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Retention of aggregated LDL by cultured human coronary artery endothelial cells
BBRC Biochemical and Biophysical Research Communications 321 (2004) 728–735 www.elsevier.com/locate/ybbrc
Retention of aggregated LDL by cultured human coronary artery endothelial cells Bin Zhao a, Wei Huang a, Wei-Yang Zhang a, Itsuko Ishii b, Howard S. Kruth a,* a
Section of Experimental Atherosclerosis, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, MD 20892, USA b Department of Clinical Pharmacology, Graduate School of Pharmaceutical Sciences, Chiba University, Chiba, Japan Received 25 June 2004 Available online 22 July 2004
Abstract Aggregated LDL (AgLDL) accumulates within the subendothelial space of developing atherosclerotic lesions. We were interested to learn whether endothelial cells can interact with AgLDL. Incubation of endothelial cells with AgLDL resulted in apparent cholesterol retention. Microscopic examination revealed that cholesterol retention resulted mainly from endothelial cell surface attachment of AgLDL. Little AgLDL entered endothelial cells consistent with the small amount of endothelial cell degradation of AgLDL. Although endothelial cell retention of AgLDL was inhibited by LDL, AgLDL retention was not blocked by lactoferrin, C7 anti-LDL receptor monoclonal antibody, or receptor-associated protein, suggesting that LDL receptor family members did not mediate this retention. Surface retention of AgLDL depended on microtubule function and could be regulated by the protein kinase C activator, PMA. Treatment of endothelial cells with PMA either before or during, but not after incubation with AgLDL, inhibited retention of AgLDL. Our findings show that endothelial cells can retain AgLDL but internalize and metabolize little of this AgLDL. Thus, it is unlikely that endothelial cells can transport AgLDL out of atherosclerotic lesions, but it is likely that retention of AgLDL affects endothelial function. Published by Elsevier Inc. Keywords: Endothelial cells; Aggregated LDL; PMA; Cholesterol; Atherosclerosis
As the major carrier of cholesterol in the plasma, LDL is believed to be the source of cholesterol that accumulates in vessel wall atherosclerotic lesions [1]. Endothelial cells are the first vessel wall cell to interact with LDL and these cells function in the transport of LDL into the subendothelial space. Aggregated LDL (AgLDL) has been found in the subendothelial space of early developing atherosclerotic lesions and may be present in plasma [2,3]. LDL potentially aggregates in the subendothelial space due to interaction with glycosaminoglycans or exposure to phospholipase A2 and sphingomyelinase, enzymes secreted by vessel wall cells [4,5]. Also, LDL may aggregate secondary to oxidation
*
Corresponding author. Fax: 1-301-402-4359. E-mail address: [email protected] (H.S. Kruth).
0006-291X/$ - see front matter. Published by Elsevier Inc. doi:10.1016/j.bbrc.2004.07.017
of LDL another process thought to occur within atherosclerotic lesions [6,7]. Endothelial cell processing of lipoproteins and cholesterol may be important to plaque lipid accumulation as endothelial cells overlying plaques have been implicated in both deposition and removal of cholesterol from the vessel wall [1,8]. In this regard, it is important to note that vessel wall cholesterol accumulation is much less than can be predicted from the actual uptake and delivery of lipoprotein cholesterol to the vessel wall [9]. This observation and kinetic studies showing that LDL exits the vessel wall at the vessel lumen suggest that endothelial cells may function to remove lipoproteins/ cholesterol from the vessel wall [10]. Previous studies have shown that aggregation of LDL stimulates LDL uptake by macrophages [11] and may be one mechanism by which lesion macrophages
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accumulate LDL-derived cholesterol. While many studies have examined macrophage interaction with AgLDL, there has been much less investigation of endothelial cell interaction with AgLDL [12]. Because AgLDL is a potential source of lipid that could be transported by vascular endothelial cells, it is important to our understanding of atherosclerotic plaque lipid accumulation to determine whether AgLDL interaction with endothelial cells can lead to AgLDL uptake by endothelial cells. Endothelial uptake of AgLDL is a first step that would be required if endothelial cells function in transport of AgLDL either into or out of the vessel wall. Although our findings show endothelial retention of AgLDL is a process mediated by protein kinase C that can be regulated, little AgLDL enters or is degraded by these cells. Thus, it is unlikely that AgLDL is substantially transported by endothelial cells.
Materials and methods Reagents. Human LDL and HDL were purchased from Intracel, and human 125I-LDL was obtained from Biomedical Technologies. Sphingomyelinase (EC 3.1.4.12) from Bacillus cereus, heparinase III (EC 4.2.2.8) from Flavobacterium heparinum, chondroitinase ABC (EC 4.2.2.4) from Proteus vulgaris, lactoferrin, and BSA were obtained from Sigma. C7 mouse anti-LDL receptor monoclonal antibody was purified from supernatant of the hybridoma cell line 1691-CRL obtained from ATCC. Isotype-matched mouse control monoclonal IgG2b antibody was purchased from Southern Biotechnology Associates. Mouse monoclonal anti-a-tubulin and mouse IgG1 negative control were obtained from Molecular Probes and Dako, respectively. Alexa Fluor 488 goat anti-mouse IgG (H + L) secondary antibody was also obtained from Molecular Probes. Fetal bovine serum and EGM-2 culture medium were obtained from Gibco and Clonetics, respectively. PMA and nocodazole were obtained from Calbiochem. Human recombinant receptor-associated protein (RAP) was obtained from Innovative Research. Preparation of aggregated LDL and b-VLDL. Sphingomyelinaseaggregated LDL (SmAgLDL) was prepared by modification of previously published methods [13,14]. LDL was dialyzed overnight against buffer (5 mM Hepes, 2 mM CaCl2, 5 mM MgCl2, and 140 mM NaCl, pH 7.4). Then, sphingomyelinase was added to the LDL at an activity of 50 mU/mg LDL protein. After incubation for 24 h at 37 °C, SmAgLDL was collected by centrifugation at 16,000g for 10 min. After washing the pelleted SmAgLDL once with DulbeccoÕs phosphatebuffered saline (DPBS) plus Mg2+ and Ca2+, the SmAgLDL was resuspended in DPBS plus Mg2+ and Ca2+. Note that the aggregates produced here are about 2 lm in diameter, and thus, are much larger than the SmAgLDL originally described by Xu and Tabas [13] that is 0.1 lm in diameter. The reason for the difference in size most likely is due to the longer time of incubation of LDL with sphingomyelinase used in our study. Vortexed aggregated LDL (VxAgLDL) was prepared following the method of Khoo et al. [15]. Two hundred microliters of LDL (5 mg/ml for unlabeled LDL and 1–3 mg/ml for 125I-LDL) was placed into a 1.5ml silicone-coated polypropylene tube and vortexed (VWR vortex mixer) at the maximal speed for 1 min. Then, VxAgLDL was pelleted by centrifugation at 16,000g for 10 min. Supernatant was vortexed and centrifuged 2 more times to increase the yield of VxAgLDL. All pellets were resuspended in DPBS plus Mg2+ and Ca2+ and combined. The protein contents of both types of AgLDL were measured with the Lowry method using BSA as a standard [16].
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b-VLDL (d < 1.006 g/ml) was isolated from the plasma of rabbits that had been fed a 1% cholesterol diet for 12 weeks [17]. All lipoproteins used to inhibit AgLDL association with endothelial cells were dialyzed against EBM-2 culture medium before use. Cell culture. Human coronary artery endothelial cells (lot numbers 7F-0259, 7F-0496, and 7F-0051 obtained from Clonetics) were cultured in EGM-2 culture medium containing 10% FBS. Sixth to eighth passage cells were seeded in six-well plates (ICN Biomedical) at a density of 2500 cells/cm2. Medium was changed every day after the third day. It generally took 7 days for cells to become confluent. Elutriated human monocytes were cultured in 12-well plates as described previously [18] and used for experiments after differentiating for 2 weeks into macrophages. For experiments, confluent cell cultures (about 0.3 mg protein/well and 0.2 mg protein/well for macrophages and endothelial cells, respectively) were first rinsed three times with serum-free medium. Then, cultures were incubated 1 day with the indicated concentration of AgLDL or LDL and the reagents specified in each experiment. Assay of cholesterol and protein contents of cells. After incubations, cells were rinsed three times each with DPBS plus Mg2+, Ca2+, and 0.2% BSA, and then DPBS plus Mg2+ and Ca2+. Next, cells were harvested by scraping into 1 ml of distilled water. Lipids were extracted from an aliquot of cell suspension using the Folch method [19]. Cholesterol was determined with the fluorometric method of Gamble et al. [20]. Protein content was measured on another cell suspension aliquot using the Lowry method as described above. Assay of 125I-lipoprotein cell-association and degradation. Cell-association and degradation of 125I-lipoproteins by the cells were determined according to the methods of Goldstein et al. [21]. 125I-LDL was used at a specific activity of 200 lCi/mg protein. Lipoprotein degradation was quantified by measurement of trichloroacetic acid-soluble (TCA) organic iodide radioactivity in supernatants of media samples that were centrifuged at 16,000g for 10 min. Values obtained in the absence of cells were <5% of those values obtained with cells. These control values were subtracted. Cell-associated 125I-lipoprotein was determined by rinsing cells three times with DPBS plus Ca2+, Mg2+, and 0.2% BSA, and then three times with DPBS plus Ca2+ and Mg2+. Next, cells were dissolved overnight in 0.1 N NaOH at 37 °C. Aliquots of cell samples were assayed for 125I radioactivity with a gamma counter. Values were subtracted for 125I radioactivity determined for wells incubated with 125 I-lipoproteins but without cells. These values were <1% of the cell-associated 125I-lipoprotein. Microscopic analysis. Endothelial cells were seeded into two-well plastic slide chambers (Lab-Tek) at the same density (2500 cells/cm2) used for chemical studies above. After incubations with AgLDL, cells were viewed by phase microscopy or stained with ruthenium red to distinguish extracellular from intracellular membranes and prepared for electron microscopy as described previously [22]. Nocodazole disruption of endothelial cell microtubules was monitored by immunofluorescence staining of tubulin in endothelial cells incubated with and without 10 lM nocodazole for 24 h. Following incubations, cells were rinsed three times with DPBS plus Ca2+ and Mg2+, fixed with 4% paraformaldehyde overnight, incubated for 10 min with 0.5% Triton X-100, then 30 min in DPBS plus Ca2+ and Mg2+ with 1% BSA, and finally with 0.1 lg/ml mouse monoclonal antia-tubulin antibody or control mouse IgG1 at 4 °C overnight. Cells were rinsed three times with DPBS plus Ca2+ and Mg2+ and incubated with 1 lg/ml of Alexa Fluor 488 secondary goat anti-mouse (H + L) antibody for 2 h. Then cells were rinsed three times and mounted with glycerol-gelatin. All incubations were carried out at room temperature unless specified otherwise. Antibodies were diluted in PBS plus 1% BSA. Statistical analysis. All data are presented as means ± SD. The means were determined from three culture wells for each data point. The standard deviation is not shown for line graphs when its value was less than the symbol height.
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Results Endothelial cells retain AgLDL After a 1-day incubation with 200 lg/ml SmAgLDL, both macrophages and endothelial cells showed apparent cholesterol accumulation (Fig. 1). Endothelial cell total cholesterol increased from 81 ± 6 to 799 ± 51 nmol/mg cell protein, and macrophage total cholesterol increased from 76 ± 4 to 508 ± 43 nmol/mg cell protein during incubation with SmAgLDL. Native LDL produced no or only a small amount of cholesterol retention in the two cell types. When endothelial cells were incubated with varying concentrations of SmAgLDL (highest tested was 200 lg/ml) for 1 day, total cholesterol increased linearly from 90 ± 3 at baseline up to 522 ± 57 nmol/mg protein (Fig. 2A). Endothelial cells incubated up to 2 days with 100 lg/ml SmAgLDL showed a time-dependent increase in total cholesterol (Fig. 2B). In contrast to SmAgLDL-induced cholesterol accumulation, native LDL showed very little concentration- or time-dependent increase in endothelial cell cholesterol accumulation (Fig. 2). We also tested another type of AgLDL, VxAgLDL, to see if endothelial cells showed cholesterol accumulation with this AgLDL. This was the case. Endothelial cell total cholesterol increased about the same amount at 550 ± 27 nmol/mg cell protein and 662 ± 9 nmol/mg cell protein during a 1-day incubation with 100 lg/ml VxAgLDL and SmAgLDL, respectively.
Fig. 1. SmAgLDL interaction with endothelial cells and macrophages. Confluent cultured human coronary artery endothelial cells and 2week-old cultured human monocyte-derived macrophages were incubated 1 day with 200 lg/ml LDL or LDL aggregated by treatment with sphingomyelinase (SmAgLDL) with and without nocodazole (10 lM). Then, total cholesterol contents of cultures were determined. Total cholesterol increased 8.9 and 6.7 times compared with the no addition controls for endothelial cells and macrophages, respectively. Nocodazole inhibited cholesterol retention by 78% for endothelial cells, but had no effect on macrophage cholesterol retention.
Fig. 2. Concentration- and time-dependence of SmAgLDL interaction with endothelial cells. (A) Endothelial cells were incubated with varying concentrations of SmAgLDL and LDL for 1 day. (B) Endothelial cells were incubated with 100 lg/ml SmAgLDL and LDL for 1 or 2 days. Following incubations, cholesterol content of the endothelial cells was determined.
Endothelial cell metabolism of
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I-AgLDL
To investigate whether endothelial cells metabolize AgLDL, endothelial cells were incubated with 50 lg/ ml125I-SmAgLDL. After a 1-day incubation, cell-association and degradation of 125I-SmAgLDL and 125I-LDL by endothelial cells were compared (Fig. 3). Cell-association of 125I-SmAgLDL was 21 times greater than cellassociation of 125I-LDL. Endothelial cells accumulated 6.3 times more total (i.e., cell-association plus degradation) 125I-SmAgLDL than 125I-LDL, and degraded
Fig. 3. Endothelial cell metabolism of 125I-LDL and 125I-SmAgLDL. Endothelial cells were incubated with 50 lg/ml 125I-LDL or 125ISmAgLDL for 1 day without and with nocodazole (10 lM). Endothelial cells took up only very small amounts of 125I-LDL. Endothelial cell uptake (i.e., cell-association plus degradation) of 125I-SmAgLDL was increased 6.3 times compared with uptake of 125I-LDL. Endothelial cells degraded only 23 ± 2% of 125I-SmAgLDL compared with 78 ± 1% of 125I-LDL that they degraded. Cell-association of 125ISmAgLDL was nocodazole-sensitive, but degradation of 125ISmAgLDL was not nocodazole-sensitive.
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23 ± 2% and 78 ± 1% of the total 125I-SmAgLDL and 125 I-LDL, respectively, that they took up. Thus, most of the 125I-SmAgLDL associated with endothelial cells remained undegraded, while most of the 125I-LDL they took up was degraded. Interaction of AgLDL with endothelial cells results mainly in surface attachment of AgLDL Previously, we observed that undegraded AgLDL is stored in surface-connected compartments of macrophages [23]. Thus, it was of interest to learn where undegraded AgLDL was retained by endothelial cells. After a 1-day incubation with 100 lg/ml SmAgLDL, endothelial cell cultures showed many clusters of surfaceattached SmAgLDL when viewed by phase microscopy (Figs. 4A and B). These clusters were approximately 50–100 lm in diameter, which was about 25–50 times the diameter of the added aggregates of SmAgLDL. No 50–100 lm clusters of SmAgLDL were observed in the culture medium or in wells not containing endothelial cells, suggesting that the clusters of SmAgLDL formed in association with the cell monolayer surface. Electron microscopy revealed some localization of SmAgLDL within ruthenium-red labeled surface-connected compartments, but most SmAgLDL was associated with the endothelial cell surface (Fig. 4C).
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AgLDL retention by endothelial cells is microtubuledependent Nocodazole (10 lM), an agent that disrupts microtubules, inhibited endothelial cell cholesterol retention by 78 ± 2%, but did not inhibit macrophage cholesterol retention (Fig. 1). Consistent with this, nocodazole decreased cell-association of 125I-SmAgLDL in endothelial cells by 52 ± 8%, but did not affect endothelial cell degradation of 125I-SmAgLDL (Fig. 3). On the other hand, nocodazole showed no effect on 125I-LDL cell-association in endothelial cells, but inhibited endothelial cell degradation of 125I-LDL by 52 ± 4%. Immunofluorescence staining of tubulin in endothelial cells incubated with and without nocodazole confirmed that nocodazole treatment disrupted microtubule formation (data not shown). Retention of AgLDL with endothelial cells can be regulated with PMA Because protein kinase C activation with PMA stimulates degradation of AgLDL retained within macrophage surface-connected compartments [24], we examined the effect of PMA on SmAgLDL interaction with endothelial cells. Incubation of endothelial cells with SmAgLDL (100 lg/ml) for 1 day in the presence of PMA (0.1 lg/ml) decreased retention of SmAgLDL by 79 ± 2% (measured by cholesterol retention in Fig. 5A). Pretreatment of endothelial cells with PMA for 1 day followed by incubation of endothelial cells with SmAgLDL without PMA for 1 day similarly showed inhibition (80 ± 1%) of SmAgLDL retention (Fig. 5B). In this case, the added SmAgLDL remained in the medium unchanged from its original size. If endothelial cells were treated with PMA after they had already retained SmAgLDL during a 1-day incubation, endothelial cells retained 88 ± 7% of the SmAgLDL that was retained without PMA treatment (Fig. 5A). PMA affected VxAgLDL retention similar to its effect on SmAgLDL retention (data not shown). Thus, PMA inhibited AgLDL retention when endothelial cells were pretreated with PMA or incubated with PMA and SmAgLDL together, but PMA could not release AgLDL already retained by endothelial cells. Role of LDL receptors in AgLDL retention
Fig. 4. Microscopic analysis of SmAgLDL interaction with endothelial cells. Endothelial cells were incubated without (A, phase micrograph) or with 100 lg/ml SmAgLDL (B and C, phase and electron micrographs, respectively) for 1 day. Focal attachment of SmAgLDL to endothelial cells is shown in (B) (arrows). (C) Endothelial cells were sectioned parallel to the culture surface. SmAgLDL is shown attached to the plasma membrane surface (arrowheads) and within (arrows) a surface-connected compartment of the endothelial cell. The membrane of the surface-connected compartment is darkened by ruthenium red. Bar in (B) also applies to (A) and is 100 lm; bar in (C) is 2 lm.
LDL inhibited endothelial cell retention of SmAgLDL but had no effect on macrophage retention of SmAgLDL (Fig. 6A). A 5-fold weight excess of LDL added to endothelial cells during (but not when added only before) incubation with SmAgLDL inhibited endothelial retention of SmAgLDL by 87 ± 2%. This finding suggested the possibility that retention was mediated by an LDL receptor family member. Incubation of
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Fig. 5. Effect of PMA on SmAgLDL interaction with endothelial cells. Endothelial cells were treated with 0.1 lg/ml PMA either during or after (A) and before (B) incubation for 1 day with 100 lg/ml SmAgLDL. Then, total cholesterol content of the endothelial cells was determined. PMA treatment before and during incubation with SmAgLDL inhibited SmAgLDL interaction with cells. In contrast, PMA post-treatment did not affect SmAgLDL that had already associated with the cells.
Fig. 6. Effect of LDL and HDL on SmAgLDL interaction with endothelial cells and macrophages. Endothelial cells and macrophages were incubated 1 day with 100 lg/ml SmAgLDL and the indicated concentrations of native LDL (A) or HDL (B). (A) Endothelial cells and macrophages incubated 1 day without LDL had cholesterol contents of 70 ± 1 and 74 ± 1 nmol cholesterol/mg cell protein, respectively. (B) Endothelial cells incubated 1 day without HDL had a cholesterol content of 88 ± 3 nmol cholesterol/mg cell protein.
endothelial cells with C7 mouse monoclonal antibody (100 lg/ml) that blocks LDL binding to the classical LDL receptor [25] showed no inhibition of endothelial retention of 125I-SmAgLDL or 125I-VxAgLDL (50 lg/ ml) during a 1-day incubation (data not shown). At the same time, the C7 monoclonal antibody did inhibit endothelial cell degradation of native 125I-LDL (50 lg/ ml) by 74 ± 4%. Other LDL receptor family members, megalin and LRP [26], also did not mediate endothelial retention of AgLDL as lactoferrin (100 lg/ml) and receptor-associated protein (0.9 lM), inhibitors of these receptors, showed no inhibition of SmAgLDL (100 lg/ml) retention during a 1-day incubation. Also, glycosaminoglycans did not contribute substantially to AgLDL retention. Endothelial cells were pretreated with heparinase (6 U/ml), chondroitinase ABC (5 U/ml), or the two glycosaminoglycan-digesting enzymes together for 3 h. Then, endothelial cells were incubated 1 day with 100 lg/ml SmAgLDL in the presence of chlorate (50 mM) to block synthesis of new sulfated glycosaminoglycans [27,28]. These pretreatments did not decrease retention of SmAgLDL. b-VLDL, a lipoprotein containing apoE, also inhibited endothelial retention of AgLDL. A 10-fold protein
weight excess of either b-VLDL or LDL inhibited endothelial cell-association and total uptake of 125ISmAgLDL (50 lg/ml) by more than 95% during a 24-h incubation. Interestingly, a 5-fold weight excess of HDL added to endothelial cells during incubation with SmAgLDL inhibited endothelial retention of SmAgLDL by 62 ± 3% (Fig. 6B).
Discussion Our results show that like macrophages, endothelial cells retain AgLDL. However, in contrast to macrophages, most AgLDL is retained on the surface of the endothelial cells without being internalized into surface-connected compartments [23]. Other differences exist between endothelial cell and macrophage retention of AgLDL. Retention of AgLDL by endothelial cells was dependent on microtubule function (i.e., showed inhibition by nocodazole), while macrophage retention of AgLDL in surface-connected compartments does not depend on microtubule function [23]. Lastly, PMA, an activator of protein kinase C, inhibited endothelial cell retention of AgLDL, but has no effect on macrophage retention of AgLDL. Rather, PMA stimulates macro-
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phage degradation of AgLDL retained in macrophage surface-connected compartments [24]. PMA could not reverse endothelial cell retention of AgLDL after retention had already occurred. This suggests that PMA regulated a binding site that mediated only the initial but not later retention of the AgLDL, or that retention of the AgLDL stabilized the site to which it was bound. The former possibility is similar to what has been suggested for the mechanism of chylomicron remnant uptake by the liver. In that case, chylomicron remnants bind to heparin sulfate glycosaminoglycans and/or hepatic lipase before they are transported to other LDL receptor family members that mediate their uptake by the liver [29]. Although retention of AgLDL was inhibited by monomeric LDL, known LDL receptor family members did not apparently mediate AgLDL retention. C7 monoclonal antibody, which blocks LDL receptor binding, did not inhibit endothelial cell AgLDL retention. PMA treatment decreases ligand binding activity of the VLDL receptor in endothelial cells and monocytes, and the expression of other LDL receptor family members, such as LRP in macrophages [30,31]. However, it is unlikely that these receptors mediated endothelial retention of AgLDL because the VLDL receptor does not bind LDL [26] which competed AgLDL binding to endothelial cells. Also, RAP, which blocks ligand binding to most LDL receptor family members, and lactoferrin, an inhibitor of two other receptors that bind LDL, megalin and LRP [26], did not affect endothelial cell retention of AgLDL. This distinguishes AgLDL retention by human vascular endothelial cells and uptake of AgLDL by human vascular smooth muscle cells that is mediated by LRP [32]. The apoB48 receptor, which is expressed on endothelial cells, binds b-VLDL but does not bind LDL and thus is not a good candidate receptor for mediating endothelial retention of AgLDL [33,34]. However, our findings that both LDL and HDL inhibited endothelial retention of AgLDL suggest the possibility that human scavenger receptor class B type I, hSR-BI, may have mediated this interaction. This is because hSR-BI binds both LDL and HDL and is expressed on endothelial cells [35,36]. The results reported by Tabas and colleagues [13,37] may be relevant to the finding here that interaction of AgLDL with endothelial cells enhanced aggregation of the AgLDL (i.e., larger LDL aggregates formed during incubation with endothelial cells). These investigators incubated vascular smooth muscle cells, fibroblasts, and endothelial cells with native LDL, and found that LDL became aggregated on the surface of the cells when lipoprotein lipase and sphingomyelinase were added to the incubation. All those cells showed a relatively large amount of cell-association and a relatively small amount of degradation
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of the LDL as we have observed for endothelial metabolism of the pre-formed AgLDL used here. They suggested that lipoprotein lipase could bridge between sphingomyelinase-induced small LDL aggregates and extracellular glycosaminoglycans and thus promote aggregation of the LDL. In our culture system, it is possible that endothelial cells or their extracellular matrix provides a component that functions like the lipoprotein lipase bridging molecule in the Tabas study, further cross-linking the pre-formed AgLDL causing it to form even larger aggregates. However, extracellular glycosaminoglycans did not mediate AgLDL retention, as retention was not affected by treatment of endothelial cells with heparinase and chondroitinase ABC. Endothelial cell retention of subendothelial AgLDL could result in its transport from the subendothelial space to the luminal surface. Studies indicate that an undefined endothelial efflux pathway for LDL exists [10,38]. Consistent with this possibility, lipid particle aggregates similar to AgLDL are released from the luminal surface of coronary endothelial cells in cholesterol-fed swine [8]. In this regard, it would be of interest to study endothelial retention and transport of AgLDL deposited underneath cultured endothelial cells. Unfortunately, we have not yet found a suitable way to present AgLDL to the subendothelial space of endothelial cells. We have carried out experiments in which we adhered AgLDL to the culture surface and then added endothelial cells to the culture (unpublished data). In these experiments, the endothelial cells initially adhered only to bare areas of the culture well. Later, the endothelial cells contacted and retained the AgLDL on the top surface of the endothelial cell similar to when we added AgLDL to already cultured endothelial cells. In summary, we have found interaction of AgLDL with human coronary endothelial cells mainly resulted in surface attachment with only some uptake and degradation. Endothelial retention of AgLDL was dependent on microtubules and was inhibited by activating endothelial cells with PMA. The endothelial cell site that mediated AgLDL retention is not any of the known LDL receptors. Our findings suggest that while endothelial cells can retain AgLDL and this could influence endothelial function, it is unlikely that endothelial cells can clear AgLDL from atherosclerotic lesions by sequestration and transport of AgLDL.
Acknowledgments We thank Janet Chang and Rani Rao for help in carrying out experiments; and the Department of Transfusion Medicine, Clinical Center, National Institutes of Health, for providing elutriated monocytes.
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Retention of aggregated LDL by cultured human coronary artery endothelial cells Bin Zhao a, Wei Huang a, Wei-Yang Zhang a, Itsuko Ishii b, Howard S. Kruth a,* a
Section of Experimental Atherosclerosis, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, MD 20892, USA b Department of Clinical Pharmacology, Graduate School of Pharmaceutical Sciences, Chiba University, Chiba, Japan Received 25 June 2004 Available online 22 July 2004
Abstract Aggregated LDL (AgLDL) accumulates within the subendothelial space of developing atherosclerotic lesions. We were interested to learn whether endothelial cells can interact with AgLDL. Incubation of endothelial cells with AgLDL resulted in apparent cholesterol retention. Microscopic examination revealed that cholesterol retention resulted mainly from endothelial cell surface attachment of AgLDL. Little AgLDL entered endothelial cells consistent with the small amount of endothelial cell degradation of AgLDL. Although endothelial cell retention of AgLDL was inhibited by LDL, AgLDL retention was not blocked by lactoferrin, C7 anti-LDL receptor monoclonal antibody, or receptor-associated protein, suggesting that LDL receptor family members did not mediate this retention. Surface retention of AgLDL depended on microtubule function and could be regulated by the protein kinase C activator, PMA. Treatment of endothelial cells with PMA either before or during, but not after incubation with AgLDL, inhibited retention of AgLDL. Our findings show that endothelial cells can retain AgLDL but internalize and metabolize little of this AgLDL. Thus, it is unlikely that endothelial cells can transport AgLDL out of atherosclerotic lesions, but it is likely that retention of AgLDL affects endothelial function. Published by Elsevier Inc. Keywords: Endothelial cells; Aggregated LDL; PMA; Cholesterol; Atherosclerosis
As the major carrier of cholesterol in the plasma, LDL is believed to be the source of cholesterol that accumulates in vessel wall atherosclerotic lesions [1]. Endothelial cells are the first vessel wall cell to interact with LDL and these cells function in the transport of LDL into the subendothelial space. Aggregated LDL (AgLDL) has been found in the subendothelial space of early developing atherosclerotic lesions and may be present in plasma [2,3]. LDL potentially aggregates in the subendothelial space due to interaction with glycosaminoglycans or exposure to phospholipase A2 and sphingomyelinase, enzymes secreted by vessel wall cells [4,5]. Also, LDL may aggregate secondary to oxidation
*
Corresponding author. Fax: 1-301-402-4359. E-mail address: [email protected] (H.S. Kruth).
0006-291X/$ - see front matter. Published by Elsevier Inc. doi:10.1016/j.bbrc.2004.07.017
of LDL another process thought to occur within atherosclerotic lesions [6,7]. Endothelial cell processing of lipoproteins and cholesterol may be important to plaque lipid accumulation as endothelial cells overlying plaques have been implicated in both deposition and removal of cholesterol from the vessel wall [1,8]. In this regard, it is important to note that vessel wall cholesterol accumulation is much less than can be predicted from the actual uptake and delivery of lipoprotein cholesterol to the vessel wall [9]. This observation and kinetic studies showing that LDL exits the vessel wall at the vessel lumen suggest that endothelial cells may function to remove lipoproteins/ cholesterol from the vessel wall [10]. Previous studies have shown that aggregation of LDL stimulates LDL uptake by macrophages [11] and may be one mechanism by which lesion macrophages
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accumulate LDL-derived cholesterol. While many studies have examined macrophage interaction with AgLDL, there has been much less investigation of endothelial cell interaction with AgLDL [12]. Because AgLDL is a potential source of lipid that could be transported by vascular endothelial cells, it is important to our understanding of atherosclerotic plaque lipid accumulation to determine whether AgLDL interaction with endothelial cells can lead to AgLDL uptake by endothelial cells. Endothelial uptake of AgLDL is a first step that would be required if endothelial cells function in transport of AgLDL either into or out of the vessel wall. Although our findings show endothelial retention of AgLDL is a process mediated by protein kinase C that can be regulated, little AgLDL enters or is degraded by these cells. Thus, it is unlikely that AgLDL is substantially transported by endothelial cells.
Materials and methods Reagents. Human LDL and HDL were purchased from Intracel, and human 125I-LDL was obtained from Biomedical Technologies. Sphingomyelinase (EC 3.1.4.12) from Bacillus cereus, heparinase III (EC 4.2.2.8) from Flavobacterium heparinum, chondroitinase ABC (EC 4.2.2.4) from Proteus vulgaris, lactoferrin, and BSA were obtained from Sigma. C7 mouse anti-LDL receptor monoclonal antibody was purified from supernatant of the hybridoma cell line 1691-CRL obtained from ATCC. Isotype-matched mouse control monoclonal IgG2b antibody was purchased from Southern Biotechnology Associates. Mouse monoclonal anti-a-tubulin and mouse IgG1 negative control were obtained from Molecular Probes and Dako, respectively. Alexa Fluor 488 goat anti-mouse IgG (H + L) secondary antibody was also obtained from Molecular Probes. Fetal bovine serum and EGM-2 culture medium were obtained from Gibco and Clonetics, respectively. PMA and nocodazole were obtained from Calbiochem. Human recombinant receptor-associated protein (RAP) was obtained from Innovative Research. Preparation of aggregated LDL and b-VLDL. Sphingomyelinaseaggregated LDL (SmAgLDL) was prepared by modification of previously published methods [13,14]. LDL was dialyzed overnight against buffer (5 mM Hepes, 2 mM CaCl2, 5 mM MgCl2, and 140 mM NaCl, pH 7.4). Then, sphingomyelinase was added to the LDL at an activity of 50 mU/mg LDL protein. After incubation for 24 h at 37 °C, SmAgLDL was collected by centrifugation at 16,000g for 10 min. After washing the pelleted SmAgLDL once with DulbeccoÕs phosphatebuffered saline (DPBS) plus Mg2+ and Ca2+, the SmAgLDL was resuspended in DPBS plus Mg2+ and Ca2+. Note that the aggregates produced here are about 2 lm in diameter, and thus, are much larger than the SmAgLDL originally described by Xu and Tabas [13] that is 0.1 lm in diameter. The reason for the difference in size most likely is due to the longer time of incubation of LDL with sphingomyelinase used in our study. Vortexed aggregated LDL (VxAgLDL) was prepared following the method of Khoo et al. [15]. Two hundred microliters of LDL (5 mg/ml for unlabeled LDL and 1–3 mg/ml for 125I-LDL) was placed into a 1.5ml silicone-coated polypropylene tube and vortexed (VWR vortex mixer) at the maximal speed for 1 min. Then, VxAgLDL was pelleted by centrifugation at 16,000g for 10 min. Supernatant was vortexed and centrifuged 2 more times to increase the yield of VxAgLDL. All pellets were resuspended in DPBS plus Mg2+ and Ca2+ and combined. The protein contents of both types of AgLDL were measured with the Lowry method using BSA as a standard [16].
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b-VLDL (d < 1.006 g/ml) was isolated from the plasma of rabbits that had been fed a 1% cholesterol diet for 12 weeks [17]. All lipoproteins used to inhibit AgLDL association with endothelial cells were dialyzed against EBM-2 culture medium before use. Cell culture. Human coronary artery endothelial cells (lot numbers 7F-0259, 7F-0496, and 7F-0051 obtained from Clonetics) were cultured in EGM-2 culture medium containing 10% FBS. Sixth to eighth passage cells were seeded in six-well plates (ICN Biomedical) at a density of 2500 cells/cm2. Medium was changed every day after the third day. It generally took 7 days for cells to become confluent. Elutriated human monocytes were cultured in 12-well plates as described previously [18] and used for experiments after differentiating for 2 weeks into macrophages. For experiments, confluent cell cultures (about 0.3 mg protein/well and 0.2 mg protein/well for macrophages and endothelial cells, respectively) were first rinsed three times with serum-free medium. Then, cultures were incubated 1 day with the indicated concentration of AgLDL or LDL and the reagents specified in each experiment. Assay of cholesterol and protein contents of cells. After incubations, cells were rinsed three times each with DPBS plus Mg2+, Ca2+, and 0.2% BSA, and then DPBS plus Mg2+ and Ca2+. Next, cells were harvested by scraping into 1 ml of distilled water. Lipids were extracted from an aliquot of cell suspension using the Folch method [19]. Cholesterol was determined with the fluorometric method of Gamble et al. [20]. Protein content was measured on another cell suspension aliquot using the Lowry method as described above. Assay of 125I-lipoprotein cell-association and degradation. Cell-association and degradation of 125I-lipoproteins by the cells were determined according to the methods of Goldstein et al. [21]. 125I-LDL was used at a specific activity of 200 lCi/mg protein. Lipoprotein degradation was quantified by measurement of trichloroacetic acid-soluble (TCA) organic iodide radioactivity in supernatants of media samples that were centrifuged at 16,000g for 10 min. Values obtained in the absence of cells were <5% of those values obtained with cells. These control values were subtracted. Cell-associated 125I-lipoprotein was determined by rinsing cells three times with DPBS plus Ca2+, Mg2+, and 0.2% BSA, and then three times with DPBS plus Ca2+ and Mg2+. Next, cells were dissolved overnight in 0.1 N NaOH at 37 °C. Aliquots of cell samples were assayed for 125I radioactivity with a gamma counter. Values were subtracted for 125I radioactivity determined for wells incubated with 125 I-lipoproteins but without cells. These values were <1% of the cell-associated 125I-lipoprotein. Microscopic analysis. Endothelial cells were seeded into two-well plastic slide chambers (Lab-Tek) at the same density (2500 cells/cm2) used for chemical studies above. After incubations with AgLDL, cells were viewed by phase microscopy or stained with ruthenium red to distinguish extracellular from intracellular membranes and prepared for electron microscopy as described previously [22]. Nocodazole disruption of endothelial cell microtubules was monitored by immunofluorescence staining of tubulin in endothelial cells incubated with and without 10 lM nocodazole for 24 h. Following incubations, cells were rinsed three times with DPBS plus Ca2+ and Mg2+, fixed with 4% paraformaldehyde overnight, incubated for 10 min with 0.5% Triton X-100, then 30 min in DPBS plus Ca2+ and Mg2+ with 1% BSA, and finally with 0.1 lg/ml mouse monoclonal antia-tubulin antibody or control mouse IgG1 at 4 °C overnight. Cells were rinsed three times with DPBS plus Ca2+ and Mg2+ and incubated with 1 lg/ml of Alexa Fluor 488 secondary goat anti-mouse (H + L) antibody for 2 h. Then cells were rinsed three times and mounted with glycerol-gelatin. All incubations were carried out at room temperature unless specified otherwise. Antibodies were diluted in PBS plus 1% BSA. Statistical analysis. All data are presented as means ± SD. The means were determined from three culture wells for each data point. The standard deviation is not shown for line graphs when its value was less than the symbol height.
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Results Endothelial cells retain AgLDL After a 1-day incubation with 200 lg/ml SmAgLDL, both macrophages and endothelial cells showed apparent cholesterol accumulation (Fig. 1). Endothelial cell total cholesterol increased from 81 ± 6 to 799 ± 51 nmol/mg cell protein, and macrophage total cholesterol increased from 76 ± 4 to 508 ± 43 nmol/mg cell protein during incubation with SmAgLDL. Native LDL produced no or only a small amount of cholesterol retention in the two cell types. When endothelial cells were incubated with varying concentrations of SmAgLDL (highest tested was 200 lg/ml) for 1 day, total cholesterol increased linearly from 90 ± 3 at baseline up to 522 ± 57 nmol/mg protein (Fig. 2A). Endothelial cells incubated up to 2 days with 100 lg/ml SmAgLDL showed a time-dependent increase in total cholesterol (Fig. 2B). In contrast to SmAgLDL-induced cholesterol accumulation, native LDL showed very little concentration- or time-dependent increase in endothelial cell cholesterol accumulation (Fig. 2). We also tested another type of AgLDL, VxAgLDL, to see if endothelial cells showed cholesterol accumulation with this AgLDL. This was the case. Endothelial cell total cholesterol increased about the same amount at 550 ± 27 nmol/mg cell protein and 662 ± 9 nmol/mg cell protein during a 1-day incubation with 100 lg/ml VxAgLDL and SmAgLDL, respectively.
Fig. 1. SmAgLDL interaction with endothelial cells and macrophages. Confluent cultured human coronary artery endothelial cells and 2week-old cultured human monocyte-derived macrophages were incubated 1 day with 200 lg/ml LDL or LDL aggregated by treatment with sphingomyelinase (SmAgLDL) with and without nocodazole (10 lM). Then, total cholesterol contents of cultures were determined. Total cholesterol increased 8.9 and 6.7 times compared with the no addition controls for endothelial cells and macrophages, respectively. Nocodazole inhibited cholesterol retention by 78% for endothelial cells, but had no effect on macrophage cholesterol retention.
Fig. 2. Concentration- and time-dependence of SmAgLDL interaction with endothelial cells. (A) Endothelial cells were incubated with varying concentrations of SmAgLDL and LDL for 1 day. (B) Endothelial cells were incubated with 100 lg/ml SmAgLDL and LDL for 1 or 2 days. Following incubations, cholesterol content of the endothelial cells was determined.
Endothelial cell metabolism of
125
I-AgLDL
To investigate whether endothelial cells metabolize AgLDL, endothelial cells were incubated with 50 lg/ ml125I-SmAgLDL. After a 1-day incubation, cell-association and degradation of 125I-SmAgLDL and 125I-LDL by endothelial cells were compared (Fig. 3). Cell-association of 125I-SmAgLDL was 21 times greater than cellassociation of 125I-LDL. Endothelial cells accumulated 6.3 times more total (i.e., cell-association plus degradation) 125I-SmAgLDL than 125I-LDL, and degraded
Fig. 3. Endothelial cell metabolism of 125I-LDL and 125I-SmAgLDL. Endothelial cells were incubated with 50 lg/ml 125I-LDL or 125ISmAgLDL for 1 day without and with nocodazole (10 lM). Endothelial cells took up only very small amounts of 125I-LDL. Endothelial cell uptake (i.e., cell-association plus degradation) of 125I-SmAgLDL was increased 6.3 times compared with uptake of 125I-LDL. Endothelial cells degraded only 23 ± 2% of 125I-SmAgLDL compared with 78 ± 1% of 125I-LDL that they degraded. Cell-association of 125ISmAgLDL was nocodazole-sensitive, but degradation of 125ISmAgLDL was not nocodazole-sensitive.
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23 ± 2% and 78 ± 1% of the total 125I-SmAgLDL and 125 I-LDL, respectively, that they took up. Thus, most of the 125I-SmAgLDL associated with endothelial cells remained undegraded, while most of the 125I-LDL they took up was degraded. Interaction of AgLDL with endothelial cells results mainly in surface attachment of AgLDL Previously, we observed that undegraded AgLDL is stored in surface-connected compartments of macrophages [23]. Thus, it was of interest to learn where undegraded AgLDL was retained by endothelial cells. After a 1-day incubation with 100 lg/ml SmAgLDL, endothelial cell cultures showed many clusters of surfaceattached SmAgLDL when viewed by phase microscopy (Figs. 4A and B). These clusters were approximately 50–100 lm in diameter, which was about 25–50 times the diameter of the added aggregates of SmAgLDL. No 50–100 lm clusters of SmAgLDL were observed in the culture medium or in wells not containing endothelial cells, suggesting that the clusters of SmAgLDL formed in association with the cell monolayer surface. Electron microscopy revealed some localization of SmAgLDL within ruthenium-red labeled surface-connected compartments, but most SmAgLDL was associated with the endothelial cell surface (Fig. 4C).
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AgLDL retention by endothelial cells is microtubuledependent Nocodazole (10 lM), an agent that disrupts microtubules, inhibited endothelial cell cholesterol retention by 78 ± 2%, but did not inhibit macrophage cholesterol retention (Fig. 1). Consistent with this, nocodazole decreased cell-association of 125I-SmAgLDL in endothelial cells by 52 ± 8%, but did not affect endothelial cell degradation of 125I-SmAgLDL (Fig. 3). On the other hand, nocodazole showed no effect on 125I-LDL cell-association in endothelial cells, but inhibited endothelial cell degradation of 125I-LDL by 52 ± 4%. Immunofluorescence staining of tubulin in endothelial cells incubated with and without nocodazole confirmed that nocodazole treatment disrupted microtubule formation (data not shown). Retention of AgLDL with endothelial cells can be regulated with PMA Because protein kinase C activation with PMA stimulates degradation of AgLDL retained within macrophage surface-connected compartments [24], we examined the effect of PMA on SmAgLDL interaction with endothelial cells. Incubation of endothelial cells with SmAgLDL (100 lg/ml) for 1 day in the presence of PMA (0.1 lg/ml) decreased retention of SmAgLDL by 79 ± 2% (measured by cholesterol retention in Fig. 5A). Pretreatment of endothelial cells with PMA for 1 day followed by incubation of endothelial cells with SmAgLDL without PMA for 1 day similarly showed inhibition (80 ± 1%) of SmAgLDL retention (Fig. 5B). In this case, the added SmAgLDL remained in the medium unchanged from its original size. If endothelial cells were treated with PMA after they had already retained SmAgLDL during a 1-day incubation, endothelial cells retained 88 ± 7% of the SmAgLDL that was retained without PMA treatment (Fig. 5A). PMA affected VxAgLDL retention similar to its effect on SmAgLDL retention (data not shown). Thus, PMA inhibited AgLDL retention when endothelial cells were pretreated with PMA or incubated with PMA and SmAgLDL together, but PMA could not release AgLDL already retained by endothelial cells. Role of LDL receptors in AgLDL retention
Fig. 4. Microscopic analysis of SmAgLDL interaction with endothelial cells. Endothelial cells were incubated without (A, phase micrograph) or with 100 lg/ml SmAgLDL (B and C, phase and electron micrographs, respectively) for 1 day. Focal attachment of SmAgLDL to endothelial cells is shown in (B) (arrows). (C) Endothelial cells were sectioned parallel to the culture surface. SmAgLDL is shown attached to the plasma membrane surface (arrowheads) and within (arrows) a surface-connected compartment of the endothelial cell. The membrane of the surface-connected compartment is darkened by ruthenium red. Bar in (B) also applies to (A) and is 100 lm; bar in (C) is 2 lm.
LDL inhibited endothelial cell retention of SmAgLDL but had no effect on macrophage retention of SmAgLDL (Fig. 6A). A 5-fold weight excess of LDL added to endothelial cells during (but not when added only before) incubation with SmAgLDL inhibited endothelial retention of SmAgLDL by 87 ± 2%. This finding suggested the possibility that retention was mediated by an LDL receptor family member. Incubation of
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Fig. 5. Effect of PMA on SmAgLDL interaction with endothelial cells. Endothelial cells were treated with 0.1 lg/ml PMA either during or after (A) and before (B) incubation for 1 day with 100 lg/ml SmAgLDL. Then, total cholesterol content of the endothelial cells was determined. PMA treatment before and during incubation with SmAgLDL inhibited SmAgLDL interaction with cells. In contrast, PMA post-treatment did not affect SmAgLDL that had already associated with the cells.
Fig. 6. Effect of LDL and HDL on SmAgLDL interaction with endothelial cells and macrophages. Endothelial cells and macrophages were incubated 1 day with 100 lg/ml SmAgLDL and the indicated concentrations of native LDL (A) or HDL (B). (A) Endothelial cells and macrophages incubated 1 day without LDL had cholesterol contents of 70 ± 1 and 74 ± 1 nmol cholesterol/mg cell protein, respectively. (B) Endothelial cells incubated 1 day without HDL had a cholesterol content of 88 ± 3 nmol cholesterol/mg cell protein.
endothelial cells with C7 mouse monoclonal antibody (100 lg/ml) that blocks LDL binding to the classical LDL receptor [25] showed no inhibition of endothelial retention of 125I-SmAgLDL or 125I-VxAgLDL (50 lg/ ml) during a 1-day incubation (data not shown). At the same time, the C7 monoclonal antibody did inhibit endothelial cell degradation of native 125I-LDL (50 lg/ ml) by 74 ± 4%. Other LDL receptor family members, megalin and LRP [26], also did not mediate endothelial retention of AgLDL as lactoferrin (100 lg/ml) and receptor-associated protein (0.9 lM), inhibitors of these receptors, showed no inhibition of SmAgLDL (100 lg/ml) retention during a 1-day incubation. Also, glycosaminoglycans did not contribute substantially to AgLDL retention. Endothelial cells were pretreated with heparinase (6 U/ml), chondroitinase ABC (5 U/ml), or the two glycosaminoglycan-digesting enzymes together for 3 h. Then, endothelial cells were incubated 1 day with 100 lg/ml SmAgLDL in the presence of chlorate (50 mM) to block synthesis of new sulfated glycosaminoglycans [27,28]. These pretreatments did not decrease retention of SmAgLDL. b-VLDL, a lipoprotein containing apoE, also inhibited endothelial retention of AgLDL. A 10-fold protein
weight excess of either b-VLDL or LDL inhibited endothelial cell-association and total uptake of 125ISmAgLDL (50 lg/ml) by more than 95% during a 24-h incubation. Interestingly, a 5-fold weight excess of HDL added to endothelial cells during incubation with SmAgLDL inhibited endothelial retention of SmAgLDL by 62 ± 3% (Fig. 6B).
Discussion Our results show that like macrophages, endothelial cells retain AgLDL. However, in contrast to macrophages, most AgLDL is retained on the surface of the endothelial cells without being internalized into surface-connected compartments [23]. Other differences exist between endothelial cell and macrophage retention of AgLDL. Retention of AgLDL by endothelial cells was dependent on microtubule function (i.e., showed inhibition by nocodazole), while macrophage retention of AgLDL in surface-connected compartments does not depend on microtubule function [23]. Lastly, PMA, an activator of protein kinase C, inhibited endothelial cell retention of AgLDL, but has no effect on macrophage retention of AgLDL. Rather, PMA stimulates macro-
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phage degradation of AgLDL retained in macrophage surface-connected compartments [24]. PMA could not reverse endothelial cell retention of AgLDL after retention had already occurred. This suggests that PMA regulated a binding site that mediated only the initial but not later retention of the AgLDL, or that retention of the AgLDL stabilized the site to which it was bound. The former possibility is similar to what has been suggested for the mechanism of chylomicron remnant uptake by the liver. In that case, chylomicron remnants bind to heparin sulfate glycosaminoglycans and/or hepatic lipase before they are transported to other LDL receptor family members that mediate their uptake by the liver [29]. Although retention of AgLDL was inhibited by monomeric LDL, known LDL receptor family members did not apparently mediate AgLDL retention. C7 monoclonal antibody, which blocks LDL receptor binding, did not inhibit endothelial cell AgLDL retention. PMA treatment decreases ligand binding activity of the VLDL receptor in endothelial cells and monocytes, and the expression of other LDL receptor family members, such as LRP in macrophages [30,31]. However, it is unlikely that these receptors mediated endothelial retention of AgLDL because the VLDL receptor does not bind LDL [26] which competed AgLDL binding to endothelial cells. Also, RAP, which blocks ligand binding to most LDL receptor family members, and lactoferrin, an inhibitor of two other receptors that bind LDL, megalin and LRP [26], did not affect endothelial cell retention of AgLDL. This distinguishes AgLDL retention by human vascular endothelial cells and uptake of AgLDL by human vascular smooth muscle cells that is mediated by LRP [32]. The apoB48 receptor, which is expressed on endothelial cells, binds b-VLDL but does not bind LDL and thus is not a good candidate receptor for mediating endothelial retention of AgLDL [33,34]. However, our findings that both LDL and HDL inhibited endothelial retention of AgLDL suggest the possibility that human scavenger receptor class B type I, hSR-BI, may have mediated this interaction. This is because hSR-BI binds both LDL and HDL and is expressed on endothelial cells [35,36]. The results reported by Tabas and colleagues [13,37] may be relevant to the finding here that interaction of AgLDL with endothelial cells enhanced aggregation of the AgLDL (i.e., larger LDL aggregates formed during incubation with endothelial cells). These investigators incubated vascular smooth muscle cells, fibroblasts, and endothelial cells with native LDL, and found that LDL became aggregated on the surface of the cells when lipoprotein lipase and sphingomyelinase were added to the incubation. All those cells showed a relatively large amount of cell-association and a relatively small amount of degradation
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of the LDL as we have observed for endothelial metabolism of the pre-formed AgLDL used here. They suggested that lipoprotein lipase could bridge between sphingomyelinase-induced small LDL aggregates and extracellular glycosaminoglycans and thus promote aggregation of the LDL. In our culture system, it is possible that endothelial cells or their extracellular matrix provides a component that functions like the lipoprotein lipase bridging molecule in the Tabas study, further cross-linking the pre-formed AgLDL causing it to form even larger aggregates. However, extracellular glycosaminoglycans did not mediate AgLDL retention, as retention was not affected by treatment of endothelial cells with heparinase and chondroitinase ABC. Endothelial cell retention of subendothelial AgLDL could result in its transport from the subendothelial space to the luminal surface. Studies indicate that an undefined endothelial efflux pathway for LDL exists [10,38]. Consistent with this possibility, lipid particle aggregates similar to AgLDL are released from the luminal surface of coronary endothelial cells in cholesterol-fed swine [8]. In this regard, it would be of interest to study endothelial retention and transport of AgLDL deposited underneath cultured endothelial cells. Unfortunately, we have not yet found a suitable way to present AgLDL to the subendothelial space of endothelial cells. We have carried out experiments in which we adhered AgLDL to the culture surface and then added endothelial cells to the culture (unpublished data). In these experiments, the endothelial cells initially adhered only to bare areas of the culture well. Later, the endothelial cells contacted and retained the AgLDL on the top surface of the endothelial cell similar to when we added AgLDL to already cultured endothelial cells. In summary, we have found interaction of AgLDL with human coronary endothelial cells mainly resulted in surface attachment with only some uptake and degradation. Endothelial retention of AgLDL was dependent on microtubules and was inhibited by activating endothelial cells with PMA. The endothelial cell site that mediated AgLDL retention is not any of the known LDL receptors. Our findings suggest that while endothelial cells can retain AgLDL and this could influence endothelial function, it is unlikely that endothelial cells can clear AgLDL from atherosclerotic lesions by sequestration and transport of AgLDL.
Acknowledgments We thank Janet Chang and Rani Rao for help in carrying out experiments; and the Department of Transfusion Medicine, Clinical Center, National Institutes of Health, for providing elutriated monocytes.
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