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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 2
Oxidizedd lipoproteins degrade the
endotheliall surface
layers-Implicationss for
platelet-endotheliall cell adhesion
Hanss Vink, Alina A. Constantinescu, and Jos A.E. Spaan
488 Chapter 2. Hans Vink—Ox-LDL degrades the endothelial surface layer
Abstract t
Backgroundd Flowing erythrocytes and platelets are separated from the
lu-minall endothelial cell (EC) surface by a 0.5 um wide spacing named the en-dotheliall surface layer. We hypothesized that disruption of the EC surface layerr by oxidized low density lipoproteins (OX-LDL) contributes to athero-genicc increases in vascular wall adhesiveness.
Methodss and Results The hamster cremaster muscle preparation was used
forr intravital microscopic observation of the distance of red blood cells to thee capillary EC surface. Moderately oxidized LDL was prepared by expo-suree of native LDL to CuS04 for 6 hours. The dimension of the EC surface
layerr averaged 0.6 0.1 um during control, but bolus intravenous injection off OX-LDL (0.4 mg/100 g BW) transiently diminished the EC surface layer by 600 % within 25 minutes which correlated with a transient increase in the numberr of platelet-endothelial adhesions. Combined administration of su-peroxidee dismutase and catalase completely blocked the effect of OX-LDL onn the dimension of the EC surface layer and inhibited platelet-endothelial celll adhesion.
Conclusionss Oxygen derived free radicals mediate disruption of the EC surfacee layer and increased vascular wall adhesiveness by OX-LDL.
2.12.1 Introduction 49
2.11 Introduction
Damagee to the endothelial cell (EC) glycocalyx appears to be the earliest detectablee injury to the vascular wall during development of atherosclero-siss and is associated with increased vascular permeability and adhesive-nesss [1,2]. Recently, Vink and Duling [3] have shown that measurement off the relative positions of flowing blood cells and the EC membrane in skeletall muscle capillaries provides dimensional information on the in vivo endotheliall surface layer, which includes the EC glycocalyx and associated plasmaa proteins. Moreover, it was demonstrated that light-dye induced generationn of oxygen derived free radicals disrupted the endothelial sur-facee layer which resulted in localized adhesion of platelets and erythrocytes too the vascular wall [3]. In the present study we tested the hypothesis that oxidizedd low density lipoproteins (OX-LDL) modulate the endothelial sur-facee layer in a similar fashion.
2.22 Methods
2.2.11 Animal preparation
Malee Golden hamsters (n - 19, body weight: 139 7g) were anesthetized withh intraperitoneal pentobarbital sodium (7omg/kg BW) and the trachea wass cannulated to ensure a patent airway. The left femoral vein was cannu-latedd for continuous infusion of 0.9 % saline (0.5 ml/h) containing 10 mg/ml pentobarbitall sodium. The hamster was placed on a Plexiglas platter and thee right cremaster muscle was prepared for visualization of the microcir-culationn as previously described [3]. The cremaster muscle was continu-ouslyy superfused at ^ml/min with a bicarbonate-buffered physiological saltt solution (composition in mM: 131.9 NaCl, 4.6 KC1, 2.0 CaCl2,1.2 MgS04,
200 NaHC03). The supervision solution was gas-equilibrated with 5 % C02
andd 95 % N2 to obtain a pH of 7.35 to 7.45 and the solution was maintained
att 34°C. Succinylcholine do"5 M, Sigma) was added to the superfusion so-lutionn to reduce spontaneous skeletal muscle contractions. Body temper-aturee was maintained at 37~38°C with conducted heat. These procedures weree in accordance with institutional guidelines.
2.2.22 Intravital microscopy
Thee cremaster muscle was observed with an intravital microscope (Olym-puss BHM) and a cooled ICCD video camera (GenlV ICCD, Princeton Instru-ments).. The tissue was transilluminated with a Hg lamp (100 W) equipped withh a 435 nm bandpass interference filter (blue light) using an aplanar,
500 Chapter 2. Hans Vink—Ox-LDL degrades the endothelial surface layer achromaticc condensor set at numerical aperture (NA) 1.2 (U-AAC, Olym-pus).. All preparations were examined with a x6o water immersion objec-tivee lens (Olympus, UPlanApo NA 1.2 w or LUMPlanFL NA 0.9) and a tele-scopicc tube to give a final object-to-camera magnification of X250. Images weree displayed on a Philips CM 8833-II video monitor and recorded using aa SVHS video tape recorder (jvc BR-S611E) and a time coding interface unit (jvcc SA-F911E) for further image analysis.
2.2.33 Data analysis
Videoo images were digitized using a frame grabber (DT3152, PCI local bus) andd Image-Pro Plus software {Image-Pro Plus version 3.0, Media Cybernet-ics,, Silver Spring, PA, US A). An onscreen caliper using a 1 mm/0.01 mm stagee micrometer was used for all calibrated dimensional measurements. Thee anatomical capillary diameter was estimated by positioning digital caliperss at the inside of the capillary wall. Observed micro vessel diameters rangedd between 3-10 um, indicating that in addition to true capillary blood vesselss the observed population of microvessels has probably included ter-minall arterioles and/or capillary venules. Platelets that remained stuck to thee endothelium for at least 2 video frames in the presence of continuously flowingg erythrocytes were counted off-line by slow motion video play back.
2.2.44 Experimental protocols
Alll experimental protocols started 45-60 min after completion of the ham-sterr cremaster preparation. Measurements of capillary dimensions were madee starting 10-15 minutes prior to injection of either native (n = 4) or oxidizedd low density lipoproteins (n - 7) at o.4mg/ioog BW. Human LDL (Sigma,, L 2139) was dialyzed against PBS (phosphate-buffered saline) for 244 hours at 4°C at pH 7.4 without EDTA. LDL was oxidized by addition of CuSOO at a concentration of 7.5 uM for 6h at 37°C and this reaction was stoppedd by addition of 0.01 mM EDTA; native (normal) LDL was stored for 6hh at 4°C. Finally, both normal and oxidized LDL were dialyzed for 48 h in PBSS + 0.01 % EDTA at 4°C. Protein concentrations were determined accord-ingg to Lowry. LDL samples were stored at 4°C. Based on approximately 5 ml plasmaa volume in a ïoog hamster, the initial systemic OX-LDL concentra-tionn is < 0.4 m g / 5 ml, or < 8mg/dl, which appears to be clinically relevant basedd on recent measurements in atherosclerotic patients, reporting OX-LDL concentrationss between i - 6 m g / d l [4]. To test for the involvement of oxy-genn derived free radicals, additional OX-LDL experiments (n = 8) were per-formedd in the presence of superoxide dismutase and catalase as described previouslyy [3].
2.j2.j Results 51
2 . 2 . 55 Statistics
Dataa on the dimension of the spacing between erythrocytes and luminal en-dotheliall cell m e m b r a n e (RBC-EC gap) are presented as means SE. RBC-EC g a pp values following injection of either normal or oxidized LDL were com-paredd with their respective controls (pre-injection values) using a paired t-testt (two-way) to test for significance at P < 0.05.
2.33 Results
Figuress 1A-C show the g a p between flowing red blood cells and the lu-minall endothelial cell m e m b r a n e as a measure of the dimension of the en-dotheliall surface layer. Injection of moderately oxidized LDL (figure 2.1B), butt not normal LDL (figure 2.1 A), resulted in a transient decrease of more thann 50 % in the distance of flowing blood cells to the endothelial surface, whichh could be completely blocked by administration of SOD + catalase (fig-uree 2.1C). N u m b e r s of adhering platelets counted in 10 m i n u t e intervals are depictedd on the time axis. Only 1 platelet w a s seen adhering spontaneously too the endothelial cell surface in a total of 4 experiments after injection of normall LDL. In contrast, in 7 out of 7 OX-LDL experiments 1 or more platelets weree seen sticking to the endothelium, giving a total of 15 platelet-EC adhe-sionss in 7 experiments with OX-LDL. N o adhering platelets were observed inn the presence of SOD + catalase. Panels 2 A - C depict examples of the tran-sientt decrease in RBC-EC gap dimension o, 24 a n d 70 minutes after bolus
injectionn of OX-LDL.
2.44 Discussion
Althoughh early electron microscopic studies revealed already decades ago thatt carbohydrate rich endothelial surface structures form the interface be-tweenn blood and the luminal endothelial m e m b r a n e [5], relatively few stud-iess have attempted to associate modulation of endothelial cell function with modificationn of the endothelial surface layer, which is defined as the endo-theliall cell glycocalyx including associated plasma proteins.
Treatmentt of erythrocyte surface charge with polycations [6] or enzy-maticc treatment of the endothelial surface layer [7,8] has been s h o w n to modulatee red cell flow through capillary blood vessels and to stimulate platelet-endotheliall cell adhesion. Studies on transvascular exchange de-monstratedd that adsorption of albumin a n d other plasma proteins to the endotheliall cell glycocalyx confers filter like properties to the endothelial surfacee layer and reduces microvascular hydraulic conductivity and solute
5 2 2 ChapterChapter 2. Hans Vink—Ox-LDL degrades the endothelial surface layer 1.0 0 0.8H H RBC-ECC 0.6 Gap p (Mm)) 0.4 0.2 2 0.0 0 00 20 40 60 80
Timee after injection of norm-LDL (min)
1.0 0 0.8 8 RBC-ECC 0.6 Gap p (|im)) 0.4 0.2 2 0.0 0 00 20 40 60 80 Timee after injection of Ox-LDL (min)
1.0 0 0.8 8 RBC-ECC 0.6 Gap p (Hm)) 0.4 0.2 2 0.0 0 00 20 40 60 80 Timee after injection of Ox-LDL (min)
inn the presence of SOD / CAT
Figuree 2.1: Panels A, B and C show the dimension of the gap between flowing
redred blood cells and the luminal endothelial cell membrane, as a measure of the di-mensionmension of the endothelial surface layer, before and after injection of either normal (panel(panel A), oxidized LDL (panel B), or oxidized LDL together with superoxide dis-mutasemutase and catalase (panel C). Platelets observed to adhere to the endothelial cell surfacesurface within 10 minute intervals are depicted on the time axis as spheres.
2.4-2.4- Discussion 53
21:11:27:08 8
Figuree 2.2: Examples of light microscopic images of red blood cells (RBCs)
flow-inging through a hamster cremaster muscle capillary during an Ox-LDL experiment shoivingshoiving the gap between flowing RBCs and the luminal endothelial cell membrane (EC).(EC). Panels A, B and C show the transient decrease in RBC-EC gap dimension afterafter 0, 24. and yo minutes following bolus injection of Ox-LDL, respectively. The calibrationcalibration bar in panel A represents 2 pm.
544 Chapter 2. Hans Vink—Ox-LDL degrades the endothelial surface layer permeabilityy [9-11]. Enzymatic disruption of the endothelial cell glycoca-lyxx increased adhesion of blood cells to the vascular wall [8] and abolished floww dependent dilation due to impaired endothelial production of nitric oxidee (NO) 112,13]. These studies demonstrate that the endothelial sur-facee layer is essential for several aspects of endothelial function, including controll of transvascular exchange, providing vessels with an anti-adhesive innerr lining, and flow dependent dilation.
Inn the present study we used a recently developed light microscopic techniquee [3] to demonstrate for the first time that a clinically relevant dose off OX-LDL [4] reduces the in vivo dimension of the endothelial surface layer andd simultaneously increases platelet-endothelial cell adhesion. These ef-fectss were completely inhibited by administration of SOD and catalase, in-dicatingg that increased amounts of oxygen derived free radicals mediated OX-LDLL induced degradation of the endothelial surface layer and conse-quentt loss of endothelial anti-adhesive properties. Similarly, Lehr et al. [14] demonstratedd that increased adhesion of leukocytes to the endothelium of smalll arterioles and venules after systemic bolus injection of OX-LDL could bee prevented by vitamin C or SOD. Furthermore, Liao and Granger [15] showedd that SOD prevented OX-LDL induced albumin leakage and leuko-cytee adhesion to vascular endothelium.
Oxygenn derived free radicals such as superoxide anion may degrade thee endothelial surface layer and induce adhesion of platelets by inactivat-ingg paracrine anti-platelet substances such as NO. Alternatively, oxygen radicalss may have unmasked constitutive endothelial adhesion molecules suchh as PECAM by removing glycocalyx associated plasma proteins from the endotheliall cell surface. Re-adsorption of plasma substances to the theliall cell glycocalyx might then explain the rapid recovery of the endo-theliall surface layer following its disruption by bolus injection of OX-LDL. InIn agreement with this possibility, our reported layer recovery time (20-300 min) agrees well with published data on plasma protein reconstitution off the EC surface layer following vascular perfusion with a protein-poor mediumm [16]. However, reconstitution of the EC surface layer by newly synthesizedd or preformed proteoglycans and/or glycosaminoglycans must alsoo be considered and additional studies need to be designed to distin-guishh between these mechanisms of surface layer repair.
References s
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