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(Circulation Research. 1997;80:810-818.)
© 1997 American Heart Association, Inc.

Articles

Induction of P-Selectin by Oxidized Lipoproteins

Separate Effects on Synthesis and Surface Expression

Devendra K. Vora, Zhuang-Ting Fang, Stephanie M. Liva, Timothy R. Tyner, Farhad Parhami, Andrew D. Watson, Thomas A. Drake, Mary C. Territo, , Judith A. Berliner

From the Departments of Pathology (Z.-T.F., T.R.T., F.P., A.D.W., T.A.D., J.A.B.) and Medicine (D.K.V., S.M.L., M.C.T.), University of California, Los Angeles.

Correspondence to Devendra Vora, MD, Sam Nassi Fellow in Cardiology, Department of Medicine/Cardiology, 10833 Le Conte Ave, 47-123 CHS, University of California, Los Angeles, CA 90024.

	Abstract

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Abstract Leukocyte binding to the endothelium is one of the earliest events in the occurrence of atherosclerosis. Leukocyte adhesion molecules involved in this process have not been definitely identified. We have found that treatment of human aortic endothelial cells (HAECs) with minimally modified low-density lipoprotein (MM-LDL) for 24 hours caused a 2- to 3-fold increase of P-selectin protein, with little change in P-selectin surface expression. A 15-minute histamine treatment of cells exposed to MM-LDL caused a 50% to 100% increase in P-selectin surface expression compared with cells not treated with the lipoprotein. This increase resulted in a 2-fold increase in binding of leukocytes to the endothelium. Immunostaining of permeabilized HAECs after MM-LDL treatment also revealed a highly reproducible increase in intracellular P-selectin associated with rod-shaped structures, typical of Weibel-Palade bodies. Oxidized phospholipids were shown to be mainly responsible for the action of MM-LDL. This increased P-selectin expression was associated with MM-LDL–induced cAMP elevation. Like histamine, highly oxidized low-density lipoprotein, especially the oxidized fatty acids, caused immediate redistribution of P-selectin to the cell surface followed by reinternalization. Immunohistochemical staining showed that endothelial cells on human fatty streak lesions expressed increased levels of P-selectin compared with nonlesion areas. These studies suggest that P-selectin may play an important role in early recruitment of mononuclear cells to the subendothelium in human atherosclerosis and that oxidized lipoproteins may contribute to the increased expression of this molecule by increasing intracellular stores and causing redistribution to the cell surface.

Key Words: atherosclerosis • oxidized low-density lipoprotein • minimally modified low-density lipoprotein • P-selectin • endothelial cell

	Introduction

Top Abstract Introduction Materials and Methods Results Discussion References

 
An important early event in the initiation of atherosclerosis in humans and animals is adhesion of mononuclear cells to the endothelium of large arteries, probably induced by subendothelial lipids.^{1 2 3} The adherent mononuclear cells subsequently transmigrate and accumulate in the intima, where the monocytes differentiate into macrophages and form foam cells. These monocytes/macrophages contribute to the formation and progression of the atherosclerotic lesion.^{4 5} This initial localized mononuclear cell recruitment is most likely caused by specific leukocyte adhesion molecules expressed by the endothelial cells at those sites. Leukocyte recruitment to the subendothelium is a multistep process that involves rolling, activation, firm adhesion, and transmigration.⁶ The molecules involved in this monocyte-specific recruitment in early human atherosclerosis and their mechanism of induction have not been well studied.

Oxidized LDL has been found to be present in fatty streaks.^{7 8} Probucol, an antioxidant, has been shown to reduce lesion formation in hypercholesterolemic rabbits.^{9 10} Hence, it is likely that oxidized lipids play an important role in the initiation of atherosclerosis. Oxidation of LDL has been shown to alter its various biological properties, as reviewed previously.¹¹ We have used the interaction of HAECs with MM-LDL as a model to study very early events that might be occurring in early atherogenesis. Our group has shown that treatment of endothelial cells for 4 to 24 hours with MM-LDL, but not native LDL or OX-LDL, induced monocyte binding to HAECs in vitro.¹² Additionally, mRNA levels for other molecules also present in fatty streaks, such as MCP-1 and MCSF, were found to be increased in HAECs in response to MM-LDL.^{13 14} OX-LDL has been shown to be chemotactic for SMCs,¹⁵ to increase intracellular free Ca²⁺ in SMCs¹⁶ and cardiac myocytes,¹⁷ to cause macrophage foam cell formation, and to be cytotoxic for dividing cells.¹⁸ Recently, we have focused on examining the expression of various leukocyte adhesion molecules induced by MM-LDL in HAECs. Our group has shown that E-selectin, VCAM-1, and ICAM-1 mRNA and protein levels are not induced by MM-LDL in HAECs in culture.¹⁹ Others have suggested that VCAM-1 may be one of the adhesion molecules involved in this early adhesion process, as evidenced by the presence of VCAM-1 in lesions from rabbits fed an atherogenic diet.²⁰ However, it has not yet been convincingly documented that endothelial cells in human atherosclerotic lesions consistently express VCAM-1, although some studies have documented variable expression.²¹

The initial rolling of the leukocytes on endothelial cells was shown to be necessary for eventual extravascular migration.^{22 23 24} Rolling is brought about by a family of molecules termed selectins, but the molecules involved in rolling of mononuclear cells being recruited to early atherosclerotic lesions are not known. We previously reported that MM-LDL induced P-selectin mRNA severalfold in rabbit aortic endothelial cells.²⁵ Others have reported that P-selectin expression is increased in endothelial cells overlying human atherosclerotic plaques.²⁶ P-Selectin is a member of the selectin family of leukocyte adhesion molecules, which bring about rolling of leukocytes on endothelial cells during inflammation.²⁷ P-Selectin is constitutively expressed in endothelial cells, is localized in the Weibel-Palade bodies,^{28 29} and is rapidly mobilized to the surface of endothelial cells in response to agents such as histamine, phorbol, hydrogen peroxide, and thrombin.^{30 31} P-Selectin has been shown to support rolling of neutrophils, monocytes, and some types of T lymphocytes.²⁷ In the present study, we report our finding that MM-LDL induces intracellular accumulation of P-selectin, which can then be released to the surface by OX-LDL. We also show that P-selectin expression is increased in endothelial cells of human fatty streak lesions, suggesting that this induction of P-selectin by mildly oxidized LDL might be important for early monocyte recruitment during atherogenesis.

	Materials and Methods

Top Abstract Introduction Materials and Methods Results Discussion References

 
Cell Preparation
HAECs at passages 5 through 8 were used for all studies. These were isolated and cultured as previously reported.³² Monocytes were isolated by a modification of the Recalde method.³³ Polymorphonuclear cells were prepared from human blood by a previously described method.^{34 35}

Lipoprotein Preparation
MM-LDL was obtained by treatment of LDL with soybean lipoxygenase and phospholipase A2 or by mild iron oxidation.³⁶ All active preparations contained 2 to 6 nmol thiobarbituric acid–reactive substance per milligram cholesterol. All lipoproteins were stored in PBS containing EDTA (0.3 mmol/L) and butylated hydroxytoluene (0.1 mmol/L). Several different preparations with similar activities were used in these studies. We have observed, as reported previously,¹² that endothelial cells from different donors exhibit varying susceptibility to the action of MM-LDL. For the present study, HAECs were exposed to 125 µg/mL of MM-LDL in media, which consistently stimulated cell lines from a variety of donors. OX-LDL was obtained by dialysis in 0.15 mol/L NaCl (pH 6.5) containing 6 µmol/L CuSO₄ for 24 to 48 hours (thiobarbituric acid–reactive substance, 16 to 20 nmol/L per milligram cholesterol). After oxidation, the LDL was further dialyzed in PBS (pH 7.4) containing 0.01% EDTA. All LDL, MM-LDL, and OX-LDL preparations were tested for LPS using the chromogenic limulus assay³⁷ ; the preparations used for these studies contained a final concentration of <25 pg/mL LPS. For testing of OX-LDL subfractions, lipids were extracted with chloroform/methanol and fractionated by solid-phase chromatography on aminopropyl columns into polar lipids, fatty acids, cholesterol, cholesteryl esters, and triglycerides.³⁸ Cells were treated with selected subfractions after resuspension in PBS or PBS+ethanol. Ethanol was present at a final concentration of 0.05% in the medium. Cells were treated with the lipid subfractions at a concentration equivalent to 50 or 100 µg/mL of LDL protein.

Northern Analysis
HAECs were incubated with MM-LDL (125 µg/mL) for 24 hours and LPS (1 ng/mL) for 6 hours. Total RNA was isolated from cells using the guanidine thiocyanate/phenol method.³⁹ A Northern blot analysis was performed as previously described.³⁶ Tubulin and human P-selectin cDNA (kindly provided by R. McEver, Oklahoma City, Okla) were used as probes.

Antibodies and Reagents
Antibody to P-selectin (AC 1.1) was obtained from Becton Dickinson (BD 550014). Antibody to PECAM-1 (Becton Dickinson, BD 550023) was used as an endothelial cell marker. Antibody to integrin IIb/IIIa (5B12), obtained from Dako, was used as a marker for platelets. A blocking antibody to P-selectin (GA 6)⁴⁰ was kindly provided by Dr Bruce Furie, Boston, Mass. An antibody to CD19 (M740) from Dako was used as a control antibody. Histamine sulfate (Sigma Chemical Co) and H8 (Calbiochem) were used in some experiments.

Antibody Binding Assay
Pretreated HAECs were washed three times with medium 199 containing 0.1% BSA. Primary P-selectin antibody (AC 1.1) diluted 1:5000 in medium was added, and the cells were incubated on ice for 2 hours. The cells were then washed three times with PBS containing 0.1% BSA and fixed with 4% paraformaldehyde for 30 minutes. The cells were then washed three times with PBS containing 0.05% Tween 20 and 0.1% BSA before being treated with peroxidase-labeled goat anti-mouse secondary antibody for 1 hour at room temperature.

Unbound antibody was removed by washing, O-phenylenediamine was added, and absorbance was read at 492 nm on a Titertek Multiscan MCC/340. Data are expressed as percent increase in OD of treated cells compared with untreated cells. This percent increase in P-selectin expression was similar for comparable incubation conditions, whereas the absolute OD of untreated cells varied from 0.1 to 0.45. Data were calculated as (OD of treated cells-OD of untreated cells/OD of untreated cells)x100. P values were calculated using the model-1 ANOVA test.

Leukocyte Adhesion Assay
Pretreated HAECs in 48-well plates were rinsed twice with media, and neutrophils or monocytes were added at 10⁵ cells per well. After 20 minutes of incubation at 37°C, the unbound leukocytes were removed, and the cells were fixed with 1% glutaraldehyde in PBS. The number of bound leukocytes was determined by counting a minimum of four fields per well under light microscopy.³⁶

P-Selectin Protein Assay
Pretreated and untreated HAECs in tissue culture dishes were scraped and pelleted. An equal amount of protein, measured by Micro BSA assay (Pierce), from each cell lysate was blotted onto a nitrocellulose membrane in a dot blot apparatus. The membrane was then exposed to primary antibody followed by a peroxidase-conjugated secondary antibody. The secondary antibody was detected using an Amersham ECL (chemiluminescence) assay. The amount of antibody binding was determined by densitometry. Dose-response curves for both antibody and lysate were used to establish the range of linearity, and concentrations within this range were used for analysis.

Immunocytochemistry of Cultured Cells and Lesions
Unfixed frozen sections of human coronary vessels were stained with antibodies to P-selectin (BD 550014), PECAM-1 (BD 550023), and GPIIb/IIIa (Dako 5B12).Peroxidase-conjugated secondary antibody was used. HAECs cultured on glass coverslips were treated with MM-LDL for 24 hours, permeabilized using 0.1% Triton X-100, and stained with antibody to P-selectin. Fluorescein-conjugated secondary antibody was used for visualization.

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

 
MM-LDL Regulation of P-Selectin Expression
Incubation of HAECs for 24 hours with MM-LDL (125 µg/mL) resulted in a doubling of mRNA levels for P-selectin (Fig 1). In three separate experiments, levels were increased 1.8-, 2-, and 2.1-fold after tubulin normalization. In HAECs, cholera toxin (1 µg/mL) (data not shown) and LPS (1 ng/mL) increased P-selectin mRNA to an extent similar to MM-LDL (Fig 1, left). A similar increase in P-selectin protein was seen in HAECs treated with MM-LDL for 24 hours. For these studies, human anti–P-selectin monoclonal antibody, AC 1.1, and a chemiluminescence assay system were used. Densitometric analysis showed a 2- to 2.5-fold increase in P-selectin protein in MM-LDL–treated HAECs compared with control cells (Fig 1, right). A Western blot was performed on HAEC lysate, which confirmed that the antibody AC 1.1 bound to only a single 140-kD protein, the characteristic size of P-selectin.

View larger version (19K): [in this window] [in a new window]   Figure 1. MM-LDL induces P-selectin mRNA (left) and protein (right) in HAECs. Left, HAECs were incubated at 37°C with 1 ng/mL LPS (L) for 6 hours, with 125 µg/mL of MM-LDL (M) for 24 hours, and with no additives (C). Northern blot analysis was performed using RNA extracted from the cells and probed with 32P-labeled human P-selectin and -tubulin cDNA. The blots were scanned, and densitometry was performed. A representative blot of three of three experiments is shown. Right, HAECs were incubated at 37°C with (mm-ldl) and without (con) 125 µg/mL of MM-LDL for 24 hours. The cells were scraped from 100-mm dishes, pelleted, and sonicated. Whole-cell extract (10 µg of protein per slot) was placed on nitrocellulose and probed with anti–P-selectin antibody (AC 1.1, 1 µg/mL) (P). Extracts (1 µg per slot) were simultaneously probed with anti-tubulin antibody (1 µg/mL) (T). Antibody staining was detected with an ECL system (Amersham). Three separate experiments were performed, and a representative blot is shown. The blots were scanned, and densitometry was performed.

HAECs treated with MM-LDL or native LDL for 10 minutes, 6 hours, and 24 hours did not express P-selectin on the cell surface as determined by surface ELISA (Fig 2). We hypothesized that although P-selectin protein levels were increased by MM-LDL, the protein was accumulating intracellularly and that the expression on the cell surface would require an additional stimulus, such as histamine or thrombin. A dose-response curve and a time course for histamine-induced P-selectin surface expression were obtained. Maximal release of the P-selectin to the surface was induced by a histamine concentration of 10^-5 mol/L in medium at 15 minutes. On the basis of these findings, 10^-5 mol/L histamine for 15 minutes was used to release P-selectin to the surface. Cells pretreated with MM-LDL but not native LDL (Fig 2) showed a significant increase in histamine-induced P-selectin surface expression over control cells (Fig 3). There was significantly more P-selectin released by histamine in cells treated with MM-LDL for 24 hours compared with 6 hours, and levels remained elevated in cells treated with MM-LDL for 48 hours. Interestingly, there was a small but significant increase in P-selectin surface expression in cells exposed for 48 hours to MM-LDL compared with control cells, even in the absence of histamine (Fig 3, solid bars). We also observed a highly reproducible increase in fluorescence in MM-LDL–treated cells compared with control cells, with staining localized to rod-shaped structures typical of Weibel-Palade bodies (Fig 4).

View larger version (32K): [in this window] [in a new window]   Figure 2. Effect of native LDL and MM-LDL on histamine-induced P-selectin surface expression. HAECs were incubated in the absence (con) or presence of 125 µg/mL of LDL or MM-LDL in media for 10 minutes (L10m and m10m, respectively), 6 hours (L6 and m6, respectively), and 24 hours (L24 and m24, respectively). The lipids were then removed, and the cells were washed three times with media. Half the lipid-treated wells were exposed to 10-5 mol/L histamine sulfate for 15 minutes (hi+), as were half of the untreated cells (con); the rest were not treated with histamine (hi-). An ELISA was performed to detect surface expression of P-selectin. The data are representative of three of three experiments, and the values from a representative experiment are presented as percent increase in OD of P-selectin expression compared with untreated cells. Values are mean±SEM (n=3). *P<.001 for significant increase compared with control+histamine.

View larger version (34K): [in this window] [in a new window]   Figure 3. Effect of prolonged MM-LDL treatment on P-selectin surface expression in the presence (hi+) or absence (hi-) of histamine. HAECs were incubated in the absence or presence of 125 µg/mL of MM-LDL in media for 6 hours (m6), 24 hours (m24), and 48 hours (m48). MM-LDL was then removed, and the cells were washed three times with media. Half the MM-LDL–treated wells were treated with 10-5 mol/L histamine sulfate for 15 minutes (hi+), as were some untreated cells (con). An ELISA was performed to detect surface expression of P-selectin. The data are representative of three of three experiments, and the values from a representative experiment are presented as percent increase in OD of P-selectin compared with untreated cells. Values are mean±SEM (n=3). *P<.001 for significant increase compared with cells treated with histamine alone. +P<.01 for significant increase compared with untreated cells.

View larger version (59K): [in this window] [in a new window]   Figure 4. Immunofluorescence of P-selectin in MM-LDL–treated HAECs. HAECs were untreated (left) or treated for 24 hours with 125 µg/mL of MM-LDL (right). The cells were then permeabilized and incubated with P-selectin antibody AC1.1. An FITC-labeled secondary antibody was used to detect the bound primary antibody (magnification x1200). The data are representative of three of three experiments. Each experiment was performed in triplicate.

Since MM-LDL–treated cells appeared to accumulate P-selectin in the histamine-releasable Weibel-Palade bodies, which are known to have a relatively long half-life,⁴¹ we hypothesized that this P-selectin might be available for a considerable period, even after removal of the MM-LDL stimulus. To test this hypothesis, we treated HAECs with MM-LDL for 6 hours and then removed the MM-LDL and incubated these HAECs for a further period of 18 hours; finally, histamine was added for 15 minutes to release P-selectin. We found that MM-LDL–pretreated cells had a significantly higher level of histamine-induced P-selectin expression compared with untreated cells (histamine, 38±3; MM-LDL+histamine, 75±4; and MM-LDL removed+histamine, 55+5 O.D.; P<.01).

Functionality of P-Selectin Protein Accumulated in MM-LDL–Treated Cells
To test if the induced P-selectin protein was functional, HAECs either untreated or treated with MM-LDL for 24 hours were exposed to 10^-5 mol/L histamine sulfate, and the binding of neutrophils to the monolayer was examined. There was a 3-fold increase in neutrophil binding to MM-LDL–treated cells compared with untreated cells (Fig 5). This binding could be blocked by anti–P-selectin antibody (GA 6) but not by an irrelevant IgG (M740) antibody, suggesting that the induced P-selectin was functional and that the increased binding seen after MM-LDL treatment was due to P-selectin and not other adhesion molecules. MM-LDL also caused an increase in monocyte binding to HAECs in response to histamine, which was inhibited by anti–P-selectin antibody (GA 6) (data not shown). However, this was difficult to evaluate, since the level of monocyte binding to MM-LDL–treated cells was increased significantly (compared with control cells) even in the absence of histamine, as previously reported by our group.¹²

View larger version (34K): [in this window] [in a new window]   Figure 5. Functionality of MM-LDL–induced P-selectin. HAECs were incubated in the absence (con) or presence (m24) of 125 µg/mL of MM-LDL for 24 hours. MM-LDL was then removed, and half of the wells were treated with 10-5 mol/L histamine (hi+); hi- indicates the absence of histamine. The cells were then incubated for 10 minutes with freshly isolated neutrophils either in the presence of a blocking antibody to P-selectin (P-AB) or an irrelevant IgG (I-AB). The data are representative of two of two experiments, and the values from a representative experiment are expressed as neutrophil binding per field. Values are mean±SEM (n=4). *P<.001 for significant decrease compared with MM-LDL+histamine–treated cells.

The Role of cAMP in Regulating P-Selectin Levels
It has been shown that most of the biological activities of cholera toxin are mediated via induction of cAMP.⁴² Our group has shown that MM-LDL induces an increase in cAMP levels in HAECs.³⁶ To determine whether cAMP could increase P-selectin protein, HAECs were treated for 6 and 30 hours with cholera toxin, and histamine-releasable P-selectin surface expression was measured (Fig 6). The effect of cholera toxin was similar to that of MM-LDL. As discussed above, cholera toxin also caused accumulation of P-selectin mRNA, suggesting that cAMP is involved in the induction of P-selectin by MM-LDL. To determine whether cAMP might be involved in the accumulation of P-selectin protein induced by MM-LDL, we treated HAECs with MM-LDL for 24 hours in the presence or absence of the cAMP inhibitor H8.³⁶ H8 reduced the level of histamine-induced P-selectin surface expression to levels similar to those of control cells treated with histamine (Fig 7).

View larger version (51K): [in this window] [in a new window]   Figure 6. Effect of cholera toxin on histamine-induced P-selectin surface expression. HAECs were incubated with or without 400 ng/mL of cholera toxin for 6 hours (CT6) and 30 hours (CT30). Cholera toxin was then removed, and half the wells were treated with 10-5 mol/L histamine (hi+). An ELISA was performed to detect surface expression of P-selectin. The data are representative of three of three experiments, and the values from a representative experiment are expressed as percent increase in OD of P-selectin expression over untreated cells. Values are mean±SEM (n=3). *P<.001 for significant increase compared with histamine-treated cells.

View larger version (19K): [in this window] [in a new window]   Figure 7. Protein kinase A inhibitor H8 inhibits MM-LDL induction of P-selectin. HAECs pretreated with 60 µmol/L of H8 for 1 hour (H8+) were incubated with (mm24) or without (con) 125 µg/mL of MM-LDL for 24 hours. MM-LDL was then removed, and half the cells were treated with 10-5 mol/L histamine (hi). An ELISA was performed to detect surface expression of P-selectin. The data are representative of three of three experiments, and the values from a representative experiment are expressed as percent increase in OD of P-selectin expression over untreated cells. Values are mean±SEM (n=3). *P<.0001 for significant decrease in the presence of H8 compared with cells incubated with MM-LDL+histamine in the absence of H8 (H8-).

OX-LDL Triggers P-Selectin Release
OX-LDL is present in atherosclerotic lesions and, like histamine, has been shown to strongly increase Ca²⁺ flux in certain cell types.^{16 17} We found that treatment of HAECs with OX-LDL caused surface expression of P-selectin at 15 minutes, which progressively decreased over the next 2 hours (Fig 8). The amount of release varied for different preparations. Unlike MM-LDL, treatment of HAECs for 6 to 12 hours with OX-LDL did not increase histamine-releasable P-selectin expression (data not shown). This lack of increase was not due to toxicity, as judged by the morphology of the cells.

View larger version (47K): [in this window] [in a new window]   Figure 8. OX-LDL–induced P-selectin surface expression. HAECs were either untreated or treated with 50 µg/mL OX-LDL for 15 minutes (ox15m), 1 hour (ox1h), and 2 hours (ox2h) or with 10-5 mol/L histamine for 15 minutes (hi+15m). An ELISA was performed to detect surface expression of P-selectin. The data are representative of three of three experiments, and the values from two representative experiments are expressed as percent increase in OD of P-selectin expression over untreated cells. EXP-1 and EXP-2 indicate experiments 1 and 2, respectively. Each experiment was performed using preparations of OX-LDL from different donors. Values are mean±SEM (n=3). *P<.005 for significant increase compared with untreated cells.

Effect of Lipid Subfractions on P-Selectin Expression
The effect of lipid subfractions of MM-LDL and OX-LDL on the induction of P-selectin surface expression was examined. HAECs were treated for 24 hours with phospholipid, fatty acid, cholesterol, cholesteryl ester, and triglyceride subfractions obtained from MM-LDL (at levels obtained from 125 µg/mL of MM-LDL), and then these cells were exposed to histamine for 15 minutes and the surface expression of P-selectin was determined (Table). Both the fatty acid and phospholipid fractions were active in inducing histamine-releasable P-selectin; the phospholipid fraction had higher activity. Subfractions of OX-LDL were tested for the ability to cause the release of P-selectin to the surface (Table). HAECs were treated for 15 minutes with subfractions obtained from OX-LDL (at levels obtained from 50 µg of OX-LDL). Only the fatty acid fraction of OX-LDL was found to cause a significant release of P-selectin.

View this table: [in this window] [in a new window]   Table 1. Effect of Subfractions of MM-LDL and OX-LDL

P-Selectin Expression in Atherosclerotic Lesions
To test whether P-selectin expression is actually increased in early atherosclerotic lesions, cross sections of fatty streaks from three vessels and fibrofatty lesions from 10 different coronary vessels of diameter >3 mm taken from heart transplant recipients were examined (the total number of lesions was 13). In all but one of these vessels, the amount of P-selectin over the lesion area was increased compared with that over the nonatherosclerotic lesion areas with thin and adaptively thickened intima. Both the intensity of staining and the percentage of the endothelium containing visible P-selectin were increased. Staining for PECAM-1 showed that endothelium was present in the corresponding lesion and nonlesion areas. P-Selectin staining of the endothelium was not due to platelets adhering to the vessel wall, since P-selectin staining was seen in the absence of staining for GPIIb/IIIa, a platelet marker protein, whereas platelets in another area of the section were stained with this marker (data not shown). A representative vessel with an early fatty streak is presented in Fig 9. A low-magnification micrograph of the vessel stained with anti–PECAM-1 and anti–P-selectin shows that anti–PECAM-1 (Fig 9A) stained endothelial cells in both the lesion and the nonatherosclerotic lesion areas, confirming the presence of the endothelium on the entire inner surface of the artery wall. In contrast, intense P-selectin (Fig 9B) staining was seen only in the lesion area. This is demonstrated in more detail in the higher-magnification micrographs of the nonatherosclerotic lesion areas with thin and adaptively thickened intima (Fig 9C) and the lesion-containing areas (Fig 9D) that stained for PECAM-1 and of the nonatherosclerotic lesion (Fig 9E) and lesion (Fig 9F) areas that stained for P-selectin.

View larger version (89K): [in this window] [in a new window]   Figure 9. Staining of a section of left coronary artery through a region of fatty streak. A, Low magnification of the area of a lesion and nonatherosclerotic lesion areas (with thin and adaptively thickened intima) stained with anti–PECAM-1 (magnification x120). B, Low magnification of area of a lesion and nonatherosclerotic lesion areas (with thin and adaptively thickened intima) stained with anti–P-selectin (magnification x120). C, Higher magnification of the nonatherosclerotic lesion areas stained with anti–PECAM-1 (magnification x750). D, Higher magnification of center of the early atherosclerotic lesion stained with antibody to PECAM-1 (magnification x750). E, Higher magnification of the nonatherosclerotic lesion areas with anti–P-selectin (magnification x750). F, Higher magnification of center of the early atherosclerotic lesion area with anti–P-selectin (magnification x750).

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

 
Recently, increased expression of P-selectin has been observed in endothelial cells overlying human atherosclerotic lesions.²⁶ However, the inducers of P-selectin in the setting of atherosclerosis have not been identified. In these studies, we have demonstrated that MM-LDL increases P-selectin mRNA and protein accumulation in cultured HAECs. It has been previously shown that the effects of MM-LDL on monocyte binding and MCP-1 mRNA accumulation were mediated by an increase in cAMP.³⁶ The data in these studies showed that cholera toxin, which acts by elevating cAMP, also induced the formation of P-selectin (Fig 6) and that H8, a protein kinase A inhibitor, is able to block effects of MM-LDL on P-selectin expression, suggesting that cAMP plays an important role in this particular action of MM-LDL.

The present study shows that MM-LDL causes progressive accumulation of P-selectin intracellularly over a period of 24 hours and that P-selectin levels remain elevated for a further period of 24 hours in the continual presence of MM-LDL (Fig 3). P-Selectin was also found to be available for release 18 hours after the removal of MM-LDL. Using an immunohistochemical technique, Subramanian et al⁴¹ have shown that reinternalized P-selectin persists in the Weibel-Palade bodies for at least 18 hours. The effect of MM-LDL appears to be mediated by the phospholipids present in MM-LDL. We have previously reported that oxidized palmitoyl arachidonyl phosphatidylcholine is the component of MM-LDL responsible for inducing monocyte binding.⁴³

OX-LDL did not induce P-selectin mRNA expression or intracellular accumulation (data not shown), suggesting that during the process of LDL oxidation, a lipid is formed (present in MM-LDL) that is biologically active in inducing P-selectin formation. This lipid then gets further oxidized in OX-LDL and loses some of its biological activity. We have previously noted a decrease in the effect on monocyte binding and tissue factor synthesis by MM-LDL that has been further oxidized.^{12 44}

In contrast to MM-LDL (Fig 2, 10 minutes), OX-LDL caused a rapid release of P-selectin on the HAEC surface similar to the action of histamine (Fig 8). This release was not due to toxicity, since OX-LDL had no apparent cytotoxic effect on HAECs until after a 12-hour treatment (data not shown). It has been suggested that histamine mediates the release of the Weibel-Palade bodies through an increase in intracellular Ca²⁺.⁴⁵ OX-LDL, like histamine, is known to cause an immediate rise in intracellular Ca²⁺ in SMCs¹⁶ and cardiac myocytes,¹⁷ suggesting that the mechanism of P-selectin release by OX-LDL might be mediated by Ca²⁺. The rapid release of P-selectin by OX-LDL seen in the present study is in contrast to the recently published observations of Gebuhrer et al,⁴⁶ who have noted that 1 hour of treatment with copper-oxidized LDL was required for P-selectin surface mobilization in HUVECs, suggesting that a mechanism unlike that of histamine is involved. The difference between the two studies may be due to different biological characteristics of HAECs and HUVECs and/or due to different experimental conditions used by the two groups. In the present study, the fatty acid fraction of OX-LDL induced P-selectin release. Lehr et al⁴⁷ have observed leukocyte rolling and adhesion to hamster venular endothelium within 15 minutes of intravenous OX-LDL administration. They have further shown that this phenomenon of adhesion is mediated by oxidized LDL–induced P-selectin expression on the endothelial cells.⁴⁸

It is conceivable that in humans the early recruitment of the mononuclear cells to the subendothelium in the fatty streak involves rolling on P-selectin, which is induced by minimally oxidized LDL and subsequently triggered to translocate to the surface of endothelial cells by the action of OX-LDL. Consistent with this hypothesis, we noticed that incubation of MM-LDL with HAECs for 48 hours did release a small but significant amount of P-selectin to the surface (Fig 3). This was probably due to further tissue oxidation of MM-LDL by endothelial cells, leading to the formation of OX-LDL. It should be emphasized that this release is small and probably reflects surface expression of P-selectin on a small percentage of the HAECs in the well that are in direct contact with highly oxidized molecules of LDL. Since P-selectin is available for mobilization after long periods of time, the increase in P-selectin protein by MM-LDL and its release by OX-LDL could occur at separate times in lesion development. Recently, both interleukin-3⁴⁹ and interleukin-4⁵⁰ have been shown to induce P-selectin formation and surface expression in HUVECs. It should be noted that the induction of P-selectin by MM-LDL in HAECs is modest compared with the amount of staining seen in endothelial cells overlying fatty streaks (Fig 9). It is possible that MM-LDL might be more potent in vivo or that after the initial recruitment of mononuclear cells induced by oxidized lipids in the subendothelium, other cytokines, such as interleukin-3, interleukin-4, and tumor necrosis factor,⁵¹ may further increase the expression of P-selectin. TNF was found not to induce P-selectin in HAECs (authors' unpublished observation, 1994). Thus far, of the known inducers of P-selectin only oxidized LDL has been shown to be present in early human atherosclerotic lesions, suggesting its potential importance at this stage.

Using both monocyte and neutrophil adhesion assays, we have shown that the MM-LDL–induced P-selectin in HAECs was functional. P-Selectin brings about rolling of both mononuclear cells and neutrophils; however, very few neutrophils are observed in atherosclerotic lesions.^{1 2 3} If P-selectin is important in early atherosclerosis, this discrepancy needs to be addressed. One explanation for the lack of neutrophils in the atheromatous lesions containing P-selectin is that molecules mediating activation and firm adhesion of neutrophils are lacking. We have previously shown that MM-LDL actually downregulates E-selectin expression.³⁶ Recently, an additional mechanism for mononuclear cell rolling via VCAM-1 and ₄-integrin interaction has been described, which could also play an important role in atherogenesis, although this rolling can occur only at very low shear stress levels generally seen only in venules.^{52 53}

In conclusion, we have shown that in cultured HAECs, MM-LDL induced accumulation of P-selectin, which was released to the surface as a functional molecule by products of further oxidation of LDL(OX-LDL). We have shown that endothelial cells of overly fatty streak lesions of human coronary arteries express significantly increased amounts of P-selectin and have confirmed that P-selectin is increased in fibrofatty lesions. These observations suggest that P-selectin plays an important role in the process of early mononuclear recruitment during the initial stages of atherogenesis.

	Selected Abbreviations and Acronyms

 

ELISA = enzyme-linked immunosorbent assay HAEC = human aortic endothelial cell HUVEC = human umbilical vein endothelial cell ICAM-1 = intercellular adhesion molecule 1 LDL = low-density lipoprotein LPS = lipopolysaccharide MCP-1 = monocyte chemotactic protein 1 MCSF = monocyte colony stimulating factor MM-LDL = minimally modified LDL OD = optical density OX-LDL = highly oxidized LDL PECAM-1 = platelet endothelial cell adhesion molecule 1 SMC = smooth muscle cell VCAM-1 = vascular cell adhesion molecule 1

	Acknowledgments

 
This study was supported by National Institutes of Health grants HL-30568 and HL-07412 and by a grant from the Laubisch Fund. We would like to thank Deborah Schwartz and Cynthia Harper for excellent technical assistance. We also thank Denisa Wagner for helpful advice.

	Footnotes

 
Presented at the 67th Scientific Sessions of the American Heart Association, Dallas, Tex, November 14-17, 1994, in abstract form (Circulation. 1994;90[suppl I]:I-83.).

Received December 12, 1996; accepted March 17, 1997.

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