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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 3
Elevatedd capillary tube hematocrit
reflectss degradation of the
glycocalyxx by oxidized low density
lipoproteins s
Alinaa A. Constantinescu, Hans Vink, and Jos A.E. Spaan
Abstract t
Proteoglycanss and plasma proteins bound to the endothelial cell glycoca-lyxx are essential for vascular function, but at the same time they lower cap-illaryy tube hematocrit by reducing capillary volume available to the flow-ingg blood. Because oxidized low-density lipoproteins (OX-LDL) reduce the effectivee thickness of the glycocalyx (Circulation 101:1500-1502, 2000), we designedd the present study to determine whether this is caused by patho-logicall degradation of glycocalyx constituents or by increased glycocalyx deformationn by elevated shear forces of flowing blood. Capillaries of ham-sterr cremaster muscle were examined using intravital microscopy after sys-temicc administration of 1) normal-LDL (n = 4), 2) moderately-oxidized LDL (m-Ox-LDL,, 6h oxidation with C11SO4, n = 7), 3) severely-oxidized LDL (S-OX-LDL,, 18 h oxidation, n - 5), and 4) ITL-OX-LDL and superoxide dismu-tasee and catalase (n=8). Capillary tube hematocrit increased from 3 too 0.37 0.05 and from 0.15 0.01 to 0.31 0.03 after m-Ox-LDL and s-Ox-LDL,, respectively, and these changes were paralleled by increases in RBC flux fromm 9 to 3 and from i to 16.3 2 cells/sin the absence off changes in anatomic capillary diameter, RBC velocity, as a measure for thee shear forces on the glycocalyx, was not affected by OX-LDL, while tissue pretreatmentt with superoxide dismutase and catalase completely abolished thee effects of OX-LDL on glycocalyx thickness, capillary hematocrit and RBC flux.. We conclude that elevation of capillary tube hematocrit by OX-LDL reflectss degradation of the endothelial glycocalyx by oxygen-derived free radicals. .
3.ii Introduction 59
3.11 Introduction
Capillaryy tube hematocrit is defined as the instantaneous volume fraction off a capillary blood vessel that is filled with red blood cells (RBCS). Capillary tubee hematocrit ranges from 20-50% of large vessel (systemic) hematocrit [1-4]] and an increasing amount of evidence relates low values of capillary tubee hematocrit to the presence of the endothelial cell glycocalyx, a thick endotheliall surface layer extending 0.5 um into the capillary lumen, thereby reducingg functionally perfused capillary volume [1,5]. Local perfusion of microvascularr beds with heparinase or stimuli elevating organ blood flow [1,, 3] increases capillary tube hematocrit, indicating that both enzymatic degradationn as well as shear-dependent compression of the endothelial cell glycocalyxx by red blood cells may increase functionally perfused capillary volume. .
Thee endothelial glycocalyx consists of specific proteoglycans and gly-coproteinss attached to the endothelial cell membrane, which bind a large numberr of plasma proteins that are essential for vascular function [6-9]. Degradationn of the endothelial glycocalyx has been related to increased ac-cumulationn of plasma macromolecules into the vascular wall [10,11] and impairedd protein binding and associated loss of endothelial function [12, 13].. Recently, we reported that clinically relevant levels of atherogenic ox-idizedd lipoproteins (OX-LDL) diminish the effective thickness of the endo-theliall cell glycocalyx as estimated from the distance of flowing red blood cellss to the luminal capillary endothelial surface [14L The reduction of en-dotheliall cell-RBC distance was paralleled by increased platelet-endothelial celll adhesion and could be prevented by administration of superoxide dis-mutasee (SOD) and catalase, suggesting that the effective decrease in calyxx thickness resulted from free radical-mediated modification of glyco-calyxx structures rather than from increased glycocalyx compression due to alteredd hemodynamic forces on the endothelial cell surface.
Inn the present study, we hypothesized that atherogenic levels of Ox-LDLL increase capillary tube hematocrit as a result of glycocalyx degrada-tion.. Capillary tube hematocrit was determined as a measure for func-tionallyy perfused capillary volume and capillary red cell velocity was mea-suredd to monitor possible changes in hemodynamic forces on the endothe-liall surface. Although our study was undertaken in skeletal muscle tis-sue,, degradation of the endothelial glycocalyx and associated loss of vas-cularr endothelial function by atherogenic stimuli such as OX-LDL is likely too affect other tissues as well. With respect to possible glycocalyx damage byy OX-LDL, the myocardium may be of special interest due to the estab-lishedd risk that OX-LDL imposes on the coronary vasculature in relation to endotheliall dysfunction during development of atherosclerosis. In this
re-gard,, electron microscopic studies provide evidence of coronary endothe-liall glycocalyx disruption in ischemia-reperfusion [15], dietary cholesterol challengee [16,17] and hypoxia [18]. In addition, consistent with reports on thee limiting role of the glycocalyx for skeletal muscle capillary hematocrit, examinationn of histological sections of myocardial tissue indicated simi-larr low and variable values of capillary hematocrit in the coronary circula-tionn [19]. Therefore, monitoring changes in skeletal muscle capillary tube hematocritt as a measure for perfused capillary volume may be the best tool availablee today to detect dynamic changes of endothelial glycocalyx thick-nesss reflecting altered levels of proteoglycan bound proteins and associated vascularr dysfunction in response to elevated plasma levels of atherogenic lipoproteins. .
3.22 Materials and Methods
3.2.11 Animal preparation
Malee golden hamsters (n = 24, body weight: 139 7 g) were anesthetized withh intraperitoneal pentobarbital sodium (7omg/kg BW; 35mg/ml) and thee trachea was cannulated to ensure a patent airway. The left femoral veinn was cannulated for continuous infusion of 0.9% saline (0.5 ml/h) con-tainingg i o m g / m l pentobarbital sodium to replace fluid loss and to main-tainn anesthesia. The hamster was placed on a plexiglas platter and the rightt cremaster muscle was prepared for visualization of the microcircula-tionn as previously described [3]. The muscle was continuously superfused att 5 ml/min with a bicarbonate-buffered physiological salt solution (com-position:: 131.9 mM NaCl, 4.6 mM KC1, 2.0 mM CaCl2/ 1.2 mM MgS04, and
200 mM NaHC03 which was gas-equilibrated with 5 % C02 and 95 % N2 to
obtainn a pH of 7.35 to 7.45. Succinylcholine (io~5 M, Sigma) was added too the superfusion solution to reduce spontaneous skeletal muscle contrac-tions.. The cremaster muscle was maintained at 34°C by controlling the temperaturee of the superfusate solution, while esophageal temperature was maintainedd between 37-38°C with conducted heat. All procedures were performedd in accordance with the institutional guidelines for animal wel-fare. .
3.2.22 Intravital microscopy
Microvesselss of the cremaster muscle were examined with an intravital mi-croscopee (Olympus BHM) and a cooled intensified charge-coupled device videoo camera (GenlV ICCD, Princeton Instruments). The tissue was trans-illuminatedd with a Hg lamp (100 W) equipped with a 435 nm bandpass
in-3-22 Materials and Methods 61 terferencee filter (blue light) using an aplanar, achromatic condensor set at numericall aperture (NA) 1.2 (U-AAC, Olympus). The tissue was examined withh a x6o water immersion objective lens (Olympus, UplanApo NA 1.2 or LUMPlanFLL NA 0.9) and a telescopic tube, which yielded a final X250 mag-nificationn from the object to the camera. Images were displayed on a Philips CMM 8833-II video monitor and recorded using a SVHS videotape recorder (JVCC BR-S611E) and a time coding interface unit (JVC SA-F911E) for further imagee analyses.
3.2.33 Lipoprotein preparation and oxidation
Humann LDL (Sigma, L 2139) was dialyzed against PBS (phosphate-buffered saline)) for 24 h at 4°C at pH 7.4 and the final product of this dialysis, which didd not undergo further treatment, was considered normal-LDL (n-LDL). Incubationn of lipoproteins after dialysis with 7.5 umol/1 CuS04 at 37°C was
performedd for 6 h in order to obtain moderately-oxidized LDL (ITL-OX-LDL), andd for 18 h in order to obtain severely-oxidized LDL (S-OX-LDL). In both cases,, the oxidative reaction was stopped by addition of 0.01 mmol/I EDTA andd OX-LDL was further dialyzed for 48 h in PBS + 0.01% EDTA at 4°C. The degreee of LDL oxidation was determined by analyzing the content of thio-barbituricc acid-reactive substances (TBARS) of the sample expressed as mal-onedialdehydee equivalents (MDA eq) [20]. TBARS values averaged i nmoll MDA eq/100 ug protein for n-LDL, 2 nmol MDA eq / ï o o u g pro-teinn for m-Ox-LDL, and 2.32 0.14 nmol MDA eq / ï o o u g protein for s-Ox-LDL.. The protein concentration of each sample was determined according too the Lowry method [21] and was taken into account when determining thee lipoprotein dose to be injected systemically. Normal-LDL samples, as welll as oxidized-LDL samples, were stored at 4°C until used.
3.2.44 Oxygen-derived free radicals
Too test whether oxygen-derived free radicals, such as superoxide anion and hydrogenn peroxide, were involved in the OX-LDL effect, the hamster cre-masterr vessels were pretreated with superoxide dismutase (SOD) (EC 1.15.1.1, Sigma)) and catalase (EC 1.11.1.6, Sigma), SOD and catalase were continu-ouslyy infused systemically (SOD, 29U/min; cat, 7.5 U/min) and added to thee superfusate of the cremaster muscle (SOD, 5oU/ml; catalase, 5oU/ml). Inn addition, SOD and catalase were administered as a systemic bolus (SOD, 250U/0.11 ml saline; catalase, 250U/0.1 ml saline) before lipoprotein injec-tion. .
3.2.55 Experimental protocols
Afterr surgery had been completed, 45-60 min were allowed for the hamster cremasterr muscle to recover and reach the physiological steady state. The experimentall protocols started with selection of one capillary blood ves-sell per animal that allowed proper visualization of the capillary wall and RBCC borders. The selected capillary was recorded on the videotape starting 10-155 m m before lipoprotein administration in order to provide a baseline condition.. Lipoproteins were administered as a bolus injection through the femorall vein in four distinct protocols, one protocol being used per animal ass follows: 1) n-LDL in control experiments (n = 4), 2) m-Ox-LDL (n - 7), 3)) S-OX-LDL (n = 5), and 4) FH-OX-LDL in the presence of SOD and catalase (nn = 8). Each capillary was examined and recorded for up to 60-80 min afterr lipoprotein injection. A part of the experimental protocols described heree provided the data on the thickness of the endothelial surface layer reportedd in Circulation 101: 1500-1502, 2000, as follows: n-LDL (4 experi-ments),, m-Ox-LDL (5 out the 7 experiments) and m-Ox-LDL in the presence off SOD and catalase (8 experiments). We performed for the present study 22 additional experiments in the protocol involving injection of m-Ox-LDL, sincee it was not possible to determine capillary hematocrit in all m-Ox-LDLL experiments reported previously [14]. The lipoprotein dose injected intravenouslyy was 0.4 mg/100 g BW hamster, which is a dose previously re-portedd to induce endothelial dysfunction after systemic administration to rodentss [22,23] and was reported to reduce the effective thickness of the endotheliall glycocalyx [14]. Taking into consideration a plasma volume of 3-55 ml/1 oog BW hamster, the plasma concentration of injected lipoproteins wouldd be 0.08^0.13 mg/ml. This should provide a clinically relevant dose off OX-LDL, in accordance with OX-LDL concentrations reported in coronary arteryy disease (i-6mg/dl) [24].
3.2.66 Data analysis
Videoo images were digitized using a frame grabber (DT3152, PCI Local Bus) andd Image-Pro Plus version 3.0 software (Media Cybernetics, Silver Spring, PA).. We used an onscreen caliper with a 1 mm/0.1 mm stage micrometer forr all calibrated dimensional measurements. The anatomic capillary di-ameter,, the width of RBCS, and the dimension of the gap between capillary walll and RBCS were measured from bright field images as described by Vink andd Duling [5]. The anatomic capillary diameter was measured by posi-tioningg digital calipers at the inside of the capillary wall. The functional capillaryy diameter was measured using the width of RBCS by positioning digitall calipers at the RBC borders. The gap between capillary wall and RBC
3.33.3 Results 63
(EC-RBCC gap), calculated by subtracting the functional capillary radius from thee anatomic capillary radius, was used as a measure for the dimension of thee endothelial glycocalyx. Capillary tube hematocrit (H) w a s calculated fromm measurements of the anatomical capillary diameter ( Da) , the flux of
RBCSS (F) a n d the velocity of RBCS (V) in each capillary, using the formula:
HH = — — — MCV (3.1)
VV Ti/4 Da
wheree MCV is the mean corpuscular volume of hamster RBCS (61 u m3) . Duringg slow-motion video playback, RBC flux w a s determined by the timee necessary for at least 50 RBCS to pass through a certain point chosen insidee the capillary segment and w a s calculated as cells/s. The velocity of RBCSS in the capillary w a s determined by measuring the length of a capillary segmentt and dividing it by the time required for red blood cells to traverse thiss segment.
Forr each capillary, all parameters were determined by 30 measurements att every 10 min d u r i n g the examination period. These measurements were averagedd for each individual capillary and the resulting values were again averagedd between all capillaries from each experimental protocol, yielding thee final values reported here.
3.2.77 Statistical analysis
Dataa are presented as m e a n s SEM for each g r o u p of experiments. Capil-laryy hematocrit, RBC flux and velocity values, a n d EC-RBC gap values weree compared with their respective controls (pre-injection values) using aa paired t-test (two-way) to test for significance at p < 0.05.
3.33 Results
Capillaryy hematocrit w a s measured in vessels with diameters between 4.2 andd 9.7 p m , the mean diameter being 6.4 0.3 p m (n = 24). In all four protocols,, the anatomic diameter of the vessels remained constant d u r i n g thee time course of our experiments (figure 3.1).
Capillaryy hematocrit increased transiently, reaching a m a x i m u m value inn the interval 20-25 m m after injection of m-Ox-LDL and S-OX-LDL, a n d returnedd to the baseline value approximately 60 min after injection (fig-uree 3.2). In the control experiments, capillary hematocrit remained constant afterr n-LDL (baseline value 0.13 0.02, 20 min value 0.12 0.02, p = 0.82). Administrationn of m-Ox-LDL increased capillary hematocrit to 233 40 % off the baseline value, i.e. from 0.16 0.03 before injection to 0.37 0.05
—— — n-LDL —— m-Ox-LDL — * —— s-Ox-LDL
—— m-Ox-LDL + SOD + catalase
HH 1 , 1 1 1 1 1 1
00 20 40 60 T i m ee after lipoprotein injection (min)
Figuree 3.1: Capillary anatomic diameter did not change after administration of eithereither normal-LDL (n-LDL), 6h-oxidized LDL (m-Ox-LDL), i8h-oxidized LDL (s-Ox-LDL)(s-Ox-LDL) or m-Ox-LDL in the presence of SOD and catalase.
(pp < 0.01). Similarly, administration of S-OX-LDL increased capillary hema-tocritt to 196 % of baseline value, from 0.15 1 to 0.31 3 ( p < o . o i ) . Combinedd administration of m-Ox-LDL with SOD and catalase to prevent d a m a g ee by oxygen-derived free radicals inhibited the effect of OX-LDL on capillaryy hematocrit (0.16 0.02 at t = o and 0.16 0.02 at 25 min, p = 0.94). Thee dimension of the endothelial glycocalyx w a s estimated by measure-mentt of the g a p b e t w e e n the endothelium and red blood cells (EC-RBC gap) (figuree 3.3). We reported before that the endothelial glycocalyx decreased too half of the control value after m-Ox-LDL, but remained constant after n-LDLL a n d m-Ox-LDL in the presence of SOD and catalase [14]. In the ex-perimentss in which capillary tube hematocrit was determined, administra-tionn of m-Ox-LDL r e d u c e d the EC-RBC g a p to 53 12% of the control value, i.e.. from 0.65 0.13 p m to 0.33 0.09 p m (p < 0.05), and, consistently, s-Ox-LDLL decreased the EC-RBC gap to 59 % of the control value 20-25 min after administration,, i.e. from 0.55 0.02 p m to 0.32 0.03 p m (p < 0.01).
Inn order to d e t e r m i n e whether the changes in capillary hematocrit were associatedd with changes in RBC flux in capillaries (figure 3.4) or with changes inn RBC velocity (figure 3.5), we determined the evolution of these
parame-j.jj.j Results 65 c c o o o o o. . U U 0.5-1 1 —— n-LDL —— m-Ox-LDL —— s-Ox-LDL
T —— m-Ox-LDL + SOD + catalase
T i m ee after lipoprotein injection (min)
Figuree 3.2: Capillary tube hematocrit increased to 233 40% of control after
administrationadministration of 6h-oxidized LDL (m-Ox-LDL) (*, p < 0.01) and to ig6 25% ofof control after administration of i8h-oxidized LDL (s-Ox-LDL) (#, p < 0.01),
butbut remained constant in response to normal-LDL (n-LDL) or m-Ox-LDL in the presencepresence of SOD and catalase.
terss after lipoprotein injection, RBC flux, as a measure for capillary RBC per-fusion,, increased after m-Ox-LDL from 8.7 1.9 cells/s to 13.8 3 cells/s (pp < 0.05), and after S-OX-LDL from 10.7 2.1 cells/s to 16.3 3.2 cells/s (pp < 0.05). N o significant changes were found after n-LDL (12.5 4 cells/s att t = o and 13.1 6.6 cells/s at t = 20 min, p = 0.81) or after administration off m-Ox-LDL in the presence of SOD and catalase (7.2 0.9 cells/s at t = o a n dd 6.4 0.8 cells/s at t = 25 min, p = 0.07). RBC velocity did not change significantlyy after m-Ox-LDL (from 122 46 p m / s before administration to 755 12 p m / s at 23 min after administration, p = 0.34), nor did it change significantlyy after S-OX-LDL (from 112 23 p m / s to 97 32 p m / s , p = 0.31). Similarlyy no changes in RBC velocity were found after n-LDL in the control experimentss (from 172 44 p m / s to 169 64 p m / s 20 min after administra-tion,, p = 0.89), nor after m-Ox-LDL in the presence of SOD and catalase (from 1077 14 p m / s to 97 13 p m / s 25 min after administration, p = 0.39).
Timee after lipoprotein injection (min)
Figuree 3.3: The dimension of EC-RBC gap as a measure of the endothelial
gly-cocalyxcocalyx decreased to 53 12% of control after administration of 6h-oxidized LDL (m-Ox-LDL)(m-Ox-LDL) (*, p < 0.05) and to 59 6% of control after administration of i8h-oxidizedoxidized LDL (s-Ox-LDL) (#, p < 0.01).
3.44 Discussion
Wee determined the effect of oxidized low density lipoproteins (OX-LDL) on capillaryy tube hematocrit as well as capillary red cell velocity to test the hypothesiss that OX-LDL decreases the effective glycocalyx dimension by re-movall of endothelial surface structures [14] independently of shear induced glycocalyxx deformation. Capillary tube hematocrit increased in parallel withh a transient decrease in glycocalyx dimension following administration off OX-LDL in the absence of significant changes in capillary red cell veloc-ity.. These findings are in support of the hypothesis that OX-LDL increases capillaryy volume accessible to red blood cells by removal of proteoglycans orr adsorbed proteins from the endothelial surface.
3.4.11 Determinants of capillary tube hematocrit
Microscopicc observation of capillary beds in vivo shows that capillary tube hematocritt is much lower than systemic hematocrit [1-4]. Possible phe-nomenaa that account for the observed difference include microvascular net-workk events, such as phase-separation of red blood cells (RBCS) and plasma
33 4 Discussion 67
u u
m m ai i — — N N33 #
O O Z Z c c s s 2500 n 200 0 —— n - L D L —— m-Ox-LDL *—— s-Ox-LDL—— m-Ox-LDL + SOD + catalase
Timee after lipoprotein injection (min)
Figuree 3.4: RBC flux increased to 204. 50% of control after 6h-oxidized LDL
(m-Ox-LDL)(m-Ox-LDL) (*, p < 0.0;) and to 154 16% of control after i8h-oxidized LDL (s-Ox-LDL)(s-Ox-LDL) (#, p < 0.05), but did not change after normal LDL (n-LDL) or m-Ox-LDLLDL in the presence of SOD and catalase.
att upstream bifurcations or intercapillary heterogeneity of blood flow, as welll as intracapillary events, such as different velocities for RBCS and plasma insidee capillaries (Fahraeus effect) [25] or retardation of a plasma layer close too the capillary wall [3]. More recent data have implicated the endothelial celll glycocalyx as a wall structure with a considerable thickness in capil-lariess [5], which may retard intracapillary plasma flow, reduce functionally perfusedd capillary volume, and act therefore as an important determinant off capillary tube hematocrit [1,5].
Althoughh capillary filling with RBCS is low under resting conditions, capillaryy tube hematocrit may increase with adenosine and tissue metabolic activityy and may decrease by high oxygen exposure or reduced flow veloc-ityy [1, 3]. In addition to the flow-dependent variations of capillary tube hematocrit,, Desjardins and Duling reported that heparinase treatment of thee capillary endothelial glycocalyx increased capillary hematocrit two to threefoldd independently of red cell velocity [1]. They suggested that capil-laryy hematocrit increases as a result of removal of heparan-sulfate proteo-glycanss from the capillary wall. Furthermore, Pries et al. [26] reported that heparinasee treatment of microvascular networks decreased flow resistance
250 0 o o _c c 3 3 > >
u u
DC DC T33 o NN gï C Cz z
—— n-LDL ••—— m-Ox-LDL *—— s-Ox-LDL•—— m-Ox-LDL + SOD + catalase
200 40 60 Timee after lipoprotein injection (min)
Figuree 3.5: RBC velocity remained constant in response to either normal LDL (n-LDL),(n-LDL), 6h-oxidized LDL (Ox-LDL), i8h-oxidized LDL (s-Ox-LDL), or m-Ox-LDLOx-LDL in the presence of SOD and catalase.
byy 15-20 %, indicating that the endothelial surface glycocalyx contributes significantlyy to microvascular resistance.
Inn the present study, capillary anatomic diameter did not change after exposuree to OX-LDL and neither did red cell velocity. Hence, the increased capillaryy hematocrit and red cell flux indicates an increased functionally perfusedd capillary volume due to the degradation of the endothelial sur-facee layer by OX-LDL rather than a modified capillary RBC hemodynamics withh consequences on shear dependent compression of the endothelial gly-cocalyx. .
However,, OX-LDL may also affect microvascular tone, since it is reported thatt OX-LDL decreases NO production of endothelial cells [27] and impairs NOO mediated dilation of isolated coronary arterioles to flow and adeno-sinee [10]. This raises the question whether the OX-LDL effect on capillary hematocritt observed in our study might be due to a direct effect of OX-LDL onn the vascular tone of terminal arterioles, proximal to the capillary bed. Inn this way, inhibition of NO-mediated dilation could be associated with an alteredd level of capillary hematocrit. Unpublished data from our laboratory showw that inhibition of NO synthesis with the L-arginine analog, N G
-nitro-3434 Discussion 69
L-argininee methyl ester (L-NAME), does not affect capillary hematocrit at concentrationss that decrease arteriolar diameter. Furthermore, it is reported thatt arteriolar vasoconstriction to various agents actually lowers capillary tubee hematocrit [1, 3] indicating that a possible effect of OX-LDL on mi-crovascularr tone due to impaired NO activity is unlikely to have caused thee increase in capillary hematocrit observed in our study.
Nevertheless,, an effect of OX-LDL on the bioactivity of NO may have contributedd to the degradation of the endothelial glycocalyx by further dis-turbingg the balance between oxygen radical production and NO availabil-ityy at the endothelial surface. This would be consistent with the reported disruptionn of the endothelial glycocalyx as demonstrated by electron mi-croscopyy in postischemic guinea-pig hearts that involved production of oxygenn radicals, while perfusion with L-NAME in aerobic medium did not changee glycocalyx appearance [15].
3.4.22 Degradation of the endothelial glycocalyx in vivo
Thee mechanism responsible for the degradation of the endothelial glyco-calyxx in vivo remains to be elucidated. The protective effect of adminis-trationn of SOD and catalase indicates that oxygen-derived free radicals me-diatedd glycocalyx degradation by OX-LDL in our present study, as well as duringg exposure to light-dye [5]. We aimed to differentiate in the present studyy between the effects of moderately and severely oxidized lipoprotein molecules.. We found that 18 h-oxidized LDL, with a high TBARS content, did nott affect the endothelial glycocalyx differently from 6 h-oxidized LDL. This suggestss that early-stage modification of the LDL molecule, i.e. depletion of antioxidantss and peroxidation of lipids [28], may be sufficient to induce disruptionn of the endothelial glycocalyx by upsetting the balance between pro-oxidativee and anti-oxidative stresses at the endothelial surface, since SODD and catalase prevented the OX-LDL effect.
Whetherr oxygen-derived free radicals cleave proteoglycans from the endotheliall membrane or impair adsorption of plasma proteins to glyco-saminoglycanss needs to be further investigated. Previous studies using electronn microscopy report that hypoxia induces disruption of endothelial proteoglycanss [18,29] and that this effect can be prevented by administra-tionn of oxygen radical scavengers such as SOD [15]. Furthermore, it has beenn reported that OX-LDL treatment is able to disrupt heparan-sulfate pro-teoglycanss of the subendothelial matrix [30]. However, the time courses off degradation and reconstitution of endothelial glycocalyx reported here forr OX-LDL are too short to involve replacement of degraded proteoglycans, unlesss preformed proteoglycans stored intracellularly are expressed on the endotheliall surface. Therefore, it is more likely that impaired adsorption of
plasmaa proteins to the glycocalyx may have contributed to the reduction of surfacee layer dimension in response to OX-LDL. Consistent with the time coursee for the recovery of the endothelial glycocalyx reported in our study considerablee re-adsorbtion of plasma proteins to the glycocalyx following plasmaa substitution protocols has been reported to occur within 15 min [8].
3.4.33 Ox-LDL effect
OX-LDLL increases microvascular permeability [31,32], albumin leakage [23] andd leukocyte-endothelial cell adhesion [22,23,33], anc* therefore may trig-gerr development of endothelial dysfunction in atherosclerosis. Although dennedd by morphological lesions of large vessels, atherosclerosis induces pathophysiologicall changes that appear to extend also into the microcircu-lationn [34,35]. In this respect, the present study that investigated the ef-fectt of "atherogenic" concentrations of OX-LDL on capillary hematocrit and capillaryy red cell perfusion addresses the OX-LDL induced microvascular dysfunction,, since we could not find an effect of OX-LDL on systemic hem-atocritt values (unpublished data).
Thee transient effect of OX-LDL on capillary hematocrit may be accounted forr by a rapid clearance of OX-LDL from systemic circulation by hepatic up-take.. It is reported that oxidatively modified LDL, when injected systemi-cally,, is much faster cleared from the circulation than native LDL, inasmuch ass 60-80% of 125I-labelled OX-LDL is taken up by the liver in the first 10 min afterr injection in rats in parallel with the decay in OX-LDL serum concentra-tionn [36].
3.4.44 The effect of increased capillary hematocrit for tissue
oxygenn supply
Microvascularr hematocrit participates in determination of tissue oxygen supply,, together with capillary blood flow and capillary density [4]. It has beenn suggested that capillary hematocrit may have a major role particularly inn non-steady-state conditions, when the intracapillary transit time of red bloodd cells may be an important variable for tissue oxygen supply [3].
Inn our experiments, capillary hematocrit, as well as RBC flux, increased inn response to OX-LDL by twofold, while RBC velocity remained constant. Sincee oxygen is transported in RBCS, it is suitable to reason that capillary oxygenn content will increase proportionally with the OX-LDL induced in-creasee in RBC flux. However, it is difficult to assess whether and to what extendd the increased capillary oxygen content will increase oxygen trans-portt to the tissue or will increase oxygen radical formation at the capillary endotheliall surface. However, since capillary hematocrit and flux doubled
ReferencesReferences 71
inn response to OX-LDL, it is possible that the high oxygen concentration may actt as an additional source of oxygen-derived free radicals both inside the tissuee and at the capillary endothelial surface. This may further stimulate productionn of inflammatory mediators and may further affect the endothe-liall glycocalyx. Therefore, although the effect of OX-LDL was transient in ourr study presumably due to OX-LDL metabolic degradation, a continuous productionn of oxygen radicals deriving from a sustained elevation of capil-laryy hematocrit and oxygen content may be expected in a chronic situation.
3.4.55 Implications for the coronary circulation
Wee have shown in the present study that elevated capillary tube hemato-critt induced by oxidized lipoproteins implies degradation of the endothe-liall glycocalyx and in particular, considering the rapid time constant of this process,, an altered adsorbtion of plasma proteins to the endothelial surface. Adsorbtionn of plasma proteins to the cell surface proteoglycans and gly-coproteinss is intimately related to endothelial function [37]. For example, albumin,, fibrinogen, and orosomucoid adsorbed to the endothelial surface influencee vascular wall permeability for solutes and macromolecules [7-9]. Furthermore,, lipoprotein lipase bound to endothelial cell heparan sulfate proteoglycanss actively regulates lipoprotein metabolism, while proteogly-cann bound antithrombin III modulates the level of coagulation near the en-dotheliall surface. Because all these processes are affected during atheroge-nesis,, altered adsorbtion of proteins to the endothelial cell surface is likely too have a major functional impact also on the coronary circulation.
References s
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