INTRODUCTION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

THE VASCULAR WALL consists mainly of smooth muscle cells that play an important role in maintaining vascular tone and structure. In the normal vascular wall, smooth muscle cells are predominantly contractile. These cells are characterized by an abundance of myofilaments and a relatively low amount of organelles necessary for cell proliferation, i.e., free ribosome, rough endoplasmic reticulum, Golgi, etc. (5, 7, 14, 15). However, in atherosclerosis, smooth muscle cells change their phenotypic profile from contractile to synthetic. The synthetic smooth muscle cells are characterized by a significant decrease in contractile proteins and the presence of high numbers of organelles necessary for cell migration and proliferation (6, 7, 14, 15). It is unclear what factors during atherosclerosis induce the change in smooth muscle cell phenotype. Cholesterol and its carrier, low-density lipoprotein (LDL), are potentially involved because they are important risk factors for atherosclerotic disease (28, 30). Recent work has also demonstrated that oxidative modification of LDL (oxLDL) plays a crucial role in atherogenesis (29-31). OxLDL has the capacity to stimulate the migration and proliferation of smooth muscle cells from the medial layer to the intima and to morphologically transform smooth muscle cells within the intima (29). However, little attention has been paid to the direct effects that oxLDL may have on the cytoskeleton of smooth muscle cells. Recently, it has been demonstrated in vitro that oxLDL plays an important role in altering the structural organization of F-actin in endothelial cells (32). A similar alteration in F-actin was also observed in vivo in aortic endothelial cells in rabbits fed a high-cholesterol diet (9). Thus, although precedents have been set for the capacity of cholesterol and oxLDL to alter myofilament density in endothelial cells, little is known about a similar ability in smooth muscle cells where these proteins play an even more important role. The purpose of this study therefore was to investigate if oxLDL has the capacity to decrease myosin and actin content and distribution in smooth muscle cells.

	MATERIALS AND METHODS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Materials. Dulbecco's modified Eagle's medium (DMEM), fetal bovine serum, penicillin-streptomycin, and trypsin-EDTA were purchased from GIBCO-BRL. Cholesterol diet was purchased from Purina Test Diets (Richmond, IN). Antibodies for smooth muscle cell -actin, myosin, proliferating cell nuclear antigen (PCNA), and FITC-conjugated anti-mouse IgG were purchased from Sigma-Alderich (Oakville, ON). 5,5'-Dithio-bis(2-nitrobenzoic acid), phenylmethylsulfonyl fluoride, thimerosal, transferrin, selenium, ascorbate, insulin, cholesterol oxidase, and cholesterol esterase were obtained from Sigma-Aldrich. The Live/Dead Viability Assay kit was purchased from Molecular Probes (Eugene, OR).

LDL isolation and oxidation. LDL in density range of 1.019-1.063 g/ml was isolated as described previously (19). Addition of EDTA (0.1 mmol/l) prevented oxidation of LDL throughout the isolation. The LDL fraction was dialyzed against 0.15 M NaCl and 0.1 mmol/l EDTA (pH 7.4), sterile filtered (0.2-µm pore size), and stored at 4°C. The protein concentration of LDL was determined by Lowry's method (21), and cholesterol (free and esterified) was measured enzymatically as described (24). LDL was tested for the presence of malondialdehyde reactive products and oxidized cholesterol to confirm the absence of auto-oxidation of the LDL (10, 20, 26).

Vascular smooth muscle cells. Normal rabbit thoracic aorta was used to generate a primary culture of smooth muscle cells from explants (3, 11). Details regarding the culture of smooth muscle cells using the explant method are found elsewhere (17, 18). To induce differentiation, the smooth muscle cells were placed in a serum-free medium supplemented with transferrin (5 µg/ml), selenium (1 nmol/l), ascorbate (200 µmol/l), and insulin (10 nmol/l) for 5-6 days before treatment. This period was crucial for full development of contractile proteins in the cultured vascular smooth muscle cells (17).

Chronic treatment of vascular smooth muscle cells with oxLDL. Smooth muscle cells from either first or second passage were used in our experimental protocol. These cells were placed in the serum-free medium previously described for 6 days before exposure to LDL or oxLDL. In chronic experiments, vascular smooth muscle cells were exposed for up to 6 days to different concentrations of LDL or freshly prepared oxLDL (0.01 to 0.1 mg cholesterol/ml LDL). The medium was changed daily and supplemented with an aliquot of LDL or freshly oxidized LDL. The control cells were maintained in the same medium (but without LDL or oxLDL) for the same period of time as the treated cells.

Cytotoxicity. The cytotoxicity of different concentrations of oxLDL was assessed by ethidium homodimer staining of cell nuclei (Live/Dead EukoLight Viability/Cytotoxicity Assay Kit, Molecular Probes).

Immunocytochemistry. Smooth muscle cell phenotype was identified using monoclonal antibodies against smooth muscle -actin and myosin. Monoclonal anti--actin antibody is specific for the single smooth muscle -actin isoform, and monoclonal antimyosin antibody recognizes myosin heavy chain isoforms SM-1 (204 kDa) and SM-2 (200 kDa). These cells were fixed with 1% paraformaldehyde in PBS for 20 min and then incubated with primary antibodies followed by secondary antibodies conjugated to FITC. The fluorescent images of the FITC were obtained with a Bio-Rad MRC-600 ultraviolet-confocal system connected to a Nikon Diaphot 300 epifluorescence microscope. This system is equipped with an argon ion laser capable of excitation of different fluorophores at ultraviolet (351 and 363 nm) and visible (488 and 514 nm) wavelengths. The FITC fluorescence was obtained by exciting the cells with a 488-nm laser line and the emission was collected at 520 nm. All of the images were obtained through a Nikon Fluor ×40 (numerical aperature 1.3) oil immersion lens and pseudocolor autumn was used for presentation purposes.

Western blotting. After treatment of vascular smooth muscle cells with LDL or oxLDL, cells were lysed with lysis buffer (1% SDS, 100 mmol/l NaCl, 62.5 mmol/l Tris · HCl, pH 7.6, 1 mmol/l phenylmethylsulfonyl fluoride, and 10 µg/ml leupeptin) and kept on ice. The protein concentration was determined with the modified Lowry assay. Cell extracts were denatured with sample buffer (62.5 mmol/l Tris · HCl, 1% SDS, 10% glycerol, 0.01% bromphenol blue, and 20 µg/ml -mercaptoethanol) at 100°C for 5 min. The samples were separated on a 3-15% gradient SDS-polyacrylamide gel with a 4% stacking gel in the running buffer (0.025 mol/l Tris · HCl, 0.192 mol/l glycine, and 0.1% SDS). The proteins were transferred electrophoretically to nitrocellulose membrane (GIBCO-BRL) in transfer buffer (25 mmol/l Tris · HCl, 192 mmol/l glycine, and 0.05% SDS). The blots were blocked with 10% skim milk in PBST (i.e., PBS and 0.05% Tween) for 30 min at room temperature on a multimixer. The membranes were then incubated with smooth muscle-specific monoclonal antibodies against -actin or myosin (Sigma-Aldrich). These antibodies were diluted (1:10,000) in 1% skim milk and PBST and incubated at room temperature for 1 h on a multimixer. The blots were further incubated with horseradish peroxidase-conjugated goat anti-mouse IgG (Bio-Rad) diluted in 1% skim milk and PBST (1:20,000) for 1 h at room temperature on a multimixer. The actin and myosin were detected with the Pierce Super Signal detection system, and the blots were exposed to Kodak X-OMAT film. The data were analyzed by scanning the Kodak X-OMAT film with a Bio-Rad densitometer. The optical density for each band was registered, and these values were then normalized to the control group before the percent change was calculated.

Statistical analysis. Data are expressed as means ± SE. The statistical comparisons were made using one-way ANOVA, followed by the Student-Newman-Keuls test for multiple comparison. Differences between means were considered significant when P < 0.05.

	RESULTS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

LDL was modified with a Fe-ADP-free radical generating system. Under our experimental conditions, LDL was minimally oxidized. As shown in Fig. 1, the migration of oxLDL on agarose gels was modest compared with native LDL or acetylated LDL. The migration of oxLDL depends on the extent of its oxidation. -Tocopherol content was also depleted in oxLDL by a modest but significant amount (Fig. 1). The oxidation reaction also induced a significant increase in TBARS production, which is indicative of lipid peroxidation (Fig. 1). However, the amount of TBARS production (8 nmol/mg) was far below values reported by others for oxidative modification of LDL with Cu²⁺ (~50 nmol/mg).

View larger version (31K): [in this window] [in a new window]   Fig. 1.   Low-density lipoprotein (LDL) oxidation by Fe-ADP. LDL was oxidized with a solution of 50 µM FeCl3 and 0.25 mM ADP for 3 h at 37°C. A: electrophoretic mobility of oxidized LDL (oxLDL) on agarose gel: native LDL (lane a), oxLDL (lane b), and acetylated LDL (lane c) (arrow indicates origin of gel). B: alteration in -tocopherol level (n = 4) and thiobarbituric acid-reactive substances (TBARS) production (n = 8). * P < 0.05 vs. control.

OxLDL has cytotoxic effects. However, the concentration of oxLDL employed in our study had very little effect on cell integrity. As shown with ethidium homodimer staining (Fig. 2), the viability of smooth muscle cells was unaltered by oxLDL treatment. In this test, esterase activity in live cells cleaves the ester group from calcein-AM to generate a green fluorescence. Cells with compromised membrane integrity will allow infiltration of the ethidium homodimer to stain the nuclear contents with a bright red fluorescence. The control cells were 99.8 ± 0.1% alive (n = 1,357 cells counted; Fig. 2). Smooth muscle cells treated chronically for 3 days with 0.05 and 0.1 mg cholesterol/ml oxLDL were 96 ± 1% (1,321 cells counted) and 95 ± 1% alive (2,828 cells counted; Fig. 2). These data were not affected by necrotic cells lifting free from the culture surface. Cell numbers were unchanged as a function of treatment with 0.05 mg/ml oxLDL over 3 days (56.5 ± 6.8 cells were counted per field in untreated preparations compared with 55.0 ± 4.0 cells in the experimental group; n > 1,300 cells counted in total in each group).

View larger version (32K): [in this window] [in a new window]   Fig. 2.   Effect of chronic treatment with oxLDL on vascular smooth muscle cell (VSMC) integrity. VSMC were exposed for 3 days to 0, 0.05, and 0.1 mg cholesterol/ml oxLDL and stained with ethidium homodimer for cell integrity as described in MATERIALS AND METHODS. A: representative results. Live cells stained yellow/green, whereas dead cells are identified by staining of nuclei with ethidium homodimer (red fluorescence). B: number of cells was quantified in several separate experiments (1,300 to 2,800 cells were counted in each group from 3 separate experiments).

During the process of atherosclerosis, smooth muscle cells proliferate and migrate into the intima of vessels, lose their contractile apparatus, and modify into a synthetic phenotype. It is unclear what factor within the atherogenic environment is responsible for inducing the loss in smooth muscle myofilaments. As shown by Western blots in Fig. 3, the total content of both actin and myosin were altered in smooth muscle cells chronically treated with oxLDL. Smooth muscle cells treated for a period of 6 days with oxLDL showed a significant and consistent decrease in both actin and myosin content in these cells (Fig. 3). Increasing concentrations of oxLDL (0.01 to 0.05 mg cholesterol/ml) induced a pattern for the reduction in both actin and myosin, although this was statistically significant for actin only at the highest oxLDL concentration. This effect of oxLDL on actin and myosin content was also time dependent. Again, a trend to decrease both actin and myosin was observed in smooth muscle cells treated for 3 and 6 days with 0.05 mg/ml oxLDL, but this was statistically significant only at the 6-day time point (Fig. 4).

View larger version (20K): [in this window] [in a new window]   Fig. 3.   Effect of chronic treatment with oxLDL on -actin and myosin content in VSMC. Cells were treated with a range of oxLDL (0.01-0.05 mg/ml) for 6 days and then probed by Western blot for densities of smooth muscle-specific -actin and myosin. Data are means ± SE of 6 separate experiments. * P < 0.05 vs. control.

View larger version (15K): [in this window] [in a new window]   Fig. 4.   Time-dependent effect of oxLDL on -actin and myosin densities. VSMC were treated with 0.05 mg/ml oxLDL (for 0, 3, and 6 days) and cell extracts were analyzed by Western blots. Data are the means ± SE of 6 separate experiments. * P < 0.05 vs. control.

These changes were not only restricted to the content of these myofilaments within the smooth muscle cell. Surprisingly, oxLDL also induced a striking disorganization in these myofilaments within the smooth muscle cell. As shown in Fig. 5a, using confocal microscopy and immunocytochemical staining, myosin was observed as long, uninterrupted filamentous structures in control cells. However, exposure of smooth muscle cells to 0.1 mg cholesterol/ml oxLDL for 3 days induced a striking disorganization of the myosin filaments (Fig. 5, b-d). Interestingly, the myosin filaments were furled into large aggregates (as shown in Fig. 5, b-d). Smooth muscle cells that exhibited visual evidence of a change in cytoskeletal organization were counted to determine the frequency of the abnormality. Fifteen, 24, or 87% of the cells treated for 3 days with 0.025, 0.05, or 0.1 mg/ml oxLDL, respectively, exhibited evidence of some cytoskeletal disorganization similar to that depicted in Fig. 5. Because we were forced to employ different techniques (Western vs. immunocytochemistry), it is difficult to conclusively determine whether the myosin content decreased before the disorganization of cytoskeletal elements or visa versa. However, because 24% of cells exhibited cytoskeletal disorganization after 3 days of treatment with 0.05 mg/ml oxLDL and there was no significant effect of this treatment on cytoskeletal protein content (Fig. 4), it is possible to suggest that the myofibrillar disorganization may have preceded the reduction in content.

View larger version (33K): [in this window] [in a new window]   Fig. 5.   Effect of chronic treatment of VSMC with oxLDL on myosin organization and distribution. VSMC were treated with oxLDL for 3 days and immunostained with smooth muscle-specific monoclonal antimyosin antibody and FITC-conjugated secondary antibody. These images were obtained with a Bio-Rad confocal microscope. a: control smooth muscle cell. b-d: cells treated with 0.1 mg/ml oxLDL.

Chronic exposure of cells for 0, 1, 3, and 5 days to a similar concentration of native LDL (0.1 mg cholesterol/ml) failed to induce myosin aggregation (Fig. 6, a-d). Cells treated with native LDL (0.1 mg cholesterol/ml) for 1 day showed no change from control with respect to myosin organization. At day 5, these cells began to show what may be interpreted as contracture bands but clearly provided no evidence of the striking aggregation observed in oxLDL-treated cells. The effect of oxLDL (0.1 mg cholesterol/ml) on myosin was time dependent. Smooth muscle cells were treated with oxLDL for 0, 1, 3, and 5 days (Fig. 6, e-h). The cells treated with oxLDL demonstrated early signs of myosin disorganization after only 1 day of exposure. These alterations were more pronounced at day 3. At this time, myosin filaments began to clearly form into aggregates (Fig. 6g). These aggregations of myosin were more pronounced after 5 days. In some cases, large aggregates could be observed in close proximity to the plasma membrane. These appear to be in the process of being expelled from the cell (Fig. 7).

View larger version (53K): [in this window] [in a new window]   Fig. 6.   Time-dependent effects of native and oxidized LDL on myosin organization in VSMC. VSMC were treated with native LDL (0.1 mg cholesterol/ml, a-d) or oxidized LDL (e-h) and then immunostained with antimyosin antibody. Cells were treated for different periods of time with native LDL (a: control cell, b: 1 day, c: 3 days, d: 5 days) and oxLDL (e: control, f: 1 day, g: 3 days, h: 5 days).

View larger version (47K): [in this window] [in a new window]   Fig. 7.   Aggregation of myosin filaments in oxLDL-treated cells. Smooth muscle cells were treated with 0.1 mg/ml oxLDL for 3 days. Giant aggregates of myosin seem to be in process of being expelled from cell.

With use of the confocal microscope, the myosin filaments were optically sectioned in both control and oxLDL-treated smooth muscle cells. This allowed us to study the three dimensional distribution of myosin filaments in these cells. As shown in Fig. 8, a-f, the myosin filaments were intact and aligned throughout the control cells, whereas these filaments were disassembled and aggregated into a three-dimensional ball in the smooth muscle cells treated with oxLDL (Fig. 8, g-l).

View larger version (35K): [in this window] [in a new window]   Fig. 8.   Optical sectioning with confocal microscope of control and treated cells. Cells were immunostained with antimyosin and optically sectioned with a Bio-Rad confocal microscope. These sections are ~0.3 µm apart. Control (a-f) and oxLDL-treated (g-l) cells.

The effects of oxLDL were not limited to just myosin. Actin distribution was affected by oxLDL (0.1 mg cholesterol/ml) in a similar manner (Fig. 9, a-h). Whereas native LDL had little effect on actin distribution and aggregation within the cell, oxLDL had a time-dependent effect on actin organization and aggregation within the smooth muscle cells.

View larger version (69K): [in this window] [in a new window]   Fig. 9.   Time-dependent effects of native and oxidized LDL on -actin organization. VSMC were treated with native LDL (0.1 mg cholesterol/ml) (a-d) or oxidized LDL (e-h) and then immunostained with smooth muscle-specific anti--actin antibody. Cells were treated for different periods of time with native LDL (a: control cell, b: 1 day, c: 3 days, d: 5 days) and oxLDL (e: control, f: 1 day, g: 3 days, h: 5 days).

It is possible that the effects on cytoskeletal proteins were induced by a proliferative action of the oxLDL on the smooth muscle cells. The ability of oxLDL to stimulate smooth muscle cells to grow and proliferate under our experimental condition was tested by examining for the induction of PCNA. Vascular smooth muscle cells were treated with or without oxLDL (0.05 and 0.1 mg/ml) for 3 days (Fig. 10). There was no statistically significant increase in the expression of PCNA by oxLDL under our experimental conditions. This would suggest that oxLDL did not induce a proliferative response. The lack of change in cell numbers after oxLDL treatment reported earlier in RESULTS would further support this conclusion.

View larger version (26K): [in this window] [in a new window]   Fig. 10.   Effect of oxLDL on cell proliferation. Smooth muscle cells were treated with or without oxLDL (0.05 or 0.1 mg/ml) for 3 days. Total protein was then extracted from these cells and analyzed by Western blots for proliferating cell nuclear antigen (PCNA). Data are means ± SE of 4 separate experiments.

	DISCUSSION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Our data demonstrate a significant effect of oxLDL on actin and myosin densities, organization, and distribution. Chronic exposure of vascular smooth muscle cells to oxLDL resulted in a decrease in the total cellular protein content of both actin and myosin. It is well recognized that smooth muscle cells lose their contractile proteins in atherosclerotic lesions in vivo (5, 7, 14, 15). Conversely, smooth muscle cells will also reexpress contractile protein in the atheroma under conditions of lipid-lowering therapy (1). Our findings therefore identify oxLDL as one component within the atherogenic milieu that has the ability to decrease the contractile protein content as the smooth muscle cell phenotype changes.

The concentration of oxLDL used in our work was much lower than those used in other studies of oxLDL (2, 4, 8, 27). We employed lower concentrations of oxLDL over longer incubation times. The extended duration of exposure of the cells to oxLDL in the present study was used to mimic more closely the in vivo conditions. Atherosclerosis is a slow, gradual disease. It is unlikely that oxLDL is present only for a limited period of time in relatively high concentrations. Instead, it is more reasonable to propose that small amounts of oxLDL may be in proximity to cells in the subendothelial space for extended periods. It is important to emphasize that the oxLDL preparation used was minimally modified as well. Our data therefore reemphasize the potential importance of relatively low concentrations of oxLDL to induce phenotypic changes in myofilament composition within the atherogenic smooth muscle cell.

A novel and surprising finding in the present study was the identification of a striking change in the organization of the myofilaments in the smooth muscle cells. Smooth muscle cells chronically treated with oxLDL exhibited a disorganization of actin and myosin into large, ball-shaped aggregates. These findings are not without precedent in another cell type. For example, Colangelo and co-workers (9) recently showed that F-actin organization was altered in rabbits on a high-cholesterol diet. Furthermore, cultured human endothelial cells treated with ~200 µg cholesterol/ml oxLDL resulted in alterations in F-actin organization (32). However, the changes in F-actin organization were relatively minor in comparison to the large, striking aggregations of these filaments observed in the present study within vascular smooth muscle cells chronically treated with oxLDL. The higher content of contractile proteins and their linear alignment in smooth muscle cells probably accentuates the disorganizing effects of oxLDL as opposed to those observed in endothelial cells.

The mechanism whereby oxLDL induces the changes in myofilament organization is unclear. However, several possibilities exist based on evidence obtained here and previously. Native LDL was unable to induce myofilament disorganization in the present study, suggesting that an oxidized component within the oxLDL was responsible for this action. OxLDL contains oxidized species of cholesterol that have been shown to induce myofilament disorganization in endothelial cells (25). This may occur through a mitogen-activated protein (MAP) kinase-mediated effect. MAP kinase activity has been associated with actin organization in endothelial cells (13). OxLDL has been shown to stimulate MAP kinase activity in smooth muscle cells (16). This stimulation occurred through a lipid soluble component within the oxLDL. It is possible therefore that oxLDL may stimulate MAP kinase activity in the smooth muscle cells to disorganize contractile proteins under our conditions. It is unlikely that intracellular Ca²⁺ is involved as a mechanistic agent as proposed elsewhere (32). OxLDL cannot induce changes in intracellular Ca²⁺ under our incubation conditions due to alterations in sarcoplasmic reticulum function and structure (unpublished data). Thus myofilament changes due to oxLDL were unlikely to be a result of changes in intracellular Ca²⁺ levels.

In summary, our data suggest that oxLDL is capable of decreasing myofilament content within smooth muscle cells in culture. Surprisingly, this effect was preceded by a striking disorganization within the cells that ultimately resulted in the formation of giant myosin and actin aggregates. In some dramatic cases in the present study, these aggregates appeared to be in the process of being expelled from these cells. This is interesting and may have both mechanistic and clinical implications. Mechanistically, this process may explain in part the decrease in myofilament proteins observed in Western blots in the present study. It is tempting to speculate that such a process may be the cause for the presence of autoantibodies to myofilaments in the plasma of patients with coronary artery disease (23). From a clinical standpoint, it is well known that smooth muscle cells are present in the cap of an atherosclerotic plaque, and the loss of myofilaments from these cells may destabilize the plaque and contribute to rupture and thrombosis (1).