature lim-itedd to microvessels or contributes also to the formation of atherosclerotic lesionss in large arteries remains to be investigated.
7.4.22 Impaired regulation of perfused capillary volume
Thee dynamic nature of the endothelial glycocalyx has been associated with thee physiological variability of capillary tube hematocrit with the vasomo-torr state [47-49]. Capillary tube hematocrit, as a measure of functionally perfusedd capillary volume, increases by vasodilator stimuli and decreases duringg vasoconstriction [47,49,50]. It has been proposed therefore that a physiologicall variation of the endothelial glycocalyx occurs with vasomo-torr state [47,51]. We showed in chapter 6 that the increase in capillary tube hematocritt by endothelium-dependent and independent vasodilators was associatedd with a reversible increase in the penetration of anionic dextran macromoleculess of 7okDa into the luminal glycocalyx domain. Because thee thickness of the endothelial glycocalyx remained unchanged, we con-cludedd that an increase in plasma flow in the glycocalyx matrix accounted forr a more than two-fold increase in capillary tube hematocrit in response too vasodilators. Thus, while occupancy for a distance of 0.5 pm of blood vessell lumen by the endothelial glycocalyx has been shown to contribute significantlyy to microvascular blood flow resistance [52-55], the increase in plasmaa flow in the glycocalyx matrix may contribute to the reduction of floww resistance in response to vasodilators.
InIn contrast, the variability of capillary tube hematocrit and the endothe-liall glycocalyx with vasodilator stimuli was abolished in capillaries with chylomicronn deposits of hyperlipidemic ApoE3~Leiden mice. Instead, a sustainedd elevation of capillary tube hematocrit paralleled the decrease in glycocalyxx thickness for the whole range of measured red cell velocities. Furthermore,, the sieving properties of the endothelial glycocalyx were con-siderablyy altered, allowing anionic dextran of 70 kDa to penetrate the en-tiree luminal domain of the glycocalyx in control conditions, without be-ingg influenced by the administration of vasodilators. We concluded that
pathologicall glycocalyx degradation in hypercholesterolemia consists of sustainedd and severe decrease in glycocalyx thickness and sieving prop-ertiess to macromolecules, while physiological variability of the endothelial glycocalyxx is reflected in reversible increases in glycocalyx matrix perme-abilityy to large anionic macromolecules in response to vasodilators.
7.55 Perspectives for endothelial glycocalyx
investigationn in large vessels
Intravitall microscopy measurement of the endothelial glycocalyx in cre-masterr muscle capillaries offers important information regarding dynamic glycocalyxx changes in vivo in relationship with vascular endothelial func-tion.. Nevertheless, as intravital microscopy of the endothelial glycocalyx iss restricted to transparent tissues and is less accurate with increasing ves-sell diameter, additional methods have to be used for the assessment of the endotheliall glycocalyx in aorta, large vessels or myocardial tissue during developmentt of atherosclerosis.
AA new approach developed by Van den Berg et al. for stabilization of glycocalyxx structures during preparation for electron microscopy (EM) ap-pearss to solve the discrepancy regarding endothelial glycocalyx thickness measuredd by EM as compared to intravital microscopy [56]. This approach consistss of instantaneous fixation-staining of endothelial glycocalyx carbo-hydratess by Alcian Blue 8GX in a flow-controlled perfusion system, and allowedd for visualization of endothelial surface structures of 0.5 um in my-ocardiall capillaries (figure 7.1). The use of this new method in future EM studiess may allow extrapolation of glycocalyx measurements to large ves-selss and myocardial tissue.
AA confocal microscopy method for evaluation of endothelial glycocalyxx dimension and permeability properties in isolated small arteries (d -1500 um) has recently been developed by Van Haaren et al. [<jj]. This method indicatess that the FITC dextran of 148 kDa is excluded from a distance of 22 um from the endothelial surface in small mesenteric arteries. The dex-trann exclusion zone is destroyed by light-dye treatment within minutes, indicatingg that a thick endothelial surface layer contributes to the barrier permeabilityy to solutes in small arteries.
Heparinn challenge has been used recently by Van Teeffelen et al. to re-leasee plasma-derived factors bound within the endothelial glycocalyx and too assess the consequences for flow mediated arteriolar dilation [58]. It was foundd that arteriolar dilation was diminished after heparin administration, andd it was concluded that binding of plasma factors within the glycocalyx modulatess nitric oxide contribution to flow induced vasodilation.
Conclusions 141
Figuree 7.1: Electron microscopic image of a thick endothelial glycocalyx extending moremore than 0.5 urn from the endothelial surface into the lumen of rat myocardial capillariescapillaries after instantaneous fixation - staining with Alcian Blue 8GX. Courtesy ofof Dr. Bernard van den Berg, University of Amsterdam.
Thee synthesis and catabolism of membrane-attached glycocalyx struc-tures,, i.e. proteoglycans, glycosaminoglycans, glycoproteins, can be inves-tigatingg in cultured endothelial cells derived from large vessels. Using this method,, Gouverneur et al. [59] showed that exposure of endothelial cells to OX-LDLL impairs biosynthesis of membrane-bound sulfated proteoglycans.
Thus,, a variety of methods can be used complementary to intravital microscopyy in the future to provide more insight into endothelial glycoca-lyxx changes with relevance for endothelial dysfunction and development off atherosclerotic lesions in large vessels.
7.66 Conclusions
Thiss thesis investigated the in vivo domain of the endothelial glycocalyx in thee microcirculation during exposure to atherogenic factors. Although the endotheliall glycocalyx has a key position at the interface between blood andd the endothelial cells, the contribution of glycocalyx modification to en-dotheliall dysfunction has drawn less attention in the past. The studies pre-sentedd here indicate that oxidized lipoproteins and diet-induced
hypercho-lesterolemiaa degrade the endothelial glycocalyx. Glycocalyx degradation hass important consequences for vascular endothelial function, being asso-ciatedd with increased endothelial adhesiveness to leukocytes and platelets, alterationn of permselectivity to large macromolecules and subendothelial accumulationn of lipoproteins. Based on these findings it can be concluded thatt the endothelial glycocalyx is an important component of the vascular wall,, which one must not overlook when searching for new strategies to improvee vascular endothelial function and to fight atherogenesis.
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