Oxidized LDL promotes vascular endothelial cell pinocytosis via a prooxidation mechanism

^a Department of Biology, National Taiwan Normal University, Taipei, Taiwan
^b Center for General Studies, Chang Gung University, Taoyuan, Taiwan
^c Department of Physiology, College of Medicine, Chang Gung University, Taoyuan 333, Taiwan

	ABSTRACT

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Human low density lipoprotein (LDL) is prepared in the presence of antioxidants and is oxidized to different levels (measured by thiobarbituric acid reactive substance) with copper ion. The effects of unoxidized LDL and oxidized LDL (ox-LDL) on stress fiber formation, cell membrane ruffling, and pinocytosis (measured by [¹⁴C]sucrose uptake) in cultured human umbilical cord vein endothelial cells (EC) are compared. We show that at a concentration range of 100 to 200 µg cholesterol/ml, both unoxidized LDL and ox-LDL promote EC elongation and stress fiber formation, but the effect by the latter is more prominent when compared at the same dose range. In addition, ox-LDL also induces EC membrane ruffling and promotes pinocytosis. These effects are positively correlated with the extent of LDL oxidation and depend on the dose of ox-LDL. Ox-LDL-promoted membrane ruffling and pinocytosis are effectively blocked by brief preexposure of the cells to antioxidants. In contrast, stress fiber formation is not affected by antioxidant pretreatment. Although unoxidized LDL also promotes [¹⁴C]sucrose uptake, it is less potent than ox-LDL and significantly higher concentrations are required to produce a detectable effect. Unlike ox-LDL, unoxidized LDL-enhanced pinocytosis is not accompanied by the appearance of membrane ruffling; therefore, they may act via different mechanisms. Elevated pinocytosis may increase transcytotic activity of the endothelium, leading to an increased influx of plasma components such as LDL into the subendothelial space.—Chow, S.-E., Lee, R.-S., Shih, S. H., Chen, J.-K. Oxidized LDL promotes vascular endothelial cell pinocytosis via a prooxidation mechanism. FASEB J. 12, 823–830 (1998)

Key Words: membrane ruffling • EC • antioxidant • ROI • pinocytotic activity • low density lipoprotein

	INTRODUCTION

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ELEVATED LEVELS of low density lipoprotein (LDL)² cholesterol are a major risk factor for atherosclerotic disease (1). The earliest atherosclerotic lesion, the fatty streak, begins with the adherence of monocytes to a structurally intact layer of endothelial cells. The adhesion of circulating monocytes to the endothelium cannot be mimicked by exposing vascular cells to high concentrations of native LDL. However, these events can easily be observed in vitro after the oxidative modification of LDL (ox-LDL). Oxidative modification increases the atherogenicity of LDL (2–5). This theory is based on observations that oxidative modification promotes uptake and retention of circulating LDL in the arterial wall. Ox-LDL exerts a number of atherogenic effects on arterial wall cells, including the induction of colony-stimulating factors (6), monocyte chemotactic protein (7), and adhesion molecules (8, 9), hallmarks of early proatherogenic responses. It has also been reported that ox-LDL is rapidly taken up by macrophages via scavenger receptors (10) and stimulates the formation of foam cells.

In vivo evidence suggests that ox-LDL is generated in the subendothelial space of atherosclerotic lesions by prooxidant compounds released by endothelial cells (EC), smooth muscle cells, and possibly macrophages (11). Ox-LDL has thus been considered a major culprit that induces activation or dysfunction of the endothelial cells associated with the initiation of the atherosclerotic lesions (12). Alterations of the structural and/or functional integrity of the endothelial barrier allow a net influx of lipoproteins from the circulating plasma into the subendothelium. The mechanisms underlying the observed endothelial dysfunction elicited by ox-LDL have not been completely elucidated, although increased vascular oxidative stress has recently been suggested as a possible cause (13); ox-LDL may exert a prooxidant effect, resulting in alterations of the endothelial morphology and functions.

Blood-borne macromolecules are captured and internalized by vascular EC by endocytosis and/or pinocytosis. It has been shown in fibroblasts, Chinese hamster ovary cells, and epithelial cells that membrane ruffling is closely related to endocytotic activity (14–16). Membrane ruffling is also associated with increased pinocytosis, and membrane ruffles may form vesicles that pinch off and are internalized (14, 16). These membrane processes have been shown to be regulated by some actin binding proteins (17) that, in vascular EC, were shown to be activated by oxygen free radicals (18). Increased EC endocytosis is likely to be very important in the maintenance of vascular wall homeostasis and may be functionally altered in pathological events such as atherosclerosis. It has been demonstrated that the administration of antioxidant compounds [probucol, butylated hydroxytoluene (BHT), vitamin E, etc.] significantly prevents the oxidative modification of LDL and slows the progression of experimental atherosclerosis in animals (14). In the present study, we show that ox-LDL induces membrane ruffling and promotes pinocytosis in cultured vascular EC and that such effects are effectively blocked by antioxidant pretreatment, suggesting the involvement of a prooxidation mechanism.

	MATERIALS AND METHODS

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Endothelial cell culture
Endothelial cells were isolated from human umbilical cord veins as described previously (19). Cells were cultured in medium MCDB 107 supplemented with 2% fetal bovine serum (FBS) and a fibroblast growth factor-enriched fraction of porcine brain extract (1 µg/ml). Cells were incubated in an atmosphere of 5% CO₂/95% air at 37°C in a humidified incubator.

Preparation of LDL and lipoprotein-deficient serum
Low density lipoprotein (d=1.015–1.075 g/ml) was prepared from fresh human plasma by sequential ultracentrifugal flotation (20). LDL fraction was dialyzed at 4°C against 0.9% NaCl containing 0.1 mM ethylenediamine tetraacetic acid (EDTA) and 5 µM BHT (pH 7.4). Lipoprotein-deficient serum (LPDS, d>1.25 g/ml) was prepared from FBS by ultracentrifugation in sodium bromide (d=1.21 g/ml). After centrifugation, the upper half of the solution was discarded (to d=1.21 g/ml) and the bottom half (LPDS) was dialyzed extensively against phosphate-buffered saline (PBS) before use. Since the last step of LPDS preparation was extensive dialysis against PBS, it is assumed that any endogenous soluble antioxidants in the LPDS had all been removed.

Preparation of oxidized LDL
Oxidation of LDL was performed by dialysis against EDTA-free isotonic saline containing 5 µM CuSO4 at 37°C for 8 h. Oxidation was stopped by the addition of EDTA to a final concentration of 100 µM and the copper ion was removed by extensive dialysis against isotonic saline containing 0.1 mM EDTA at 4°C. LDL was sterilized by passage through a 0.22 µm filter. The extent of oxidation of the LDL preparations was measured by the thiobarbituric acid reactivity assay as described (21). Malondialdehyde (MDA) was used as a standard, and the thiobarbituric acid-reactive substance (TBAR) values were recorded as equivalents of MDA/mg LDL protein. LDL protein was determined by the method of Lowry et al. (22). In our hands, the unoxidized LDL had TBAR values of ~0.4 nmol/mg. By adjusting the oxidation time, ox-LDL was prepared with TBAR values of 3.6 to 4.9.

Determination of pinocytotic activity
Pinocytosis, expressed as nanoliters of bulk phase fluid taken up by EC, was determined by measuring the uptake of [¹⁴C]sucrose (23). EC were incubated in medium MCDB 107 supplemented with 10% LPDS alone, LPDS plus LDL, or ox-LDL (100–200 µg/ml of cholesterol) for various lengths of time; [¹⁴C]sucrose was added to a final concentration of 9 µCi/ml and the cultures were incubated for an additional 3 h. After incubation, cells were washed three times with basal medium, reincubated in basal medium for 10 min at 37°C, and rewashed twice. The cells were detached from the plates with trypsin/EDTA and dissolved in 0.1% sodium dodecyl sulfate (SDS). Cell-associated radioactivity was measured by scintillation counting. Parallel dishes were prepared and processed as in the experiments (except that no radioactive sucrose was added) and used to determine cell number.

Fluorescent staining of cytoskeleton
Endothelial cell cytoskeleton was fluorescently stained with rhodamine phalloidin, a fluorescent derivative of phallotoxin from Amanita phalloides that binds with high affinity to F-actin (24). EC were plated on 4-well chamber slides at 5 x 10⁴ per cm² and incubated for 24 h with ox-LDL, lysoPC, or oxidized cholesterol (such as 7-ketocholesterol, 25-hydroxycholesterol). After incubation, cells were washed twice with PBS, fixed with methanol:acetone (1:1) for 10 min at -20°C, and air dried. An appropriate amount of PBS rhodamine phalloidin solution (20:1, v/v) was added per well and incubated for 20 min at 37°C. Cells were then washed twice with PBS. Cell shape and cytoskeleton were examined under a Nikon Biophot fluorescent microscope. Photographs were taken using a Nikon UFX camera system and Kodak TMAX 400 film at a magnification of x400.

Materials
Medium MDCB-107 was from JRH Biosciences (Lenexa, Kans.); FBS was from GIBCO-BRL (Gaithersburg, Md.). [¹⁴C]Sucrose (specificity activity, 1.70 mCi/mg) was from Amersham (Buckinghamshire, England). Sodium bromide, SDS, and EDTA were from Merck (Darmstadt, Germany). Rhodamine phalloidin was from Molecular Probes (Eugene, Oreg.); all other chemicals were from Sigma (St. Louis, Mo.).

cis-Resveratrol, a natural antioxidant present in red wine, was synthesized as described (25) and purified by silica gel column chromatography (ethylacetate:n-hexane, 1:2, v/v) and recrystalization (ethylacetate:n-hexane, 1:1, v/v).

	RESULTS

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The morphology of EC exposed to unoxidized LDL (TBAR=0.37 nmol/mg) or ox-LDL (TBAR=4.42 nmol/mg) was compared under the same culture conditions. Cells treated with unoxidized LDL for 24 to 48 h exhibited the flattened cobblestone shape of untreated cells, but were more extended. Rhodamine phalloidin staining showed a marked increase in the stress fiber in unoxidized LDL-treated cells. Exposure of cells to ox-LDL at the same concentration ranges induced extensive cell elongation to assume a fibroblastic morphology. The average long and short axes of the control cells were 56 ± 19 µm and 18 ± 4 µm; of the unoxidized LDL-treated cells, 74 ± 7 µm and 17 ± 4 µm; and 124 ± 10 µm and 15 ± 5 µm of the ox-LDL-treated cells. In contrast to unoxidized LDL, ox-LDL exposure also caused EC membrane ruffling ( Fig. 1C, arrows); rhodamine phalloidin staining showed prominent stress fibers running parallel to the long cell axis and accumulation of F-actin under the membrane ruffles ( Fig. 1C). The extent of membrane ruffling appeared to be dose-dependently affected by ox-LDL. The changes in cell surface morphology were reproducible with different preparations of ox-LDL. To see whether one or more lipid oxidation products of ox-LDL are responsible for inducing membrane ruffling, we treated cells with various concentrations of lysophosphatidyl choline, 7-ketocholesterol, or 25-hydroxycholesterol for the same period of time and examined their morphology after rhodamine phalloidin staining. None induced membrane ruffling, but did induce stress fiber formation, especially in cells treated with 7-ketocholesterol ( Fig. 2).

View larger version (84K): [in this window] [in a new window]   Figure 1. Representative fluoresent photomicrographs of the EC cytoskeleton stained with rhodamine phalloidin. Cells were treated with LDL or ox-LDL (both at 200 µg cholesterol/ml) for 24 h or left untreated. Cells were then stained with rhodamine phalloidin and photomicrographed, as described in the text. A) Control; B) LDL-treated; C) ox-LDL treated. Original magnification, 400x.

View larger version (111K): [in this window] [in a new window]   Figure 2. Effect of oxidized lipid products of ox-LDL on the morphology of EC cytoskeleton. EC were treated with 2.5 µM lysoPC, 7-ketocholesterol, or 25-hydroxycholesterol for 24 h and stained with rhodamine phalloidin, as described. A) Control; B) lysoPC; C) 7-ketocholesterol; D) 25-hydroxycholesterol. Original magnification, 400x.

Cell membrane ruffling has been shown to be closely associated with pinocytotic activity (14–16). To see whether ox-LDL-induced membrane ruffling is associated with an increase in fluid phase pinocytosis, we compared [¹⁴C]sucrose (9 µCi/ml) uptake by vascular EC with and without unoxidized LDL or ox-LDL preexposure. Exposure of cells to unoxidized LDL for 24 to 48 h exerted little stimulation on uptake of [¹⁴C]sucrose compared to that of the control cells. As shown in Fig. 3, unoxidized LDL (TBAR=0.37 nmol/mg) at 100 µg cholesterol/ml had little effect on EC pinocytosis. When assayed at 200 µg cholesterol/ml, there was a twofold stimulation. In contrast, the [¹⁴C]sucrose uptake by ox-LDL (TBAR=4.42 nmol/mg) -exposed cells assayed under the same conditions was enhanced approximately three- to fourfold over the control. The enhanced pinocytotic activity was dose dependent on ox-LDL ( Fig. 3) and is positively related to the extents of LDL oxidation (see Fig. 3and Fig. 6B). Unoxidized LDL and ox-LDL are internalized through different types of cell surface receptors (26). To rule out that the differential pinocytotic activity observed is due to the differential activation of LDL and ox-LDL receptor cycles because of the continuous exposure of EC to these lipoproteins during the entire experimental period, cells were pretreated with LDL or ox-LDL for 24 h; the lipoprotein-containing medium was then removed and the cells were rinsed twice with basal medium. [¹⁴C]Sucrose uptake assay showed that ox-LDL-pretreated cells still exhibited higher pinocytotic activity than LDL-pretreated cells ( Fig. 4A). In another experiment, cells were pretreated with unoxidized LDL or ox-LDL for 3 h and their pinocytotic activity was compared with that of the control cells. Figure 4B shows that a brief 3 h exposure to lipoprotein had no effect on EC pinocytotic activity.

View larger version (16K): [in this window] [in a new window]   Figure 3. Effect of LDL and ox-LDL on EC pinocytosis. EC were incubated with 10% LPDS alone, 10% LPDS plus LDL, or ox-LDL at concentrations indicated for 24 h. Pinocytotic activity was measured by [14C]sucrose uptake and expressed as mean SEM from three separate experiments, each with triplicate incubations.

View larger version (33K): [in this window] [in a new window]   Figure 6. Effect of antioxidants on ox-LDL-induced EC pinocytosis. Cells were pretreated with -tocopherol (23 µM), BHT (10 µM), or cis-resveratrol (15 µM) for 30 min, as indicated, in 1% FBS. After pretreatment, the medium was replaced with the same medium but without antioxidant, and LDL (A) or ox-LDL (B) was added to a final concentration of 100 µg cholesterol/ml. Cells were then incubated for 24 h and [14C]sucrose uptake was measured, as described. Data from each experimental group were compared with the control group by analysis of variance. B) *P value less than 0.001.

View larger version (34K): [in this window] [in a new window]   Figure 4. Effect of LDL and ox-LDL preexposure on EC pinocytosis. A) EC were incubated for 24 h with 1% FBS, 1% FBS plus LDL (100 µg cholesterol/ml), or ox-LDL (100 µg cholesterol/ml). After removal of the lipoprotein-containing medium, cells were rinsed twice with 1% FBS and [14C]sucrose uptake was measured as described. B) EC were incubated with 10% LPDS, 10% LPDS plus LDL (100–200 µg cholesterol/ml), or ox-LDL (100 to 200 µg cholesterol/ml) for 3 h as indicated. Pinocytotic activity was measured by [14C]sucrose uptake. Data from each experimental groups were compared with the control group by analysis of variance. A) *P value less than 0.001; B) *P value greater than 0.05.

Because none of the ox-LDL lipid oxidation products tested are able to induce membrane ruffling and enhance pinocytosis, as is seen with the whole ox-LDL particle, it is assumed that ox-LDL may act via a prooxidation mechanism to influence cell behavior. To test this hypothesis, EC were pretreated with or without -tocopherol (23–46 µM), BHT (10 µM to 20 µM), or cis-resveratrol (15–30 µM) for 30 min before exposure for 24 h to unoxidized LDL (TBAR=0.37 nmol/mg) or ox-LDL (TBAR=4.94 nmol/mg). After treatment, cells were stained with rhodamine phalloidin, followed by membrane morphology examination, or were subjected to an [¹⁴C]sucrose uptake experiment. The results showed that the pinocytotic activity of ox-LDL treated cells was enhanced by fivefold (see Fig. 6B) and that antioxidant pretreatment effectively blocked ox-LDL-induced membrane ruffling ( Fig. 5) and pinocytotic activity ( Fig. 6B). The formation of stress fiber was not suppressed. In contrast, Fig. 6A shows that similar treatment with unoxidized LDL had little effect on pinocytosis, and pretreatment of cells with antioxidants did not alter the pinocytotic behavior of the cells.

View larger version (106K): [in this window] [in a new window]   Figure 5. Effect of antioxidants on ox-LDL-induced membrane ruffling in EC. Cells were preexposed to -tocopherol (23 µM), BHT (10 µM), or cis-resveratrol (15 µM) for 30 min or untreated in basal medium containing 1% FBS. The medium was then replaced with the same medium containing no antioxidant and ox-LDL was added to a final concentration of 200 µg cholesterol/ml. Cells were incubated an additional 24 h and then processed for fluorescent photomicrography. A) Ox-LDL (200 µg/ml). B) -Tocopherol + ox-LDL. C) BHT + ox-LDL. D) cis-Resveratrol + ox-LDL. Original magnification, 400x.

	DISCUSSION

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The most interesting finding in this study is that ox-LDL promotes stress fiber formation, membrane ruffling, and pinocytosis in cultured EC, and the latter two effects are effectively blocked by pretreatment of cells with various antioxidants. Unoxidized LDL also enhances EC pinocytosis, but it is less potent than ox-LDL and requires significantly higher concentrations to produce a comparable effect ( Fig. 3). Unoxidized LDL induced increase in pinocytosis is not accompanied by the appearance of membrane ruffling, which suggests that the acting mechanism may be different.

LDL oxidation does not occur in the presence of serum and requires trace amounts of redox-active metals such as copper and iron, suggesting that LDL oxidation in vivo is more likely to occur in the subendothelium, i.e., in the arterial walls (27). Endothelial cells, smooth muscle cells, monocytes, macrophages, and lymphocytes are all capable of oxidizing LDL, and lipoxygenase and nitric oxide have been suggested to be involved (28, 29). Oxidized LDL has been reported to promote the synthesis of adhesion molecules (such as endothelial leukocyte adhesion molecule 1, intercellular adhesion molecule 1, and vascular adhesion molecule 1) (8, 9), monocyte chemotactic protein 1 (7), colony-stimulating factors (6), protein C (30), and basic fibroblast growth factor (31) by EC. It has also been shown to impair endothelial cell-derived relaxation factor secretion (32) and to enhance endothelial cell-derived contraction factor (33) and prostaglandin secretion (34). Thus, ox-LDL has been suggested to be one of the major causative factors for the accelerated atherosclerosis and coronary heart disease in the hypercholesterolemic subjects. Nevertheless, the mechanism underlying the atherogenic nature of ox-LDL has not been fully elucidated.

Increased infiltration and retention of LDL in the subendothelium presumably subject LDL to oxidative stress, leading to its oxidation. It is therefore tempting to speculate that increased transcytosis due to increased endocytotic and/or pinocytotic activity of the endothelium under atherogenic levels of plasma LDL may be implicated in the pathogenic processes. A report has shown that high concentrations of LDL enhances the endocytotic activity of cultured EC (35). In contrast, another report (36) shows that LDL isolated in the absence of antioxidant reduces EC endocytosis. Endocytotic and pinocytotic activities were not differentiated in these reports, however, and so it was not clear whether the effect observed was due to activation of the LDL receptor pathway or to increased membrane ruffling. In the present report, we clearly show that preincubation of EC with ox-LDL induces stress fiber formation, membrane ruffling, and increased pinocytosis. Since a brief 3 h exposure of EC to ox-LDL did not enhance pinocytosis and the enhanced [¹⁴C]sucrose uptake was sustained for some time after ox-LDL removal, it is unlikely that the observed enhancing effect on pinocytosis was due to activation of the ox-LDL receptor pathway. The effect by ox-LDL is positively correlated with the oxidation levels of LDL used. Ox-LDL with a TBAR of 4.42 nmol/mg exerted more than a threefold promotion in pinocytosis ( Fig. 3), whereas ox-LDL with a TBAR of 4.94 nmol/mg showed a close to fivefold ( Fig. 6B) stimulation compared to control cells when both were assayed at 100 µg cholesterol/ml. Similar treatment with unoxidized LDL (isolated in the presence of EDTA and BHT) also induced stress fiber formation, but did not promote membrane ruffling and only slightly stimulated pinocytosis. Thus, our results are discordant with that reported by Holland et al. (35) and Børsum et al. (36). These discrepancies are probably due to differences in the ways EC were cultured and handled, the procedures used to prepare LDL and ox-LDL, or the different experimental conditions used.

Reactive oxygen intermediates (ROIs) such as hydrogen peroxide, superoxide, and hydroxyl radical are reactive oxygen radicals encountered frequently in biological systems (37). ROIs have been shown to activate numerous growth factor receptors and transcription factors, leading to altered expression of certain genes thought to be implicated in atherosclerosis (38–40). In addition to oxidized lipids and oxysterols, ox-LDL releases superoxide anion and therefore is considered a strong prooxidant (3). That ox-LDL-elicited membrane ruffling and increased pinocytotic activity in EC are blocked by preexposure to antioxidants, including -tocopherol, BHT, and cis-resveratrol, is consistent with the assumption that prooxidation mechanism is involved. Moreover, hydrogen peroxide has recently been shown by others (41) to induce EC stress fiber formation and pinocytosis, which lends further support to the idea that ox-LDL acts via a prooxidation pathway.

Retention of LDL and its subsequent oxidation in the subendothelium are believed to be an early event involved in atherogenesis. The effect of ox-LDL on monocyte adherence, foam cell formation, and EC activation have been well documented and established. But how hypercholesterolemia promotes subendothelial LDL retention/oxidation has not been fully elucidated. LDL may infiltrate into arterial wall by diffusing through endothelial cell–cell junctions and/or by transcytosis. Increased transcytosis may be a result of increased endocytosis and/or pinocytosis. Thus, elevated endocytotic and/or pinocytotic activity of the EC may disrupt the arterial wall homeostasis and favor LDL deposition and oxidation. Ox-LDL generated in the subendothelium may then promote additional transcytotic transport of plasma LDL into the arterial wall by the enhanced EC pinocytosis. These cellular changes may be an even earlier proatherogenic event elicited by hypercholesterolemia.

	ACKNOWLEDGMENTS

 
This study was supported by grants from the National Science Council, Taiwan (NSC 87–2314-B182–086), and Chang Gung Memorial Hospital (CMRP 678), respectively, to J.-K.C. The authors are grateful to Ms. Jin-Yi Chen for her assistance with graphing.

	FOOTNOTES

 
¹ Correspondence: Department of Physiology, College of Medicine, Chang Gung University, Taoyuan 333, Taiwan. E-mail: JKC508{at}cguaplo.cgu.edu.tw

Received for publication January 2, 1998. Accepted for publication February 2, 1998.

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