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(Circulation. 2000;101:1799.)
© 2000 American Heart Association, Inc.

Clinical Investigation and Reports

Enzymatically Degraded, Nonoxidized LDL Induces Human Vascular Smooth Muscle Cell Activation, Foam Cell Transformation, and Proliferation

Mariam Klouche, MD; Stefan Rose-John, PhD; Walther Schmiedt, MD; Sucharit Bhakdi, MD

From the Institute of Medical Microbiology (M.K., S.B.), Department of Internal Medicine (S.R.-J.), Section of Pathophysiology and Department of Heart and Thoracic Surgery (W.S.), University of Mainz, Germany.

Correspondence to Dr Mariam Klouche, Institute of Medical Microbiology, University of Mainz, Hochhaus am Augustusplatz, 55101 Mainz, Germany. E-mail: klouche{at}mail.uni-mainz.de

	Abstract

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Background—Enzymatic, nonoxidative modification transforms LDL to an atherogenic molecule (E-LDL) that activates complement and macrophages and is present in early atherosclerotic lesions.

Methods and Results—We report on the atherogenic effects of E-LDL on human vascular smooth muscle cells (SMC). E-LDL accumulated in these cells, and this was accompanied by selective induction of monocyte chemotactic protein-1 in the absence of effects on the expression of interleukin (IL)-8, RANTES, or monocyte inflammatory proteins-1 and -ß). Furthermore, E-LDL stimulated the expression of gp130, the signal-transducing chain of the IL-6 receptor (IL-6R) family, and the secretion of IL-6. E-LDL invoked mitogenic effects on SMC through 2 mechanisms. First, an autocrine mitogenic circuit involving platelet-derived growth factor and fibroblast growth factor-ß was induced. Second, upregulation of gp130 rendered SMC sensitive to transsignaling through the IL-6/sIL-6R activation pathway. Because E-LDL promoted release of both IL-6 and sIL-6R from macrophages, application of macrophage cell supernatants to prestimulated SMC provoked a pronounced and sustained proliferation of the cells.

Conclusions—E-LDL can invoke alterations in SMC that are characteristic of the evolving atherosclerotic lesion.

Key Words: atherosclerosis • lipoproteins • cells • muscle, smooth • enzymes

	Introduction

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Atherosclerosis is an inflammatory disease¹ in which a cascade of events involving C-reactive protein (CRP) deposition, complement activation, selective accumulation of monocytes and T-lymphocytes, activation of endothelial cells, and expression of chemokines and cytokines is elicited locally by tissue-deposited, modified lipoproteins.²

Mainstream researchers regard oxidation to be the central process that transforms LDL into an atherogenic molecule. We are advancing an alternative concept,³ however, that has evolved from the need to account for a number of findings that have not been consistent with the oxidation paradigm. We propose that LDL stranded in the connective matrix of the intima is subject to enzymatic, nonoxidative degradation by ubiquitous proteases and cholesterol esterase. Enzymatically altered LDL (E-LDL) is atherogenic because it binds CRP, thus activating complement,^{4 5} and because it provokes macrophage foam cell formation⁴ and liberation of monocyte chemotactic protein-1 (MCP-1) and interleukin (IL)-6.⁶ At high concentrations, E-LDL elicits cytotoxic effects on macrophages that may be due to apoptosis.⁶ Further, E-LDL induces upregulation of adhesion molecules on endothelial cells and promotes the selective transmigration of monocytes and T cells to the lesion.⁷ With the use of specific monoclonal antibodies, E-LDL has been shown to be present in all early human lesions.⁸ E-LDL colocalized extracellularly with CRP and with terminal complement complexes.^{5 8 9} Importantly, lipoprotein derivatives that were isolated from early atherosclerotic lesions indeed exhibited ultrastructural and biochemical features in common with E-LDL^{10 11 12} and had the capacity to activate complement.¹⁰

Because E-LDL is now emerging as a novel candidate to assume the role of the prime atherogenic species, we proceeded to investigate whether its atherogenic effects may extend to smooth muscle cells (SMC). We show that E-LDL indeed alters SMC homeostasis and identify a novel pathway that could be responsible for SMC proliferation. Thus, E-LDL can produce classic changes in SMC function that are characteristic of the developing atherosclerotic lesion.

	Methods

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Preparation and Culture of Human Vascular SMC
Human vascular SMC were cultured from pieces of human aortas obtained during aneurysm surgery (15 donors, mean age 72 years, 10 men and 5 women) by outgrowth from explanted media fragments.¹³ More than 90% of the cells were positive for -actin (clone 1A4, Sigma) and showed the typical "hill-and-valley" morphology. As a negative control, cells cultivated in DMEM supplemented with the enzyme mix used for the preparation of E-LDL were used. All experiments were performed after <5 passages of culture.

Lipoprotein Isolation and Modifications
Native human LDL was isolated,⁶ and enzymatic modification with trypsin, cholesterol esterase, and neuraminidase was undertaken as described.⁴ The ensuing E-LDL preparations comprised a population of fused LDL particles with diameters of 25 to 500 nm. The apolipoprotein B was extensively degraded, and the ratio of free to esterified cholesterol was 1:1. The concentration of free fatty acids was not determined. Phospholipids remained quantitatively associated with the E-LDL particles. For acetylation, the standard protocol of Goldstein et al¹⁴ was used. Lipid peroxidation was excluded by the absence of TBARS.¹⁵ Lipoproteins were stored at 4°C and used within 1 to 2 weeks.

Staining of Foam Cells
SMC were fixed in 10% formaldehyde for 30 minutes at room temperature and intracellular lipids were stained with a saturated solution of oil-red-O for 6 hours.

Quantification of Cholesterol Uptake
Cellular cholesterol uptake was measured by the classic method described by Goldstein et al.¹⁶ Cells were incubated with lipoproteins in the presence of [³H]oleic acid (500 nmol/L; specific activity 1.8 to 2.2x10⁷ cpm/mL) for 12 hours at 37°C. Chromatography of the extracted lipids was performed on silica gel–TLC plates (Machery & Nagel), and radioactivity was detected in a ß-counter (LKB-ß, Beckman Instruments). The oleic acid standard (Sigma) was used for calibration.

Reverse Transcription–Polymerase Chain Reaction of Chemokine and Cytokine mRNA
RNA was isolated from confluent SMC cultures by guanidine isothiocyanate–phenol–chloroform extraction as described by Chomczynski and Sacchi.¹⁷ Reverse transcription and reverse transcription–polymerase chain reaction was conducted as described.^{6 13} Samples lacking cDNA or RNA served as negative controls. Polymerase chain reaction products were run on 1% agarose gels in 1xTBE and stained with ethidium bromide. Amplification of a defined IL-6R sequence by the primers selected was ascertained by a positive signal with HepG2 cells.

Quantification of Chemokines and Cytokines
Chemokines and cytokines were determined by commercial immunoassay (IL-6, MCP-1, RANTES, MIP-1, and MIP-1ß, R&D Biosystems; IL-8, Innogenetics).

Chemotaxis Assay
Monocyte chemotaxis assays were performed as described previously.⁶ Supernatants of E-LDL–treated SMC were assayed in dilutions from 1:1 up to 1:500. As controls, cell-free medium or supernatant dilutions containing a neutralizing monoclonal anti–MCP-1 antibody (1:200) were used (clone 24822.111, IgG1; R&D Biosystems). The chemotactic index was calculated as described previously.⁶

Immunocytochemical Detection of Surface gp130 Expression
Surface expression of gp130 was analyzed on confluent SMC cultures with a PE-labeled anti-gp130 overnight at 4°C (clone AM64, IgG1; PharMingen), as described.¹³ Cells grown in the absence of lipoproteins served as controls.

Proliferation of SMC
SMC proliferation was assayed by [³H]thymidine incorporation into trichloroacetic acid (TCA; 5% wt/vol) precipitable material, as described.¹³ Cells (2x10⁴/mL) were seeded in 24-well culture plates, grown for 24 hours in complete medium, and kept in medium without additives for further 24 hours. After stimulation, cultures were pulsed with [³H]thymidine (Amersham, 2 to 10 µCi/mL) for the final 6 hours of incubation, and TCA-precipitable radioactivity was quantified in a scintillation counter (LKB-ß; Beckman). SMC cultures grown in the presence of 10% human AB-serum served as positive controls. SMC grown in medium alone or in medium containing the enzyme mix used for the preparation of E-LDL served as unstimulated controls.

Detection of Cellular Toxicity and Apoptosis
Cytotoxic effects of E-LDL on SMC were determined by quantification of the reduction of intracellular ATP.⁶ Apoptosis was detected by analysis of DNA fragmentations and by the TUNEL labeling technique, as described.⁶

Statistical Analysis
The significance of differences between control values and the stimulants was determined by the Mann-Whitney U test. Differences were considered significant at a level of P<0.05. The results are expressed as mean±SD. Analysis was performed with SPSS software.

	Results

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Foam Cell Transformation of SMC Induced by E-LDL
After incubation of human SMC with 50 µg/mL E-LDL for 6 hours, lipid staining with oil-red-O became positive. Native LDL did not invoke detectable foam cell transformation (Figure 1).

View larger version (80K): [in this window] [in a new window]   Figure 1. Foam cell transformation of human SMC after incubation with 50 µg/mL E-LDL (left) but not with native LDL (right), as shown by staining with oil-red-O.

Measurement of cholesterol-oleate confirmed cellular accumulation of the sterol. Accumulation of cholesterol-oleate commenced after 1 hour of incubation with E-LDL, was maximal after 12 hours, and did not increase thereafter. Native LDL was not amassed (Figure 2).

View larger version (14K): [in this window] [in a new window]   Figure 2. Quantification of cholesterol uptake by SMC. Cholesteryl oleate formation was measured 12 hours after incubation of SMC with native LDL, E-LDL, or acetylated (Ac) LDL (each at 25 µg/mL cholesterol). Bars indicate mean value±SD of quadruplicate determinations. *Differences were significant at P<0.02. Results were reproduced in 6 separate experiments.

Selective Induction of MCP-1 mRNA Expression by E-LDL in SMC
E-LDL stimulated a pronounced, time-dependent expression of MCP-1 mRNA in SMC that was expressed after 4 hours, peaked between 12 and 48 hours, and disappeared after 72 hours (Figure 3). MCP-1 mRNA was only weakly detectable in control cells. In contrast, expression of mRNAs encoding the chemokines IL-8, RANTES, or monocyte inflammatory protein-1 or -1ß (MIP-1, MIP-1ß) could not be detected (data not shown).

View larger version (91K): [in this window] [in a new window]   Figure 3. Kinetics of MCP-1 (A) and IL-6 (B) mRNA expression after stimulation of SMC with 50 µg/mL E-LDL. Expression of GAPDH is shown (C). M indicates DNA marker; C, unstimulated control SMC. Lanes represent time of incubation (h) with E-LDL (25 µg/mL).

Concomitantly, MCP-1 was liberated into the culture supernatants. Maximal levels of 1365±215 pg/mL (n=3) were observed after 48 hours of stimulation. Secreted MCP-1 was biologically active, and a typical bell-shaped dose-response curve with a maximal chemotactic index of 3.1 at 10 ng/mL MCP-1 was observed in chemotaxis assays. Specificity was confirmed by a neutralizing anti–MCP-1 antibody, which caused marked reduction of the chemotactic activity. Secretion of IL-8, RANTES, MIP-1, and MIP-1ß remained unaltered.

Induction of gp130 and IL-6 in SMC
Quiescent human vascular SMC expressed scant amounts of gp130 mRNA and no detectable IL-6 receptor (IL-6R) or IL-6 mRNA. Remarkably, E-LDL prominently induced gp130 mRNA. Expression of gp130 was detected after 4 hours of stimulation, peaked at 24 to 48 hours, and vanished thereafter (Figure 4). Basal gp130 expression was undetectable in more than half of the primary SMC cultures analyzed. E-LDL also promoted expression of IL-6 mRNA, which lagged 4 to 8 hours behind the induction of gp130 mRNA (Figure 3).

View larger version (46K): [in this window] [in a new window]   Figure 4. Amplification of gp130 mRNA expression in human vascular SMC by E-LDL. Time-dependent expression of gp130 mRNA after stimulation of 4 different primary SMC cultures (SMC10, SMC18, SMC19, SMC21) with 25 µg/mL E-LDL is shown. Induction of gp130 preceded that of IL-6 by 8 hours. M indicates DNA marker; , unstimulated control.

Surface expression of gp130 detected by immunocytochemistry was weak in unstimulated human vascular SMC (Figure 5, top) and greatly enhanced after stimulation with 50 µg/mL E-LDL for 8 hours (Figure 5, bottom). Prolonged exposure to E-LDL produced no further enhancement of gp130 surface expression.

View larger version (33K): [in this window] [in a new window]   Figure 5. Induction of gp130 surface protein expression by E-LDL as demonstrated by immunocytochemistry. Top, Weak basal gp130 surface expression in unstimulated SMC; bottom, enhanced gp130 expression 8 hours after exposure to 50 µg/mL E-LDL. Experiments were reproduced with 6 different SMC lines.

Effects of IL-6 and Soluble IL-6R on SMC
In atherosclerotic lesions, SMC are present in the immediate vicinity of infiltrating macrophages. E-LDL induced expression and release of IL-6 from human macrophages and promoted shedding of the soluble IL-6 receptor (sIL-6R).⁶ The next experiments were conducted to assess whether IL-6, sIL-6R, or IL-6+sIL-6R had an effect on gp130 expression on SMC and whether this effect would be influenced by the additional presence of E-LDL.

Treatment of SMC with IL-6 (250 ng/mL) alone led to an almost imperceptible induction of gp130 mRNA (Figure 6A). Similarly, sIL-6R (500 ng/mL) alone provoked only a minor effect (Figure 6B). In contrast, simultaneous stimulation with IL-6 (250 ng/mL) and sIL-6R (500 ng/mL) resulted in a sustained, dose-dependent expression of gp130 mRNA (Figure 6C). The most marked induction of gp130 mRNA was, however, observed when human vascular SMC were preincubated with 50 µg/mL E-LDL for 8 hours and then stimulated with the combination of IL-6 and sIL-6R (Figure 6D). This resulted in prominent expression of gp130 mRNA after 8 hours, which was maintained for 24 hours.

View larger version (98K): [in this window] [in a new window]   Figure 6. Differential regulation of gp130 mRNA expression by IL-6 and sIL-6R alone or in combination. Weak induction of gp130 mRNA by IL-6 (A) or sIL-6R alone (B) is shown. Marked induction of gp130 after stimulation with IL-6/sIL-6R is shown (C). Very pronounced induction of gp130 mRNA after 8-hour preincubation of SMC with 25 µg/mL E-LDL followed by stimulation with IL-6/sIL-6R is shown (D). IL-6 and sIL-6R were applied at 500 ng/mL. M indicates DNA marker; C, unstimulated control. Lanes represent time of incubation (h) with stimulants.

Toxicity of E-LDL on Human Vascular SMC
Exposure to 200 to 400 µg/mL E-LDL reduced the cellular ATP content to <10% of untreated control SMC after 48 hours (Figure 7). At E-LDL concentrations <50 µg/mL, cytotoxic effects were observed only after extended incubation (>48 hours). The cytotoxic effects were not due to apoptosis, as determined by the absence of DNA fragmentations and by negative TUNEL stainings (not shown). The presence of 4% human albumin completely abolished cytotoxicity of E-LDL (Figure 7).

View larger version (18K): [in this window] [in a new window]   Figure 7. Cytotoxic effects of E-LDL on human vascular SMC as demonstrated by measurements of cellular ATP. •, Dose-dependent cytotoxicity of E-LDL on SMC; , protective effect of 4% human albumin. Results are given as mean value±SD of 4 separate experiments.

E-LDL Stimulates SMC Proliferation Through 2 Pathways
Albumin was supplemented at 4% to abrogate the cytotoxic effects of E-LDL in the following proliferation assays. E-LDL alone induced moderate dose-dependent proliferation of human vascular SMC, which reached a maximum at 50 µg/mL E-LDL (Figure 8A). Proliferation commenced after 24 hours, and a 4-fold increase of DNA synthesis was measured at 72 hours (Figure 8B). That the increased incorporation of [³H]-thymidine indeed reflected cell multiplication was ascertained in 4 experiments by direct counting of cells after their release by trypsinization.

View larger version (19K): [in this window] [in a new window]   Figure 8. E-LDL induces proliferation of human SMC. A, Moderate dose-dependent mitogenic effect of E-LDL on SMC after 72 hours of exposure; B, time-dependent stimulation of SMC proliferation in presence of 25 µg/mL E-LDL/4% human albumin (black bars) compared with untreated control cells kept in medium with 4% human albumin (striped bars). Depicted are mean±SD of 6 separate experiments, each with triplicate cultures. *Differences were significant at P<0.05.

An antibody against platelet-derived growth factor (PDGF) reduced the E-LDL–dependent proliferative response by 60%. Blocking fibroblast growth factor (FGF)-ß activity reduced proliferation by 25%. Combined application of both antibodies was additive and reduced cell proliferation by 70%. In contrast, antibody against IL-6 was without effect (Figure 9).

View larger version (20K): [in this window] [in a new window]   Figure 9. E-LDL induced proliferative response in the presence (+) or absence (-) of blocking antibodies against PDGF (50 µg/mL), FGF-ß (10 µg/mL), or IL-6 (1:200). Proliferation was measured after incubation with 25 µg/mL E-LDL/4% human albumin for 72 hours. Results are given as mean values of [3H]thymidine incorporation ±SD of 6 separate experiments, each with triplicate cultures for each experimental condition. *Differences were significant at P<0.05.

As reported above, E-LDL induced release of IL-6 and sIL-6R from macrophages and simultaneously provoked upregulation of gp130 on SMC. It is known that IL-6/sIL-6R can stimulate cells expressing gp130.¹⁸ Therefore, in the next experiments, macrophages were incubated with E-LDL (100 µg/mL) for 72 hours, and the supernatants containing 234±65 pg/mL IL-6 and 987±122 pg/mL sIL-6R were harvested. E-LDL was removed by centrifugation, and the supernatants were incubated with SMC. When applied to nonactivated SMC, the supernatants only weakly induced cell proliferation (Figure 10A). However, if SMC were first preincubated with 25 µg/mL E-LDL for 8 hours (to induce gp130), the supernatants provoked a pronounced and sustained proliferative response that was readily apparent after 72 hours (Figure 10B). E-LDL alone was without effect.

View larger version (25K): [in this window] [in a new window]   Figure 10. Supernatants of E-LDL–stimulated macrophages induce proliferation of SMC. A, Proliferation of 3 different SMC isolates after 24-hour stimulation with different dilutions of supernatants from E-LDL–activated macrophages; B, time dependency of proliferative effect. Black bars indicate undiluted macrophage supernatants from E-LDL–stimulated macrophages; striped bars, control cells. Results are given as mean value±SD of 6 separate experiments, each with triplicate cultures. *Differences were significant at P<0.02.

	Discussion

Top Abstract Introduction Methods Results Discussion References

 
SMC proliferation is a key event in the pathogenesis of atherosclerosis, occurring subsequent to the insudation of LDL, complement activation, and the infiltration of macrophages and T-lymphocytes. Our results show that E-LDL can promote this process through 2 mechanisms. First, E-LDL directly stimulates proliferation, a property that has been described also for other lipoproteins including VLDL and oxidized LDL.^{19 20 21} We detected no proliferative responses to native LDL, which stands in some disagreement with an earlier study.²⁰ In the absence of macrophages, E-LDL–induced proliferation appeared to ensue through an autocrine pathway involving PDGF and FGF-ß, as indicated from blocking experiments with antibodies. Arterial SMC express PDGF,^{22 23} and the significance of this growth factor in atherosclerosis is supported by the restricted expression of PDGF receptors on the proliferating SMC subset in the intima,²⁴ whereas expression is absent on stationary SMC in the media.²⁵ The rat homologue of human MCP-1 has been linked both to SMC proliferation and migration,²⁶ and the capacity of E-LDL to stimulate MCP-1 production in both macrophages⁶ and SMC is of interest in this context.

The second pathway leading to an SMC-proliferative response integrates the effects of E-LDL on macrophages and on SMC and highlights the central role of IL-6 and sIL-6R. This mechanism is novel and operates through the convergence of 2 events. The first is the release of both IL-6 and its soluble receptor IL-6R from macrophages. The second is the upregulation of gp130, the signal-transducing element of the IL-6 receptor family on SMC provoked by E-LDL.

It is known that IL-6/sIL-6R complexes form spontaneously in solution after their release from cells. The complexes can subsequently bind to and activate cells that express the signal-transducing element gp130. This process, termed transsignaling, is widely operative because gp130 is constitutively expressed on many cell types.¹⁸ SMC are unusual because quiescent SMC express little gp130 but upregulate the molecule when they are stimulated with IL-6/sIL-6R.¹³ This drives the cells into a proliferative state. We have found that E-LDL is a second stimulus that causes upregulation of gp130, thus enhancing the efficiency of the transsignaling circuit in SMC. The finding satisfactorily explains why SMC proliferation occurred with a relatively long lag phase and why relatively low concentrations of IL-6/sIL-6R were required. It is of interest that in early atherosclerotic lesions, local expression of IL-6 has indeed been detected particularly in macrophage-rich regions and in SMC.^{27 28 29}

Foam cell formation is another hallmark of the atherosclerotic lesion. Although many studies have analyzed the effects of modified lipoproteins on macrophage foam cell formation,^{16 30} investigations into the pathways underlying cholesterol uptake by SMC have remained sparse. We found that E-LDL induces foam cell transformation of SMC, albeit to a smaller extent than found with macrophages. This was in line with histological studies that identify the majority of foam cells as macrophages, with SMC contributing a smaller fraction.^{31 32 33}

Selective recruitment of monocytes and T-lymphocytes into early atherosclerotic lesions probably reflects lesional expression of selective chemoattractants.^{34 35} Local producers of MCP-1 are mainly macrophages,³⁴ vascular SMC being additionally involved in the early stages.^{36 37} That E-LDL induced selective production of MCP-1 in SMC is of interest in this context. In accord with immunohistological studies, lower amounts of MCP-1 were liberated by SMC as compared with macrophages.⁶ Other lesional components including terminal complement complexes may also contribute to MCP-1 generation in SMC.³⁸ In vitro, oxidized LDL (ox-LDL) has been shown to be endowed with this capacity, a finding that might attain significance if ox-LDL were present in the lesions in sufficient amounts.³⁹

The more advanced stages of atherosclerosis are characterized by tissue necrosis. E-LDL induced cytotoxic effects on SMC that were more pronounced than those invoked by ox-LDL.²¹ However, in contrast to ox-LDL,⁴⁰ E-LDL did not recognizably trigger apoptosis of SMC. This would be in accord with immunohistochemical studies showing apoptosis of macrophages and T-lymphocytes but not of SMC in the lesions.^{41 42 43} The abolishment of E-LDL toxicity in the presence of 4% human albumin suggested that free fatty acids mediated toxic effects, as has been described for several cell types including lymphocytes, macrophages, and tumor cells.^{44 45} The other biological effects of E-LDL reported here, that is, SMC proliferation and expression of chemokines, were not affected by the addition of albumin.

In sum, our results show that E-LDL is taken up by human vascular SMC and activates these cells to acquire a proinflammatory phenotype. Of particular interest is the finding that the pathways of SMC and macrophage activation by E-LDL may merge at the level of IL-6/s-IL-6R transsignaling, which operates to drive SMC into a proliferative state. The data go further to show that E-LDL is endowed with the prime characteristics of an atherogenic lipoprotein.

	Acknowledgments

 
This work was supported by grants from the Deutsche Forschungsgemeinschaft (Bonn, Germany) and from the Stiftung Rheinland Pfalz for Innovation (Mainz, Germany). We thank Monika Hemmes for excellent technical assistance.

Received October 8, 1999; revision received November 22, 1999; accepted December 2, 1999.

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