Oxidized Low-Density Lipoprotein Retards the Growth of Proliferating Cells by Inhibiting Nuclear Translocation of Cell Cycle Proteins
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《动脉硬化血栓血管生物学》
From the Cell Biology Laboratory, Division of Stroke and Vascular Disease, St Boniface General Hospital Research Centre, Winnipeg, Manitoba, Canada; the Department of Physiology, University of Manitoba, Winnipeg, Manitoba, Canada; and the Department of Microbiology (G.Z.), University of Texas Health Science Center at San Antonio, TX.
ABSTRACT
Objective— Our study tested the hypothesis that the mitogenic effect of oxidized low-density lipoprotein (oxLDL) on vascular cells may be further enhanced by the presence of cytokines and growth factors known to be present in the atherosclerotic environment.
Methods and Results— Quiescent fibroblasts and vascular smooth muscle cells were treated with 10 or 50 μg/mL minimally-oxidized LDL in combination with serum for 24 or 48 hours. Surprisingly, these cells showed inhibited release from growth arrest and a significant reduction in the number of cells completing the cell cycle when compared with cells treated with serum alone. This was not due to an induction of apoptosis. The antiproliferative effects were not closely associated with changes in the expression of cell cycle proteins. Instead, oxLDL inhibited the translocation of cell cycle proteins cell division cycle (Cdc) 2, cyclin-dependent kinase (Cdk) 2, Cdk 4, Cyclin A, Cyclin B1, Cyclin D1, and proliferative cell nuclear antigen (PCNA) into the nucleus, as compared with separate treatments with serum alone. Kinase activation associated with specific cell cycle proteins was also inhibited by oxLDL.
Conclusions— oxLDL, in the presence of serum, has a surprising inhibitory effect on cell proliferation that occurs through an inhibition of import of cell cycle proteins into the cell nucleus.
Key Words: atherosclerosis ? cyclin ? kinase, cyclin-dependent ? cell proliferation
Introduction
Oxidized low-density lipoprotein (oxLDL) is believed to play a critical role in atherogenesis.1–4 oxLDL has numerous growth-promoting effects on cells in vitro,5 including the induction of transcription factors6,7 and the enzymes involved in mitogenesis,8,9 as well as stimulation of DNA synthesis and cell proliferation.10–12 oxLDL can stimulate vascular smooth muscle cell (VSMC) proliferation in cell culture conditions even in the absence of any other growth factors. This effect involves a stimulation of the phosphatidylinositol 3-kinase (PI3K) pathway and an induction of cell cycle proteins.13
Cell cycle proteins closely control the movement of a cell into a proliferative state.14–18 Alterations in the expression, activity, or nuclear translocation of cell cycle proteins within a cell will determine the capacity for that cell to move into the cell cycle and proliferate. Changes in the expression of a number of cell cycle proteins in the vessel wall during atherosclerosis or restenosis have been identified and are thought to represent critical cellular events that determine the proliferative potential of the cells during these pathological states.19–24
Recently, this hypothesis has been challenged.25–27 Some studies report minimal evidence in favor of accelerated cell proliferation in plaques where stimulated cell proliferation would be expected.26,27 Other plausible mechanisms have been proposed (eg, inhibited apoptosis).25 It is also possible that mitogenic factors like oxLDL may not have been studied optimally to define their proliferative potential. Previous investigations have studied the effects of oxLDL in isolation, however, in an in vivo atherosclerotic environment, vascular cells are exposed to oxLDL in the presence of a multitude of cytokines and growth factors. The purpose of the present study, therefore, was to elucidate the effects of oxLDL on cell proliferation in the presence of a variety of growth factors and cytokines found in serum. To obtain mechanistic insights, the effects of oxLDL on cell cycle proteins in this atherosclerotic environment were a focus for our study.
Methods
Cell Culture and Incubation Conditions
Confluent cultures of human neonatal fibroblasts or New Zealand White rabbit VSMCs, isolated as described,28 were passaged and then incubated for 24 hours in DMEM supplemented with 5% FBS. Cells were washed and then placed in serum-free DMEM supplemented with transferrin (5 μg/mL), selenium (1 nM), ascorbate (200 μmol/L), and insulin (10 nM) for 6 days in order to induce growth arrest. Cells were then incubated with 10 or 50 μg cholesterol/mL LDL or oxLDL in combination with FBS (5% for fibroblasts, 10% for VSMCs) for 24 or 48 hours. LDL was oxidized with a Fe-ADP free radical generating system. The method and characteristics of the minimally-modified oxLDL are reported in detail elsewhere.29,30 Typically, this preparation of oxLDL exhibits a modest increase in electrophoretic mobility, a 20% depletion of vitamin E, and a 30% increase in malondialdehyde content.30 Cholesterol concentrations were assessed prior to oxidation, and these concentrations were used for both native and oxLDL. Protein concentrations were unchanged throughout the course of the experiments. The same concentrations of Fe and ADP added to control cells in the absence of LDL had no effect (data not shown). Cultures were maintained at 37°C in humidified 5% CO2, and medium was replaced every 24 hours. Freshly prepared oxLDL was also replaced on a daily basis. Control cells were maintained in identical media (in the absence of oxLDL) for the same period of time. The duration of exposure to oxLDL was not cytotoxic to cells as determined by measurement of lactate dehydrogenase (LDH) activity in the medium31 or via ethidium homodimer staining.28 The acidic and basic fibroblast growth factors (aFGF and bFGF; Sigma) and transforming growth factor-?1 (TGF-?1, Sigma) were used at concentrations of 50 ng/mL, 10 ng/mL, and 5 ng/mL, respectively. Lipoprotein-depleted serum was prepared as described by Auge et al.10 For experiments involving inhibitors, cells were pretreated for 15 minutes with 20 μg/mL LY294002 (Sigma),32 200 ng/mL calphostin C (Sigma),8 or 3 μg/mL U73122 (Sigma)33 before exposure to oxLDL. The drugs remained in the media for the duration of the experiments.
Measurement of Cell Numbers and Cell Cycle Analysis by Flow Cytometry
Cell numbers were counted in a hemacytometer. For each condition and time point, 18 fields were counted. For cell cycle analysis, cells were trypsinized after treatment with oxLDL, fixed, and treated with RNase A (500U/mL in 1.12% sodium citrate) for 15 minutes at 37°C. DNA was stained with propidium iodide as described13 and then analyzed by flow cytometry with CellQuest software.
Apoptosis Assay
Apoptotic cells were detected using an ApoDETECT Annexin V-FITC Kit (Zymed) and visualized by confocal microscopy.
Western Blot Analysis and Assay of Kinase Activity
After treatment, cells were lysed and the cell lysate was fractionated by SDS-PAGE in a gradient gel before being transferred onto nitrocellulose membrane as described.13 Antibody reactions were detected using horseradish peroxidase-conjugated goat anti-mouse IgG (BioRad) and enhanced chemiluminescent detection reagents (Pierce). Densitometry was performed on a BioRad GS-670 Imaging Densitometer.
Immunoprecipitation of cell division cycle (Cdc) 2, cyclin-dependent kinase (Cdk) 2, and Cdk 4 was carried out as described.13 The kinase substrate was 0.2 μg/μL histone H1 (Gibco) for Cdc 2 and Cdk 2 and was 0.01 μg/μL GST-pRb (Santa Cruz) for Cdk 4. Details are found elsewhere.13
Immunocytochemistry
Cells were fixed in 50% acetone/50% methanol, then immunostained with primary antibodies to Cdc 2, Cdk 2, Cdk 4, Cyclin A, Cyclin B, Cyclin D1 (Transduction Laboratories), and proliferative cell nuclear antigen (PCNA) (Sigma). The secondary antibody was conjugated to fluorescein isothiocyanate (Sigma), observed, and quantified as described.13
Data Analysis
Results were analyzed by one-way ANOVA, followed by Dunnett’s posthoc test. A value of P<0.05 was considered significant.
Results
The effect of oxLDL on entry of cells into the cell cycle was analyzed by flow cytometry (Figure 1). Cells kept in serum-free medium for 5 to 6 days remained in a growth-arrested state (90% to 95% in G0/G1). The addition of serum to the medium caused these cells to move out of G0/G1 and into the cell cycle. Only 31% of cells maintained in 5% FBS (no oxLDL) for 24 hours remained in G0/G1. In contrast, at 24 hours, cells treated with 10 μg/mL oxLDL remained 66% arrested, and cells treated with 50 μg/mL oxLDL remained 78% arrested. Therefore, oxLDL inhibited the release of cells from growth arrest in a time- and dose-dependent manner.
Figure 1. Cell cycle entry for fibroblasts treated with serum and oxLDL. In cells maintained as described, DNA synthesis was assessed by propidium iodide staining using a FACSCalibur flow cytometer. The proportion of cells in G0/G1 following exposure to 0, 10, or 50 μg/mL oxLDL is expressed as a percentage of serum-starved control ±SEM (*P<0.05).
Total cell numbers were assessed to demonstrate that the effects of oxLDL resulted in inhibited movement through the complete cell cycle. Exposure to 10 or 50 μg/mL oxLDL in combination with serum for 24 or 48 hours resulted in significant decreases in the numbers of fibroblasts (Figure 2). VSMCs were exposed to an identical experimental protocol and the same qualitative effect was observed. Conversely, treatment of both fibroblasts and VSMCs with native LDL resulted in an increase in cell numbers under similar conditions (Figure 2).
Figure 2. Decrease in total number of fibroblasts and VSMCs following exposure to oxLDL or native LDL in combination with serum. Cells were maintained in serum-free media for 6 days before oxLDL or native LDL and serum treatment. Data represents total number of cells as counted using a hemacytometer ±SEM (*P<0.05). Error bars are too small to resolve.
The decrease in cell numbers may have been due to cell death via apoptosis. Annexin V expression was used as an apoptotic marker. No apoptotic cells were observed in the serum-treated group in the presence or absence of oxLDL over 48 hours (Figure I, available online at http://atvb.ahajournals.org). Conversely, cells treated with H2O2 displayed positive staining for annexin V.
For the purpose of comparison, we exposed quiescent fibroblasts to growth factors (aFGF, bFGF, and TGF-?1) or lipoprotein-depleted serum in combination with oxLDL. Treatment with either bFGF or TGF-?1 in combination with oxLDL produced a significant increase in cell number, while treatment with aFGF plus oxLDL diminished cell growth to an extent similar to serum (Table ). Lipoprotein-depleted serum in combination with oxLDL did not produce a significant effect: lipoprotein-depleted serum in combination with 10 μg/mL oxLDL increased cell numbers by 6%; lipoprotein-depleted serum in combination with 50 μg/mL oxLDL increased cell numbers by 4%.
Change in Cell Number After Exposure of Quiescent Fibroblasts to oxLDL in the Presence of Growth Factors
oxLDL may inhibit serum-induced proliferation through a change in the expression or distribution of cell cycle proteins. Expression of cell cycle proteins was examined by using Western blot analysis. In fibroblasts treated with serum and 10 μg/mL oxLDL for 24 hours, when cell numbers were reduced by 16% and the percentage of cells leaving G0/G1 was decreased by 35%, only the expression of Cdk 4 was significantly decreased relative to control (Figure II, available online at http://atvb.ahajournals.org). At 24 hours and in the presence of 50 μg/mL oxLDL, when cell numbers were reduced by 22% and the percentage of cells leaving G0/G1 was decreased by 47%, total cellular levels of Cdc 2, Cdk 4, Cyclin B1, and PCNA were significantly decreased relative to controls, while levels of Cyclin D1 were unchanged (Figure II). At 48 hours, when cell numbers were reduced by 20%, treatment with serum and 10 μg/mL oxLDL had no effect on the expression of any cell cycle protein (Figure II). At 48 hours, when cell numbers were reduced by 27%, treatment with serum and 50 μg/mL oxLDL significantly reduced the levels of Cdc 2, Cdk 2, Cdk 4, Cyclin B1, and PCNA relative to controls, while levels of Cyclin D1 were again unchanged (Figure II).
Due to the seeming discrepancy between the expression of the cell cycle proteins (Figure II), cell movement into the cycle (Figure 1), and the decrease in cell number (Figure 2), a change in the cellular localization of these proteins was investigated as another potential mechanism for the observed effects of oxLDL. Representative results for Cyclin D1 distribution are shown in Figure 3. After 48 hours, nuclear levels of Cyclin D1 were increased after serum treatment. However, this translocation was inhibited by both 10 and 50 μg/mL oxLDL and serum as compared with serum alone (Figure 3). This effect was in stark contrast to that observed in oxLDL treatment of cells in the absence of serum (Figure 3). Nuclear fluorescence was quantitated over a number of experiments to obtain an objective measurement of the redistribution of Cyclin D1. These measurements were also made for the 6 other cell cycle proteins (Figure III, available online at http://atvb.ahajournals.org). After 24 hours of exposure to 10 μg/mL oxLDL, significant decreases in nuclear levels of Cdc 2, Cyclin A, Cyclin D1, and PCNA were noted. After 48 hours of exposure to 10 μg/mL oxLDL, significant decreases were observed in nuclear levels of Cdk 4, Cyclin A, Cyclin D1, and PCNA. Twenty-four hours of exposure to 50 μg/mL oxLDL induced significant decreases in nuclear levels of Cdc 2, Cyclin D1, and Cyclin A. Forty-eight hours of exposure to 50 μg/mL oxLDL resulted in significant decreases in nuclear levels of Cdc 2, Cdk 2, Cdk 4, Cyclin A, Cyclin D1, Cyclin B1, and PCNA. Therefore, these data demonstrate that exposure of cells to oxLDL in the presence of serum results in decreases in nuclear levels of Cyclin D1, Cdc 2, Cdk 2, Cdk 4, Cyclin A, Cyclin B1, and PCNA. In contrast, nuclear levels of PCNA were increased in fibroblasts and VSMCs treated with native LDL and serum, though the increases (up to 6%) were not significant.
Figure 3. Representative confocal micrographs showing nuclear fluorescence of Cyclin D1 in fibroblasts after 48 hours of: no oxLDL in the presence of 5% serum (top left), 10 μg/mL oxLDL in the presence of 5% serum (middle left), and 50 μg/mL oxLDL in the presence of 5% serum (bottom left); no oxLDL under starvation conditions (top right, 10 μg/mL oxLDL under starvation conditions (middle right), and 50 μg/mL oxLDL under starvation conditions (bottom right).
The cytoplasmic retention of cell cycle proteins suggests that the cyclin/cyclin-dependent kinase complexes are inactive. We directly examined Cdc 2, Cdk 2, and Cdk4 kinase activity under our experimental conditions. Exposure of cells to 10 μg/mL oxLDL for 24 hours resulted in a 23% decrease in Cdk 2 activity in comparison to control cells (Figure 4), while exposure of cells to 50 μg/mL oxLDL for 24 hours resulted in a 14% decrease in Cdc 2 activity. Kinase activity of Cdk 4 was unchanged in oxLDL-treated cells as compared with controls.
Figure 4. Decreased kinase activity of Cdc 2 and Cdk 2 in serum-treated fibroblasts after exposure to oxLDL. Top: representative autoradiographs showing Cdc 2 activity (left) and Cdk 2 activity (right) with histone H1 as a substrate in whole cell extracts from fibroblasts treated with 0, 10, or 50 μg/mL oxLDL for 24 hours. Bottom: densitometric comparisons of Cdc 2 and Cdk 2 activity, expressed as a percentage of control ±SEM (*P<0.05, n=3 for each condition and time point).
In order to ascertain the mechanism(s) involved in oxLDL’s inhibitory effect on cell growth, a number of pharmacological inhibitors of selected signaling pathways were employed. The PI3K inhibitor LY294002 (at a concentration of 20 μg/mL) and the protein kinase C inhibitor calphostin C (at a concentration of 200 ng/mL) both failed to prevent the reduction in cell proliferation in response to oxLDL (Figure 5). However, treatment with the phospholipase (PL) C/A2 inhibitor U73122 (at a concentration of 3 μg/mL) effectively reversed the oxLDL-induced inhibition of proliferation (Figure 5). Treatment with U73122 also blocked growth in response to native LDL plus serum. Cell numbers were increased by 8% and 3% over control with 10 and 50 μg/mL native LDL, respectively; these increases in cell number were not significant. Cells treated with LY294002, calphostin C, or U73122 in the absence of oxLDL showed no evidence of cell death as compared with cells maintained in starvation medium.
Figure 5. Effect of inhibitors on cell numbers in fibroblasts exposed to oxLDL in the presence of serum. Cell counts are expressed as mean per field ±SEM (*P<0.05). Cells were pretreated with inhibitors alone for 15 minutes before exposure for 24 hours to oxLDL in combination with serum and inhibitors.
The activation of extracellular signal regulated kinase (ERK)-1 and ERK2 was evaluated in U73122-treated cells. Western blots of extracts from cells treated with serum and oxLDL in combination with U73122 showed that ERK1/ERK2 activation, as detected using a monoclonal antibody to phospho-p44/p42 mitogen-activated protein kinase (MAPK), was completely abolished in U73122-treated cells relative to controls (Figure IV, available online at http://atvb.ahajournals.org).
Discussion
The purpose of the present study was to determine the mitogenic potential of oxLDL when cells were under simultaneous mitogenic influence of other growth factors and cytokines in serum. That oxLDL reduced, rather than amplified, the proliferative response of cells to serum was unexpected.10–13 However, the observation that oxLDL functions to inhibit cell proliferation is not entirely without precedent. oxLDL has been shown by Henry’s laboratory to inhibit cell proliferation by altering the expression of mitogens.34,35 In the present study, three lines of evidence support our observation of an inhibitory effect of oxLDL on cell proliferation. First, oxLDL reduced the total number of serum-treated fibroblasts entering the cell cycle. Second, oxLDL reduced the total number of cells completing the cell cycle in serum-treated cultures. Third, oxLDL caused a decrease in nuclear levels of cell cycle proteins in serum-treated cells. These effects were not specific to cell type: both fibroblasts and VSMCs exhibited similar responses.
oxLDL has been shown to induce apoptosis.12 However, no apoptotic cells were observed under the present experimental conditions, nor did LDH levels increase after any experimental intervention (data not shown). This observation would argue strongly against cell death by necrosis. In addition, although oxLDL inhibited the proliferative effects of serum, cell numbers continued to increase, although not nearly as rapidly as in the absence of oxLDL. The most likely conclusion, therefore, is that oxLDL slowed the proliferative response through an effect on the cell cycle, not through an induction of cell death.
Therefore, we focused our mechanistic research on the cell cycle. The expression of cell cycle proteins in cells treated with oxLDL and serum was inconsistent with the observed decrease in cell entry into the cell cycle and the reduction in cell number. For example, expression of Cyclin D1 (required for movement out of G0/G1 into the cell cycle) was unchanged in cells treated with oxLDL and at all time points despite a time- and dose-dependent inhibition of release from growth arrest and a significant reduction in cell numbers. This finding does not argue in favor of a strong association between cell cycle protein expression and growth under our conditions.
Alternatively, the functional ability of the cell cycle proteins depends on their translocation to the nucleus. A dramatic reduction in the nuclear levels of all cell cycle proteins was observed in cells treated with oxLDL and serum as compared with serum alone. Significantly, the nuclear translocation of Cyclin D1 was consistently inhibited by 10 μg/mL oxLDL. The failure of the cell cycle proteins to enter the nucleus would necessarily result in the formation of fewer active cyclin/cyclin-dependent kinase complexes. Cdc 2 and Cdk 2 kinase activities were significantly reduced in cells treated with oxLDL and serum, as compared with cells treated with serum alone. Together these data define an inhibition in the nuclear translocation of cell cycle proteins as a key mechanism for the attenuated proliferative effects of oxLDL.
The surprising observation that treatment with oxLDL and serum results in a diminished, rather than enhanced, proliferative response would seem to conflict with published observations by Auge et al.10 In their experiments, the combination of oxLDL and serum produced an enhanced proliferative response in cultured bovine aortic smooth muscle cells. However, it should be noted that Auge et al used a lipoprotein-depleted serum.10 In our experiments using lipoprotein-depleted serum, we found no significant change in cell number at 24 hours. This is consistent with the results of Auge et al, who saw no significant change in cell number until 7 days in culture.10 In our investigations into the specific components of serum that may be interacting with oxLDL, we found that aFGF (but not bFGF or TGF-?1) inhibited cell growth when given in combination with oxLDL. Interestingly, the recent findings of Ananyeva et al36 suggest that oxLDL forms complexes with aFGF that inhibit the growth-promoting function of oxLDL in vitro. Thus aFGF may be responsible, at least in part, for the diminished growth observed in cells treated with oxLDL and serum.
The mechanism whereby oxLDL-mediated inhibition of nuclear translocation occurs is unclear. However, it is possible that the MAPK pathway, which can be stimulated by oxLDL growth,9,37,38 is involved. Whereas the MAPK pathway is commonly associated with cell growth,9,37,38 chronic activation (hours) of the MAPK cascade results in an inhibition of DNA synthesis, cell cycle progression, and cdk2 activity.39 Chronic activation of MAPK would be expected under our experimental conditions. MAPK activation inhibits nuclear protein import.38 Therefore, oxLDL-induced activation of MAPK would inhibit the nuclear import of cell cycle proteins. We tested this possibility directly with the use of drugs that inhibit MAPK activity. Unfortunately, both of the commonly used blockers of the MAPK pathway that we employed (PD98059 and SB203580) were cytotoxic under our extended experimental conditions (data not shown). However, U73122 is also known to be an effective blocker of the MAPK pathway.37 U73122 restored cell proliferation to control levels and negated the inhibitory effects of oxLDL (Figure 5), while completely knocking out ERK1/ERK2 activity. Therefore, it is reasonable to argue that the oxLDL-induced stimulation of the MAPK pathway was inhibited by U73122, resulting in a restoration of the cellular proliferative response. We cannot discount the possibility that U73122 is also acting as a PLC inhibitor. However, the influence of PLC activity on nuclear protein translocation is unknown. We may conclude that the depressed cell cycle protein translocation induced by oxLDL occurs at least in part via activation of the MAP kinase pathway.
The findings of this paper challenge prevailing notions about the role of oxLDL in atherogenesis. oxLDL has been suggested to participate in the development of atherosclerosis partly by promoting the growth of vascular cells.1,11,40 However, most studies investigating proliferative activity in human atherectomy tissue have found little evidence for active cell replication (typically <1%)26,27 despite the presence of oxLDL in the vascular environment.41,42 Clinically significant stenoses take several decades to develop. In the complex environment in which these plaques are formed, factors that negatively modulate the proliferative response of vascular cells (or at least slow its progression) must come into play to explain the relatively slow cell growth in atherosclerosis. Apoptosis may be one factor,25 but the present experiments demonstrate that oxLDL itself can negatively modulate the response of cells under some conditions. Its action in vivo may be far more complex than originally anticipated and may vary dramatically dependent on the proliferative state of the vasculature and the mitogenic environment.
Acknowledgments
This work was supported by a grant from the Canadian Institutes for Health Research (CIHR). M.E.Z. received a studentship from the Deer Lodge Hospital Association Memorial Fund and a Doctoral Research Award from CIHR/Heart and Stroke Foundation of Canada. G.N.P. is a CIHR Senior Scientist.
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ABSTRACT
Objective— Our study tested the hypothesis that the mitogenic effect of oxidized low-density lipoprotein (oxLDL) on vascular cells may be further enhanced by the presence of cytokines and growth factors known to be present in the atherosclerotic environment.
Methods and Results— Quiescent fibroblasts and vascular smooth muscle cells were treated with 10 or 50 μg/mL minimally-oxidized LDL in combination with serum for 24 or 48 hours. Surprisingly, these cells showed inhibited release from growth arrest and a significant reduction in the number of cells completing the cell cycle when compared with cells treated with serum alone. This was not due to an induction of apoptosis. The antiproliferative effects were not closely associated with changes in the expression of cell cycle proteins. Instead, oxLDL inhibited the translocation of cell cycle proteins cell division cycle (Cdc) 2, cyclin-dependent kinase (Cdk) 2, Cdk 4, Cyclin A, Cyclin B1, Cyclin D1, and proliferative cell nuclear antigen (PCNA) into the nucleus, as compared with separate treatments with serum alone. Kinase activation associated with specific cell cycle proteins was also inhibited by oxLDL.
Conclusions— oxLDL, in the presence of serum, has a surprising inhibitory effect on cell proliferation that occurs through an inhibition of import of cell cycle proteins into the cell nucleus.
Key Words: atherosclerosis ? cyclin ? kinase, cyclin-dependent ? cell proliferation
Introduction
Oxidized low-density lipoprotein (oxLDL) is believed to play a critical role in atherogenesis.1–4 oxLDL has numerous growth-promoting effects on cells in vitro,5 including the induction of transcription factors6,7 and the enzymes involved in mitogenesis,8,9 as well as stimulation of DNA synthesis and cell proliferation.10–12 oxLDL can stimulate vascular smooth muscle cell (VSMC) proliferation in cell culture conditions even in the absence of any other growth factors. This effect involves a stimulation of the phosphatidylinositol 3-kinase (PI3K) pathway and an induction of cell cycle proteins.13
Cell cycle proteins closely control the movement of a cell into a proliferative state.14–18 Alterations in the expression, activity, or nuclear translocation of cell cycle proteins within a cell will determine the capacity for that cell to move into the cell cycle and proliferate. Changes in the expression of a number of cell cycle proteins in the vessel wall during atherosclerosis or restenosis have been identified and are thought to represent critical cellular events that determine the proliferative potential of the cells during these pathological states.19–24
Recently, this hypothesis has been challenged.25–27 Some studies report minimal evidence in favor of accelerated cell proliferation in plaques where stimulated cell proliferation would be expected.26,27 Other plausible mechanisms have been proposed (eg, inhibited apoptosis).25 It is also possible that mitogenic factors like oxLDL may not have been studied optimally to define their proliferative potential. Previous investigations have studied the effects of oxLDL in isolation, however, in an in vivo atherosclerotic environment, vascular cells are exposed to oxLDL in the presence of a multitude of cytokines and growth factors. The purpose of the present study, therefore, was to elucidate the effects of oxLDL on cell proliferation in the presence of a variety of growth factors and cytokines found in serum. To obtain mechanistic insights, the effects of oxLDL on cell cycle proteins in this atherosclerotic environment were a focus for our study.
Methods
Cell Culture and Incubation Conditions
Confluent cultures of human neonatal fibroblasts or New Zealand White rabbit VSMCs, isolated as described,28 were passaged and then incubated for 24 hours in DMEM supplemented with 5% FBS. Cells were washed and then placed in serum-free DMEM supplemented with transferrin (5 μg/mL), selenium (1 nM), ascorbate (200 μmol/L), and insulin (10 nM) for 6 days in order to induce growth arrest. Cells were then incubated with 10 or 50 μg cholesterol/mL LDL or oxLDL in combination with FBS (5% for fibroblasts, 10% for VSMCs) for 24 or 48 hours. LDL was oxidized with a Fe-ADP free radical generating system. The method and characteristics of the minimally-modified oxLDL are reported in detail elsewhere.29,30 Typically, this preparation of oxLDL exhibits a modest increase in electrophoretic mobility, a 20% depletion of vitamin E, and a 30% increase in malondialdehyde content.30 Cholesterol concentrations were assessed prior to oxidation, and these concentrations were used for both native and oxLDL. Protein concentrations were unchanged throughout the course of the experiments. The same concentrations of Fe and ADP added to control cells in the absence of LDL had no effect (data not shown). Cultures were maintained at 37°C in humidified 5% CO2, and medium was replaced every 24 hours. Freshly prepared oxLDL was also replaced on a daily basis. Control cells were maintained in identical media (in the absence of oxLDL) for the same period of time. The duration of exposure to oxLDL was not cytotoxic to cells as determined by measurement of lactate dehydrogenase (LDH) activity in the medium31 or via ethidium homodimer staining.28 The acidic and basic fibroblast growth factors (aFGF and bFGF; Sigma) and transforming growth factor-?1 (TGF-?1, Sigma) were used at concentrations of 50 ng/mL, 10 ng/mL, and 5 ng/mL, respectively. Lipoprotein-depleted serum was prepared as described by Auge et al.10 For experiments involving inhibitors, cells were pretreated for 15 minutes with 20 μg/mL LY294002 (Sigma),32 200 ng/mL calphostin C (Sigma),8 or 3 μg/mL U73122 (Sigma)33 before exposure to oxLDL. The drugs remained in the media for the duration of the experiments.
Measurement of Cell Numbers and Cell Cycle Analysis by Flow Cytometry
Cell numbers were counted in a hemacytometer. For each condition and time point, 18 fields were counted. For cell cycle analysis, cells were trypsinized after treatment with oxLDL, fixed, and treated with RNase A (500U/mL in 1.12% sodium citrate) for 15 minutes at 37°C. DNA was stained with propidium iodide as described13 and then analyzed by flow cytometry with CellQuest software.
Apoptosis Assay
Apoptotic cells were detected using an ApoDETECT Annexin V-FITC Kit (Zymed) and visualized by confocal microscopy.
Western Blot Analysis and Assay of Kinase Activity
After treatment, cells were lysed and the cell lysate was fractionated by SDS-PAGE in a gradient gel before being transferred onto nitrocellulose membrane as described.13 Antibody reactions were detected using horseradish peroxidase-conjugated goat anti-mouse IgG (BioRad) and enhanced chemiluminescent detection reagents (Pierce). Densitometry was performed on a BioRad GS-670 Imaging Densitometer.
Immunoprecipitation of cell division cycle (Cdc) 2, cyclin-dependent kinase (Cdk) 2, and Cdk 4 was carried out as described.13 The kinase substrate was 0.2 μg/μL histone H1 (Gibco) for Cdc 2 and Cdk 2 and was 0.01 μg/μL GST-pRb (Santa Cruz) for Cdk 4. Details are found elsewhere.13
Immunocytochemistry
Cells were fixed in 50% acetone/50% methanol, then immunostained with primary antibodies to Cdc 2, Cdk 2, Cdk 4, Cyclin A, Cyclin B, Cyclin D1 (Transduction Laboratories), and proliferative cell nuclear antigen (PCNA) (Sigma). The secondary antibody was conjugated to fluorescein isothiocyanate (Sigma), observed, and quantified as described.13
Data Analysis
Results were analyzed by one-way ANOVA, followed by Dunnett’s posthoc test. A value of P<0.05 was considered significant.
Results
The effect of oxLDL on entry of cells into the cell cycle was analyzed by flow cytometry (Figure 1). Cells kept in serum-free medium for 5 to 6 days remained in a growth-arrested state (90% to 95% in G0/G1). The addition of serum to the medium caused these cells to move out of G0/G1 and into the cell cycle. Only 31% of cells maintained in 5% FBS (no oxLDL) for 24 hours remained in G0/G1. In contrast, at 24 hours, cells treated with 10 μg/mL oxLDL remained 66% arrested, and cells treated with 50 μg/mL oxLDL remained 78% arrested. Therefore, oxLDL inhibited the release of cells from growth arrest in a time- and dose-dependent manner.
Figure 1. Cell cycle entry for fibroblasts treated with serum and oxLDL. In cells maintained as described, DNA synthesis was assessed by propidium iodide staining using a FACSCalibur flow cytometer. The proportion of cells in G0/G1 following exposure to 0, 10, or 50 μg/mL oxLDL is expressed as a percentage of serum-starved control ±SEM (*P<0.05).
Total cell numbers were assessed to demonstrate that the effects of oxLDL resulted in inhibited movement through the complete cell cycle. Exposure to 10 or 50 μg/mL oxLDL in combination with serum for 24 or 48 hours resulted in significant decreases in the numbers of fibroblasts (Figure 2). VSMCs were exposed to an identical experimental protocol and the same qualitative effect was observed. Conversely, treatment of both fibroblasts and VSMCs with native LDL resulted in an increase in cell numbers under similar conditions (Figure 2).
Figure 2. Decrease in total number of fibroblasts and VSMCs following exposure to oxLDL or native LDL in combination with serum. Cells were maintained in serum-free media for 6 days before oxLDL or native LDL and serum treatment. Data represents total number of cells as counted using a hemacytometer ±SEM (*P<0.05). Error bars are too small to resolve.
The decrease in cell numbers may have been due to cell death via apoptosis. Annexin V expression was used as an apoptotic marker. No apoptotic cells were observed in the serum-treated group in the presence or absence of oxLDL over 48 hours (Figure I, available online at http://atvb.ahajournals.org). Conversely, cells treated with H2O2 displayed positive staining for annexin V.
For the purpose of comparison, we exposed quiescent fibroblasts to growth factors (aFGF, bFGF, and TGF-?1) or lipoprotein-depleted serum in combination with oxLDL. Treatment with either bFGF or TGF-?1 in combination with oxLDL produced a significant increase in cell number, while treatment with aFGF plus oxLDL diminished cell growth to an extent similar to serum (Table ). Lipoprotein-depleted serum in combination with oxLDL did not produce a significant effect: lipoprotein-depleted serum in combination with 10 μg/mL oxLDL increased cell numbers by 6%; lipoprotein-depleted serum in combination with 50 μg/mL oxLDL increased cell numbers by 4%.
Change in Cell Number After Exposure of Quiescent Fibroblasts to oxLDL in the Presence of Growth Factors
oxLDL may inhibit serum-induced proliferation through a change in the expression or distribution of cell cycle proteins. Expression of cell cycle proteins was examined by using Western blot analysis. In fibroblasts treated with serum and 10 μg/mL oxLDL for 24 hours, when cell numbers were reduced by 16% and the percentage of cells leaving G0/G1 was decreased by 35%, only the expression of Cdk 4 was significantly decreased relative to control (Figure II, available online at http://atvb.ahajournals.org). At 24 hours and in the presence of 50 μg/mL oxLDL, when cell numbers were reduced by 22% and the percentage of cells leaving G0/G1 was decreased by 47%, total cellular levels of Cdc 2, Cdk 4, Cyclin B1, and PCNA were significantly decreased relative to controls, while levels of Cyclin D1 were unchanged (Figure II). At 48 hours, when cell numbers were reduced by 20%, treatment with serum and 10 μg/mL oxLDL had no effect on the expression of any cell cycle protein (Figure II). At 48 hours, when cell numbers were reduced by 27%, treatment with serum and 50 μg/mL oxLDL significantly reduced the levels of Cdc 2, Cdk 2, Cdk 4, Cyclin B1, and PCNA relative to controls, while levels of Cyclin D1 were again unchanged (Figure II).
Due to the seeming discrepancy between the expression of the cell cycle proteins (Figure II), cell movement into the cycle (Figure 1), and the decrease in cell number (Figure 2), a change in the cellular localization of these proteins was investigated as another potential mechanism for the observed effects of oxLDL. Representative results for Cyclin D1 distribution are shown in Figure 3. After 48 hours, nuclear levels of Cyclin D1 were increased after serum treatment. However, this translocation was inhibited by both 10 and 50 μg/mL oxLDL and serum as compared with serum alone (Figure 3). This effect was in stark contrast to that observed in oxLDL treatment of cells in the absence of serum (Figure 3). Nuclear fluorescence was quantitated over a number of experiments to obtain an objective measurement of the redistribution of Cyclin D1. These measurements were also made for the 6 other cell cycle proteins (Figure III, available online at http://atvb.ahajournals.org). After 24 hours of exposure to 10 μg/mL oxLDL, significant decreases in nuclear levels of Cdc 2, Cyclin A, Cyclin D1, and PCNA were noted. After 48 hours of exposure to 10 μg/mL oxLDL, significant decreases were observed in nuclear levels of Cdk 4, Cyclin A, Cyclin D1, and PCNA. Twenty-four hours of exposure to 50 μg/mL oxLDL induced significant decreases in nuclear levels of Cdc 2, Cyclin D1, and Cyclin A. Forty-eight hours of exposure to 50 μg/mL oxLDL resulted in significant decreases in nuclear levels of Cdc 2, Cdk 2, Cdk 4, Cyclin A, Cyclin D1, Cyclin B1, and PCNA. Therefore, these data demonstrate that exposure of cells to oxLDL in the presence of serum results in decreases in nuclear levels of Cyclin D1, Cdc 2, Cdk 2, Cdk 4, Cyclin A, Cyclin B1, and PCNA. In contrast, nuclear levels of PCNA were increased in fibroblasts and VSMCs treated with native LDL and serum, though the increases (up to 6%) were not significant.
Figure 3. Representative confocal micrographs showing nuclear fluorescence of Cyclin D1 in fibroblasts after 48 hours of: no oxLDL in the presence of 5% serum (top left), 10 μg/mL oxLDL in the presence of 5% serum (middle left), and 50 μg/mL oxLDL in the presence of 5% serum (bottom left); no oxLDL under starvation conditions (top right, 10 μg/mL oxLDL under starvation conditions (middle right), and 50 μg/mL oxLDL under starvation conditions (bottom right).
The cytoplasmic retention of cell cycle proteins suggests that the cyclin/cyclin-dependent kinase complexes are inactive. We directly examined Cdc 2, Cdk 2, and Cdk4 kinase activity under our experimental conditions. Exposure of cells to 10 μg/mL oxLDL for 24 hours resulted in a 23% decrease in Cdk 2 activity in comparison to control cells (Figure 4), while exposure of cells to 50 μg/mL oxLDL for 24 hours resulted in a 14% decrease in Cdc 2 activity. Kinase activity of Cdk 4 was unchanged in oxLDL-treated cells as compared with controls.
Figure 4. Decreased kinase activity of Cdc 2 and Cdk 2 in serum-treated fibroblasts after exposure to oxLDL. Top: representative autoradiographs showing Cdc 2 activity (left) and Cdk 2 activity (right) with histone H1 as a substrate in whole cell extracts from fibroblasts treated with 0, 10, or 50 μg/mL oxLDL for 24 hours. Bottom: densitometric comparisons of Cdc 2 and Cdk 2 activity, expressed as a percentage of control ±SEM (*P<0.05, n=3 for each condition and time point).
In order to ascertain the mechanism(s) involved in oxLDL’s inhibitory effect on cell growth, a number of pharmacological inhibitors of selected signaling pathways were employed. The PI3K inhibitor LY294002 (at a concentration of 20 μg/mL) and the protein kinase C inhibitor calphostin C (at a concentration of 200 ng/mL) both failed to prevent the reduction in cell proliferation in response to oxLDL (Figure 5). However, treatment with the phospholipase (PL) C/A2 inhibitor U73122 (at a concentration of 3 μg/mL) effectively reversed the oxLDL-induced inhibition of proliferation (Figure 5). Treatment with U73122 also blocked growth in response to native LDL plus serum. Cell numbers were increased by 8% and 3% over control with 10 and 50 μg/mL native LDL, respectively; these increases in cell number were not significant. Cells treated with LY294002, calphostin C, or U73122 in the absence of oxLDL showed no evidence of cell death as compared with cells maintained in starvation medium.
Figure 5. Effect of inhibitors on cell numbers in fibroblasts exposed to oxLDL in the presence of serum. Cell counts are expressed as mean per field ±SEM (*P<0.05). Cells were pretreated with inhibitors alone for 15 minutes before exposure for 24 hours to oxLDL in combination with serum and inhibitors.
The activation of extracellular signal regulated kinase (ERK)-1 and ERK2 was evaluated in U73122-treated cells. Western blots of extracts from cells treated with serum and oxLDL in combination with U73122 showed that ERK1/ERK2 activation, as detected using a monoclonal antibody to phospho-p44/p42 mitogen-activated protein kinase (MAPK), was completely abolished in U73122-treated cells relative to controls (Figure IV, available online at http://atvb.ahajournals.org).
Discussion
The purpose of the present study was to determine the mitogenic potential of oxLDL when cells were under simultaneous mitogenic influence of other growth factors and cytokines in serum. That oxLDL reduced, rather than amplified, the proliferative response of cells to serum was unexpected.10–13 However, the observation that oxLDL functions to inhibit cell proliferation is not entirely without precedent. oxLDL has been shown by Henry’s laboratory to inhibit cell proliferation by altering the expression of mitogens.34,35 In the present study, three lines of evidence support our observation of an inhibitory effect of oxLDL on cell proliferation. First, oxLDL reduced the total number of serum-treated fibroblasts entering the cell cycle. Second, oxLDL reduced the total number of cells completing the cell cycle in serum-treated cultures. Third, oxLDL caused a decrease in nuclear levels of cell cycle proteins in serum-treated cells. These effects were not specific to cell type: both fibroblasts and VSMCs exhibited similar responses.
oxLDL has been shown to induce apoptosis.12 However, no apoptotic cells were observed under the present experimental conditions, nor did LDH levels increase after any experimental intervention (data not shown). This observation would argue strongly against cell death by necrosis. In addition, although oxLDL inhibited the proliferative effects of serum, cell numbers continued to increase, although not nearly as rapidly as in the absence of oxLDL. The most likely conclusion, therefore, is that oxLDL slowed the proliferative response through an effect on the cell cycle, not through an induction of cell death.
Therefore, we focused our mechanistic research on the cell cycle. The expression of cell cycle proteins in cells treated with oxLDL and serum was inconsistent with the observed decrease in cell entry into the cell cycle and the reduction in cell number. For example, expression of Cyclin D1 (required for movement out of G0/G1 into the cell cycle) was unchanged in cells treated with oxLDL and at all time points despite a time- and dose-dependent inhibition of release from growth arrest and a significant reduction in cell numbers. This finding does not argue in favor of a strong association between cell cycle protein expression and growth under our conditions.
Alternatively, the functional ability of the cell cycle proteins depends on their translocation to the nucleus. A dramatic reduction in the nuclear levels of all cell cycle proteins was observed in cells treated with oxLDL and serum as compared with serum alone. Significantly, the nuclear translocation of Cyclin D1 was consistently inhibited by 10 μg/mL oxLDL. The failure of the cell cycle proteins to enter the nucleus would necessarily result in the formation of fewer active cyclin/cyclin-dependent kinase complexes. Cdc 2 and Cdk 2 kinase activities were significantly reduced in cells treated with oxLDL and serum, as compared with cells treated with serum alone. Together these data define an inhibition in the nuclear translocation of cell cycle proteins as a key mechanism for the attenuated proliferative effects of oxLDL.
The surprising observation that treatment with oxLDL and serum results in a diminished, rather than enhanced, proliferative response would seem to conflict with published observations by Auge et al.10 In their experiments, the combination of oxLDL and serum produced an enhanced proliferative response in cultured bovine aortic smooth muscle cells. However, it should be noted that Auge et al used a lipoprotein-depleted serum.10 In our experiments using lipoprotein-depleted serum, we found no significant change in cell number at 24 hours. This is consistent with the results of Auge et al, who saw no significant change in cell number until 7 days in culture.10 In our investigations into the specific components of serum that may be interacting with oxLDL, we found that aFGF (but not bFGF or TGF-?1) inhibited cell growth when given in combination with oxLDL. Interestingly, the recent findings of Ananyeva et al36 suggest that oxLDL forms complexes with aFGF that inhibit the growth-promoting function of oxLDL in vitro. Thus aFGF may be responsible, at least in part, for the diminished growth observed in cells treated with oxLDL and serum.
The mechanism whereby oxLDL-mediated inhibition of nuclear translocation occurs is unclear. However, it is possible that the MAPK pathway, which can be stimulated by oxLDL growth,9,37,38 is involved. Whereas the MAPK pathway is commonly associated with cell growth,9,37,38 chronic activation (hours) of the MAPK cascade results in an inhibition of DNA synthesis, cell cycle progression, and cdk2 activity.39 Chronic activation of MAPK would be expected under our experimental conditions. MAPK activation inhibits nuclear protein import.38 Therefore, oxLDL-induced activation of MAPK would inhibit the nuclear import of cell cycle proteins. We tested this possibility directly with the use of drugs that inhibit MAPK activity. Unfortunately, both of the commonly used blockers of the MAPK pathway that we employed (PD98059 and SB203580) were cytotoxic under our extended experimental conditions (data not shown). However, U73122 is also known to be an effective blocker of the MAPK pathway.37 U73122 restored cell proliferation to control levels and negated the inhibitory effects of oxLDL (Figure 5), while completely knocking out ERK1/ERK2 activity. Therefore, it is reasonable to argue that the oxLDL-induced stimulation of the MAPK pathway was inhibited by U73122, resulting in a restoration of the cellular proliferative response. We cannot discount the possibility that U73122 is also acting as a PLC inhibitor. However, the influence of PLC activity on nuclear protein translocation is unknown. We may conclude that the depressed cell cycle protein translocation induced by oxLDL occurs at least in part via activation of the MAP kinase pathway.
The findings of this paper challenge prevailing notions about the role of oxLDL in atherogenesis. oxLDL has been suggested to participate in the development of atherosclerosis partly by promoting the growth of vascular cells.1,11,40 However, most studies investigating proliferative activity in human atherectomy tissue have found little evidence for active cell replication (typically <1%)26,27 despite the presence of oxLDL in the vascular environment.41,42 Clinically significant stenoses take several decades to develop. In the complex environment in which these plaques are formed, factors that negatively modulate the proliferative response of vascular cells (or at least slow its progression) must come into play to explain the relatively slow cell growth in atherosclerosis. Apoptosis may be one factor,25 but the present experiments demonstrate that oxLDL itself can negatively modulate the response of cells under some conditions. Its action in vivo may be far more complex than originally anticipated and may vary dramatically dependent on the proliferative state of the vasculature and the mitogenic environment.
Acknowledgments
This work was supported by a grant from the Canadian Institutes for Health Research (CIHR). M.E.Z. received a studentship from the Deer Lodge Hospital Association Memorial Fund and a Doctoral Research Award from CIHR/Heart and Stroke Foundation of Canada. G.N.P. is a CIHR Senior Scientist.
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