(Hypertension. 1997;29:326.)
© 1997 American Heart Association, Inc.
Arthur C. Corcoran Memorial Lecture |
Cholesterol Enhances Platelet-Derived Growth Factor-BB-Induced [Ca2+]i and DNA Synthesis in Rat Aortic Smooth Muscle Cells
From the Medizinische Universitäts-Poliklinik, Bonn, Germany.
Correspondence to PD Dr A. Sachinidis, Medizinische Universitäts-Poliklinik, Wilhelmstr 35–37, 53111 Bonn, Germany
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Abstract |
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In the present study, we describe possible mechanisms by which hypercholesterolemia may contribute to the development of cardiovascular diseases. Treatment of rat aortic smooth muscle cells for 20 hours with cholesterol-rich liposomes (500 µg/mL cholesterol, 100 µg/mL low-density lipoprotein) resulted in a 76±12% increase in total cholesterol content. The effects of cholesterol enrichment were examined by determination of changes in cell membrane fluidity. Fluidity of the cholesterol-enriched cell membranes was decreased at all temperatures between 15°C and 40°C. Changes in membrane fluidity in whole cell membranes represented changes in fluidity of microsomal membranes isolated by Percoll gradient ultracentrifugation. The basal [Ca2+]i and the maximal platelet-derived growth factor (PDGF)-BB-induced [Ca2+]i was elevated by 30% and 90% in cholesterol-enriched cells, respectively. In contrast, the resting pHi and the PDGF-BB-induced stimulation of the Na+/H+ exchange were not affected in cholesterol-enriched cells. The effect of PDGF-BB on [3H]thymidine incorporation in cholesterol-enriched cells was elevated by 40% in comparison with untreated cells. Our findings show that cellular cholesterol may be involved in the development of vascular diseases via modulation of the PDGF-induced increase in [Ca2+]i and DNA synthesis in vascular smooth muscle cells.
Key Words: cholesterol • vascular smooth muscle cells • platelet-derived growth factor
Abbreviations: DMEM = Dulbecco’s modified Eagle’s medium • PC = dipalmitoylphosphatidylcholine • PDGF = platelet-derived growth factor • VSMC = vascular smooth muscle cell(s)
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Introduction |
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Vascular smooth muscle cell hypertrophy and proliferation may participate in the pathophysiology of both hypertension and atherosclerosis.1,2 PDGF is a potent proliferative factor for different cell species including VSMC; thus, it is postulated that it plays an important role in the pathogenesis of atherosclerosis.3,4 Changes in cholesterol content of mammalian cell membranes modulate their physical structure and function.5,6 Several studies in the past have demonstrated that cholesterol enrichment of VSMC and cardiac cells modulates the functional activity of certain integral proteins, including ion channel activity. In this context, it has been reported that cholesterol enrichment of VSMC caused a marked increase in basal concentration of cytosolic Ca2+ ([Ca2+]i) and lowered plasma membrane fluidity.7 Furthermore, serum- and serotonin-stimulated Ca2+ uptake were potentiated in cholesterol-enriched VSMC, whereas endothelin-, vasopressin-, and thrombin-stimulated Ca2+ uptakes were not affected.8 The same group reported that in cholesterol-enriched VSMC, the activity of the L-type voltage-sensitive calcium channel current was increased.9 Moreover, decrease in total cholesterol content of embryonic cardiac cells by mevinolin resulted in an depression of the Ca2+ channel activity.10 Finally, the Na+/Ca+ exchange activity measured in cholesterol-enriched sarcolemmal vesicles was increased.11 In the last decade, many studies have shown that following stimulation of VSMC with PDGF-BB, there is an increase in [Ca2+]i and stimulation of the Na+/H+ exchange.12,13 The isoform 1 of the Na+/H+ exchanger (NHE-1) is a ubiquitously expressed membrane transport protein that has been proposed to be involved in the regulation of cell growth after cell stimulation with various growth factors.14 Until now, the potential effect of cholesterol content in VSMC on PDGF-induced cell growth has not been investigated. Because [Ca2+]i15 and pHi16 might be important signals for VSMC cell growth and thus may be involved in the pathophysiology of cardiovascular disease, we tested the hypothesis of whether cholesterol enrichment of VSMC results in an increase in PDGF-BB-induced DNA synthesis and whether it modulates the PDGF-BB-dependent effects on [Ca2+]i and pHi.
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Culture of VSMC
Rat aortic VSMC were isolated from thoracic aortas from Wistar-Kyoto rats (6 to 8 weeks old, Charles River Wiga GmbH) by enzymatic dispersion using a slight modification of the method of Chamley et al17 as described previously.18 Cells were cultured in DMEM supplemented with 10% fetal calf serum, nonessential amino acids, 100 IU/mL penicillin, and 100 µg/mL streptomycin at 37°C in the steri-cult incubator (Forma Scientific) in a humidified atmosphere of 95% air and 5% CO2. Cells (3x106) were grown in 75-cm2 flasks to confluence over 4 to 5 days. The purity of VSMC cultures was confirmed by immunocytochemical localization of smooth muscle specific
-smooth muscle actin with the use of FITC-conjugated monoclonal anti--smooth muscle actin plus a second FITC-conjugated F(ab')2 fragment of goat anti-mouse immunoglobulins. Experiments were performed using cells between passages 5 to 20.
Cholesterol Enrichment of VSMC
Cholesterol liposomes were prepared from cholesterol and PC according to Cooper et al.19 PC (48 mg) and cholesterol (96 mg) were suspended in 10 mL HEPES buffer (20 mmol/L HEPES, 16 mmol/L glucose, 130 mmol/L NaCl, 1 mmol/L MgSO4 7 H2O, 0.5 mmol/L CaCl2, Tris-base, pH 7.4) and sonicated (50 W) under nitrogen at 45°C for 45 minutes with a sonifier (Vibra Cell, Sonics Materials Inc). After sonification, the mixture was centrifuged at 21 000g to remove the undispersed lipid. Cholesterol was determined enzymatically according to instructions of the Sigma kit. Briefly, total cholesterol is measured by the use of the following enzymatic reaction. Cholesterol oxidase is added to generate H2O2. Peroxidase catalyzes the reaction of H2O2 with 4-aminoantipyrine and p-hydroxybenzene sulfonate to generate quinoneimine dye, which was quantified in a Shimadzu UV-160 spectrophotometer (Duisburg) at 500 nm. The phospholipid mass was calculated by colorimetric determination of phosphorus using the Barlett ammonium molybdate assay as described previously.20 This procedure yielded cholesterol liposomes with a cholesterol/ phospholipid molar ratio of approximately 3:1 and a concentration of 5 mg/mL cholesterol. Cholesterol-free liposomes were prepared as described above with PC alone. Liposomes were sterilized using a 0.45-µm Millipore filter. Because it has been reported that maximal cholesterol enrichment of VSMC occurs in a medium containing cholesterol-rich liposomes and LDL,7 we used cholesterol-rich medium consisting of serum-free medium (DMEM/Ham’s F-10, 1:1 vol:vol) containing cholesterol-rich liposomes (500 µg/mL) and human LDL (100 µg/mL). Control medium was of the same composition with the exception that LDL and cholesterol-rich liposomes were replaced by HEPES buffer. In some experiments, cells were treated with serum-free medium containing LDL (100 µg/mL) or cholesterol-free medium containing PC liposomes only (the same mass PC as the cholesterol-rich medium). Cholesterol enrichment was performed in confluent VSMC (75-cm2 flasks) that were preincubated (37°C in a humidified atmosphere of 95% air and 5% CO2) in serumfree medium for 24 hours. The medium was replaced by cholesterol containing media, and cells were incubated for further 20 hours. After cholesterol enrichment, cells were washed three times with PBS, then they were scraped and centrifuged at 400g. Cell pellet was dispersed in 20 volumes of chloroform/methanol (2:1 vol/vol) in a glass centrifuge tube. The cell suspension was sonicated to ensure cell disruption and allowed to stand for at least 15 minutes before denatured protein was removed by centrifugation (10 minutes, 3500 rpm). One fifth of the volume of 0.05 mol/L CaCl2 was added to the supernatant and mixed thoroughly. The upper phase was discarded. The lower phase, which consists mainly of chloroform and soluble lipid, was taken to dryness by a stream of nitrogen.21 Lipids were resolved in 100 µL chloroform, and cholesterol was quantified using the Sigma kit.
LDL Isolation
LDL (d=1.019 to 1.063 g/mL) was isolated from the plasma of normocholesterolemic subjects (serum cholesterol <6.2 mmol/L) by potassium bromide density-gradient ultracentrifugation according to Redgrave et al.22 The LDL fraction was dialyzed against 0.15 mol/L NaCl containing 1 mmol/L EDTA. No oxidation of LDL was observed as assessed by measurement of malondialdehyde by the thiobarbituric acid method as described previously.23 Quantification of LDL was performed by determination of the protein component according to the method of Bradford.24 The purity of LDL was examined with a commercially available test (Lipidophor System) following agarose electrophoresis as previously described by Wieland and Seidel.25
Measurement of [Ca2+]i
VSMC were cultured on round glass microscope slides (diameter, 12 mm) under normal tissue culture conditions until confluence. After cholesterol enrichment, cells were incubated with 2 µmol/L fura 2 pentaacetoxymethyl ester at 37°C for 20 minutes in HEPES buffer supplemented with 1% BSA (wt:vol). Just before the measurements, the cell monolayer was rinsed with HEPES buffer without BSA containing 1 mmol/L CaCl2, and the glass slide was positioned diagonally in the cuvette. Measurements were performed in HEPES buffer containing 1 mmol/L CaCl2. The Ca2+-fura 2 fluorescence was measured at 37°C in a Perkin-Elmer LS50 fluorescence spectrofluorometer at excitation wavelengths of 340 and 380 nm and at an emission wavelength of 505 nm. Maximum (Rmax) and minimum (Rmin) fluorescence was determined by adding digitonin (30 µmol/L) followed by the addition of 1% Triton X-100 (vol:vol) and Tris-base/EGTA at a final concentration of 0.1 mol/L Tris-base/25 mmol/L EGTA. Fluorescence was corrected for cellular autofluorescence. Fluorescence signals were calibrated according to Grynkiewicz et al26 using the following equation: [Ca2+]i=Kdx(R-Rmin)/ (Rmax-R)x(Sf2/Sb2). Kd for the fura 2/Ca2+ complex at 37°C is assumed to be 224 nmol/L.26 Sf2 is the 380 nm-excited fluorescence in the absence of Ca2+ (EGTA added), and Sb2 is the 380 nm-excited fluorescence in the presence of saturating Ca2+ (1 mmol/L Ca2+).
Measurement of pH1
VSMC were cultured on round glass microscope slides (diameter, 12 mm) under normal tissue culture conditions until confluence. After cholesterol enrichment, cells were incubated with the fluorescence pH indicator pentaacetoxymethylester at a concentration of 4 µmol/L for 20 minutes at 37°C in HEPES buffer (20 mmol/L HEPES, 16 mmol/L glucose, 130 mmol/L NaCl, 1 mmol/L MgSO4 7 H2O, 0.5 mmol/L CaCl2, Tris-base, pH 7.4) supplemented with 1% BSA (wt:vol). Just before the measurements, the cell monolayer was rinsed with HEPES buffer, and the glass slide was positioned diagonally in the cuvette. Measurements were performed in HEPES buffer without BSA in the Perkin-Elmer LS50 luminescence spectrofluorometer. For the fluorescence measurements, the following wavelengths were set: excitation wavelengths, 492 and 438 nm; emission wavelength, 525 nm. Calibration of BCECF fluorescence was performed in HEPES buffer in which NaCl was replaced by KCl by permeabilizing the cells with the K+/H+ ionophore nigericine (1 µg/mL) in the presence of MES as previously described (Berk et al12). The fluorescence of BCECF was linear between pHi 7.4 and 6.4.
Determination of DNA Synthesis
The effect of PDGF-BB on [3]thymidine incorporation into DNA was assessed as previously described.18 VSMC were seeded in 24-well culture plates and grown to confluence. After cholesterol enrichment of VSMC, medium was replaced by serum-free medium, and cells were stimulated immediately with PDGF-BB (50 ng/mL). After another 20 hours, [3H]thymidine (3 µCi/mL) was added. Four hours later, experiments were terminated as described previously.18 Acid-insoluble [3H]thymidine was extracted into 250 µL/dish 0.5 mol/L NaOH, and 0.1 mL of this solution was mixed with 5 mL scintillator (Packard, Ultimagold) and quantified by using the Beckman LS 3801 liquid scintillation counter. The residual solution (50 µL) was prepared for the determination of protein using the Bio-Rad protein assay according to the method of Bradford.24
Preparation of Plasma Membranes
Plasma membranes were isolated according to Brockbank and England.27 Briefly, VSMC (75-cm2 flasks) were scraped with buffer A (0.25 mol/L sucrose, 50 mmol/L imidazole, 14 mmol/L 2-mercaptoethanol, 1 mmol/L EDTA, 10 mmol/L MgCl2, pH 7.4) and were homogenized with a Polytron homogenizer. The homogenate was centrifuged at 50 000g, and the pellet was resuspended in 1 mL Percoll medium (0.17 mL Percoll, 0.3 mol/L KCl, 0.25 mol/L sucrose, 50 mmol/L imidazole, 14 mmol/L 2-mercaptoethanol, 10 mmol/L MgCl2, and 1 mmol/L EDTA, pH 7.4). This was thoroughly mixed with a further 8 mL Percoll medium and centrifuged at 10 000g for 15 minutes. Fractions of 0.75 mL were taken from the top of the tube. Aliquots from each fraction were used for 5'-nucleotidase assay (subcellular marker for plasma membranes) with 5' AMP as substrate.28 Succinate dehydrogenase (subcellular marker for mitochodria membranes) was measured with dichlorophenylindophenyl as electron acceptor.29
Fluorescence Polarization Studies
Control and cholesterol-enriched VSMC (75-cm2 flasks) were scraped off in 10 mL PBS and sonicated (50 W, 30 seconds). Total cellular membranes were labeled with the lipid soluble fluorophore DPH (10 µmol/L) after vigorous stirring of the dispersion for 3 hours at 37°C as previously described.7 The steady-state fluorescence anisotropy measurements were performed in the Perkin-Elmer LS50 luminescence spectrofluorometer equipped with polarizing filters in the excitation and emission beams at an excitation wavelength of 365 nm and an emission wavelength of 430 nm.6 Fluorescence anisotropy (r) was calculated from the degree of fluorescence polarization using the Perrin equation:
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are the emission intensities detected through an analyzer oriented parallel and perpendicular to the direction of the excitation light. Membrane fluidity was expressed in the form of Arrhenius plots (fluorescence polarization term [(r0/r)-1]-1 versus 1/ T(°K)x103. r0 is the upper limit of the r (for DPH, r0=0.362).30 Higher values of the term [(r0/r)-1]-1 indicate a lower membrane fluidity. The fluorescence anisotropy of DPH in microsomal membranes isolated from cells grown in a 75-cm2 flask was determined after collecting of microsomal membranes and labeling with 10 µmol/L DPH as described above.
Materials
Fura 2/pentaacetoxymethyl ester (fura 2/AM) and BCECF/pentaacetoxymethyl ester were obtained from Calbiochem. Anti--smooth muscle actin and DPH were obtained from Sigma Chemical Co. FITC-conjugated F(ab')2 fragment of goat anti-mouse immunoglobulins was obtained from Dako GmbH. DMEM, Ham’s F-10 and PBS were obtained from Gibco BRL. Fetal calf serum and PDGF-BB were obtained from Boehringer Mannheim. [Methyl-3H]thymidine was obtained from Amersham. X-OMAT 8x10-in films were obtained from Kodak.
Statistics
Values are expressed as the arithmetic mean±SEM or SD. Statistical analysis of the data was performed using the one-factor ANOVA-Scheffé F test (StatView 512+, version 1.0, Apple Computer Inc). Triplicate wells were analyzed for each [3H]thymidine incorporation experiment, and each experiment was performed independently at least three times. A value of P<.05 was considered to be statistically significant.
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Effect of Cholesterol-Rich and Cholesterol-Free Liposomes on Total Cellular Cholesterol
As demonstrated in Fig 1, total cholesterol content of VSMC was 7.3±0.6 µg/mg protein. VSMC treatment with cholesterol-rich liposomes (500 µg/mL cholesterol) containing 100 µg/mL LDL resulted in a 76±12% increase in total cholesterol content (mean±SEM, n=3). VSMC treatment with cholesterol-free medium (320 µg/mL PC, same mass PC as in cholesterol-rich liposomes) or with LDL (100 µg/mL) did not affect total cellular cholesterol content.
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Effect of Cholesterol-Rich Liposomes on Membrane Fluidity of VSMC
Fig 2 shows a representative Arrhenius plot of cell membrane fluidity expressed as [(r0/r)-1]-1, as a function of temperature in untreated and cholesterol-enriched cells. High levels of [(r0/r)-1]-1 indicate a lower membrane fluidity. As demonstrated, cell membrane fluidity was decreased in cholesterol-enriched VSMC at all temperatures between 15°C and 40°C. No changes in membrane fluidity were observed in cells pretreated with LDL. Because total cell membranes were used, the fluidity values obtained in these experiments represent the weight average of all cell membranes as suggested by Shinitzky and Barenholz.6 To confirm whether changes in membrane fluidity in total cell membranes also represent changes in fluidity of plasma membranes, similar experiments were performed using microsomal membranes from VSMC. Fig 3A shows the profiles of 5'-nucleotidase and succinate dehydrogenase in the fractions after Percoll density gradient centrifugation. The 5'-nucleotidase activity was found in fraction numbers 5 to 8, whereas the maximal succinate dehydrogenase activity was found in fraction numbers 9 to 12. As described previously, no activity of alkaline phosphatase or lactate dehydrogenase was found in fractions containing 5-nucleotidase, indicating that these fractions had no cytoplasmic contamination and thus contain mainly plasma membranes.27 Again, plasma membrane fluidity was decreased in cholesterol-enriched VSMC at all temperatures between 15°C and 37°C (Fig 3B) in comparison with plasma membranes isolated from untreated cells.
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Effect of PDGF-BB on [Ca2+]i in Cholesterol-Enriched VSMC
As demonstrated in Fig 4A(a), PDGF-BB (50 ng/mL) induced a maximal elevation in [Ca2+]i from approximately 50 to 200 nmol/L within 40 seconds. Stimulation of cholesterol-enriched VSMC with PDGF-BB resulted in a marked increase in [Ca2+]i from 70 to 500 nmol/L within 40 seconds (b). Thereafter, [Ca2+]i declined toward a stable value of approximately 300 nmol/L within 4 minutes. Data from separate independent experiments generated from different cell lines and passages were normalized by calculation of the mean±SEM of the percent changes of the basal value after treatment of the VSMC with cholesterol-rich liposomes, cholesterol-free liposomes, or LDL in comparison with the basal value of untreated cells (=100%). As illustrated in Fig 4B, the basal [Ca2+]i was elevated by 30% in cholesterol-enriched VSMC, whereas treatment of the VSMC with cholesterol-free liposomes or with LDL did not influence the basal [Ca2+]i. Because the maximal PDGF-BB-induced increase in [Ca2+]i occurred at 40 seconds, the effect of PDGF-BB on [Ca2+]i in cells treated with the different cholesterol-containing media was evaluated by calculating the percent change of the maximal PDGF-BB-induced [Ca2+]i in comparison with the maximal PDGF-BB-induced [Ca2+]i in untreated cells (=100%). As illustrated in Fig 4C, an approximately 90% elevation of the PDGF-BB-induced increase in [Ca2+]i was observed in cholesterol-enriched VSMC, whereas the effect of PDGF in VSMC treated with cholesterol-free liposomes or LDL was unaffected.
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Effect of PDGF-BB on pHi in Cholesterol-Enriched VSMC
Representative tracings of the time kinetic course of PDGF-BB-induced pHi changes are illustrated in Fig 5. PDGF-BB induced in the untreated cells an initial cytosolic acidification that peaked at 40 seconds and was followed by a weak alkalinization above baseline within 5 minutes (Fig 5a). The PDGF-BB-induced pHi changes in cholesterol-enriched VSMC were very similar (Fig 5b). No significant differences of the PDGF-BB-induced acidification at 2 minutes in control (0.130±0.03 pHi unit) and cholesterol-enriched cells (0.126±0.03 pHi unit, mean± SD, n=4, P>.05) were observed. Also, no significant differences in pHi at 6 minutes in control (7.51±0.08) and cholesterol-enriched cells (7.49±0.05, mean±SD, n=4, P>.05) were observed. The time of recovery to baseline in control and cholesterol-enriched cells was 4.9±1.3 and 5.1±1.1 minutes (mean±SD, n=4, P>.05), respectively. The resting value from untreated cells was 7.48±0.5 (mean±SD, n=10). The resting value in cells treated with cholesterol-rich liposomes containing LDL (100 µg/mL), cholesterol-free liposomes, and LDL (100 µg/mL) was 7.4±0.1, 7.15±0.5, and 7.6±0.4 (mean±SD, n=10), respectively. There were no significant differences in the resting pHi values in cholesterol-enriched cells in comparison with untreated cells (P>.05).
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Effect of PDGF-BB on DNA Synthesis in Cholesterol-Enriched VSMC
The effect of PDGF-BB on the [3H]thymidine incorporation in cells treated with cholesterol-rich liposomes is shown in Fig 6A (one representative experiment performed in triplicate wells). Treatment of the cells with cholesterol-rich liposomes without LDL caused a small decrease of [3H]thymidine incorporation from 79±8 (untreated cells) to 61±3 cpm/µg protein, whereas treatment of the cells with cholesterol-rich liposomes with LDL (100 µg/mL) caused an increase to 139±6 cpm/µg protein. PDGF-BB induced in untreated cells an increase in [3H]thymidine incorporation from 79±8 to 141±14 cpm/µg protein. In cells treated with cholesterol-rich liposomes without or with LDL (100 µg/mL), the effect of PDGF-BB was increased to 207±27 or 428±14 cpm/µg protein, respectively. In contrast, the effect of PDGF-BB in cells treated with cholesterol-free liposomes was reduced to 100±8 cpm/µg protein. Data generated from four separate experiments (each experiment was performed in triplicate wells) are expressed as mean±SEM (Fig 6B). The influence of the cholesterol enrichment of VSMC on their basal DNA synthesis was evaluated by calculation of the percent changes in the [3H]thymidine incorporation of the untreated cells (=100%). These results show that pre-treatment of the cells with LDL (100 µg/mL) for 20 hours resulted in a 54% increase of the [3H]thymidine incorporation. VSMC treatment with cholesterol-rich liposomes did not influence DNA synthesis. In contrast, treatment of the cells with cholesterol-free liposomes caused a 35% decrease of the [3H]thymidine incorporation. This inhibitory effect was not observed in cells that were treated with cholesterol-free liposomes containing LDL. The influence of the cholesterol enrichment of VSMC on the PDGF-BB-induced [3H]thymidine incorporation was evaluated by calculation of percent changes of the PDGF-BB-induced [3H]thymidine incorporation in untreated cells (=100%) and expressed as mean±SEM (Fig 6C). In cells treated with LDL, cholesterol-rich liposomes, and cholesterol-rich liposomes containing LDL, PDGF-BB induced an 50%, 40%, and 100% increase of the [3H]thymidine incorporation, respectively. In contrast, a 30% reduction of the effect of PDGF-BB was observed in cells treated with cholesterol-free liposomes, whereas treatment of the cells with cholesterol-free liposomes containing LDL did not influence the effects of PDGF-BB.
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Discussion |
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The present findings demonstrate that cholesterol enrichment of VSMC results in an elevation of PDGF-BB-induced DNA synthesis. This effect was associated with an increase of the PDGF-BB-induced [Ca2+]i as well as with a decrease in membrane fluidity. In contrast, resting and PDGF-BB-induced changes in pHi were not affected in these cells.
Our procedure for cholesterol enrichment of VSMC is simple but efficient. In most of the procedures, cholesterol-enrichment of VSMC occurred in the presence of lipoprotein-deficient serum.7–9 It is established that under physiological conditions, changes in the molar ratio of cholesterol to phospholipid in cell membranes result in a decrease in membrane fluidity.6 Thus, cholesterol enrichment of plasma membranes from VSMC was confirmed by determination of the membrane fluidity in total cell membranes, as well as in plasma membranes. Our findings are in concordance with the results of Gleason et al,7 who reported that cholesterol enrichment of VSMC by the use of cholesterol-rich liposomes in 1% lipoprotein-free serum results in a decrease in membrane fluidity.
Similar to Gleason et al,7 we have found that basal [Ca2+]i was elevated by 30% in cholesterol-enriched cells. The possible role of the Ca2+-influx in cholesterol-enriched VSMC in the pathophysiology of atherosclerosis is extensively discussed by Gleason et al7 and Bialecki et al.8 Both groups favored the concept of Strickberger et al31 and Phair32 that an increase in the plasma membrane calcium permeability leads to an increase in basal [Ca2+]i that is a possible mediator of the cholesterol-induced atherogenesis. One striking finding of the present study is that the maximal effect of PDGF-BB on [Ca2+]i was enhanced by 90% in cholesterol-enriched cells in comparison with the PDGF-BB effect in untreated cells. The PDGF-dependent intracellular signal transduction from the receptor into the cell involves different proteins such as the phospholipase C
, GTPase activating protein, phosphatidylinositol-3 kinase, carrying Src homology region 2 domains that are capable of binding to specific regions of the receptor containing autophosphorylated tyrosine residues.33 It is suggested that PDGF stimulates the Na+/H+ exchange by a protein kinase C-dependent pathway.34,35 In this context, it is established that activated phospholipase C leads to generation of diacylglycerol and inositoltriphosphate, which causes mobilization of Ca2+ from intracellular stores. It was suggested that both mediators, diacylglycerol and elevated [Ca2+]i, are required for activation of protein kinase C.34,35
Although the mechanisms of action whereby PDGF-BB induces Ca2+ influx in VSMC are unknown, the PDGF-BB-mediated increase in [Ca2+] is more dependent on Ca2+ influx from extracellular Ca2+ than from Ca2+ mobilization from intracellular stores.13,36,37 Furthermore, it has been demonstrated that PDGF activates the L-type Ca2+ channel in aortic VSMC38 and L-type calcium channel blockers are able to reduce the PDGF-BB-dependent increase in [Ca2+]i in VSMC.37,39 Indeed, cholesterol enrichment of VSMC increases the activity of L-type calcium channel current that may occur due to modulation of functional properties of the L-type calcium channel.9 These observations suggest that cholesterol enrichment of VSMC causes a potentiation of the PDGF-BB increase in [Ca2+] by acting at Ca2+ influx channels. From the observation that PDGF-BB promotes contraction of vascular tissues,40,41 one can hypothesize that the potentiation of the PDGF-BB increase in [Ca2+]i in cholesterol-enriched cells may result in an increased sensitivity of vascular tissues to PDGF-BB.
The observation that cholesterol enrichment of VSMC caused an approximately twofold increase of the PDGF-induced [Ca2+]i but did not influence resting and PDGF-BB-induced pHi changes suggests that (1) cholesterol enrichment of VSMC does not influence the activity of Na+/H+ exchanger and (2) increase in [Ca2+]i does not modulate the activity of the Na+/H+ exchanger. Our findings are in agreement with those from other groups suggesting that Na+/H+ exchanger activation is not essential for agonist-induced Ca2+ mobilization or the subsequent Ca2+ influx.42,43
Another remarkable finding of the present work is that the treatment of VSMC with LDL induced a 55% increase in DNA synthesis and that this effect was additive to the PDGF-BB-induced DNA synthesis. The mitogenic effect of LDL on VSMC has been studied extensively. Several laboratories reported that LDL exerts mitogenic effects on VSMC.44,45 The observation that LDL elevates [Ca2+]i and induces expression of immediate early growth response genes such as c-fos46 and egr-147 may explain the mitogenic effects of LDL. Furthermore, the PDGF-BB effect was elevated by 40% or 100% in cells enriched with cholesterol-rich liposomes or cholesterol-rich liposomes containing LDL. Since it is widely believed that [Ca2+]i plays an important role in the regulation of cell growth, the enhanced effect of PDGF-BB on DNA synthesis in cholesterol-enriched cells might be explained by the elevated PDGF-BB-induced [Ca2+]i in these cells. These pertinent findings are supported by the observation that calcium channel blockers inhibit the PDGF-BB-dependent increase in [Ca2+]i37,39 and the mitogenic effect of PDGF-BB in VSMC.39,48,49 The cellular mechanisms by which treatment of VSMC with cholesterol-free liposomes led to a 35% reduction of the basal DNA synthesis and a 30% reduction of the effect of PDGF-BB remain to be elucidated.
One may question the physiological relevance of our findings derived from cultured cells. As described by Shinitzky,50 one of the main mechanisms of modulating the cholesterol/phospholipid ratio in cell membranes is the passive distribution of cholesterol between the serum lipoproteins and the cell plasma membranes. Under physiological conditions, the cholesterol/phosphatidylcholine ratio in VSMC cell membranes is at an equilibrium. Alterations of the cholesterol/phosphatidylcholine ratio in VSMC membranes may be caused by dietary atherosclerosis,51 by pharmacological manipulations,10 or by age.30 Gleason et al7 reported that treatment of VSMC with LDL (50 µg/mL) per se caused a 29% increase in cholesterol content in VSMC. However, we could not observe a cholesterol enrichment after treatment of VSMC with LDL (100 µg/mL) per se or any modulation of the membrane fluidity. The inconsistency between our findings and the findings of Gleason et al may reflect differences in the enrichment procedure. The in vivo concentration of LDL in human plasma is eightfold higher (800 µg/mL)52 than the concentration used in our experiments (100 µg/mL). Since human plasma concentrations of LDL in hypercholesterolemia can reach values much higher than 800 µg/mL,52 it is likely that cholesterol enrichment of VSMC can occur. In this context, a close correlation between the concentration of LDL in human aortic intima and serum cholesterol level has been found.53 Hypertension and elevated levels of LDL are two of the most important risk factors for atherosclerosis and cardiovascular morbidity.54,55 Moreover, cardiovascular risk factors like hypertension and hypercholesterolemia induce an elevation of the LDL transport from blood in the rat aortic intima.56 Also, an increased transfer of LDL from blood to rat arterial vessels occurs after injection of animals with vaso-active agonists such as serotonin, angiotensin II, and catecholamines.57 It has been proposed that most of circulating LDL is transported through vascular endothelium by transcytosis via plasmalemma vesicles that deliver LDL to other cells of the vascular wall.58 Thus, it is likely that under such pathophysiological conditions, elevated LDL in the aortic intima may cause cholesterol enrichment of VSMC. According to the "response to injury hypothesis" from Ross et al,4 platelets and endothelial cells participate in the development of the atheromatous plaque by secretion of PDGF, which is a strong proliferative factor for VSMC. Another definitive factor responsible for the formation of atheromatous plaques is cholesterol transported by LDL entirely from the blood. Thus, it is conceivable that in addition to PDGF, LDL may also contribute to the formation of an atheromatous plaque via its direct mitogenic effects on VSMC or via the enhanced growth-promoting effect of PDGF in cholesterol-enriched VSMC.
In summary, our findings provide evidence that elevated cholesterol content in VSMC enhances the PDGF-BB-induced DNA synthesis and the [Ca2+]i and thus might contribute to the pathogenesis or progression of cardiovascular diseases.
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Acknowledgments |
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This work was supported by a grant of the Deutsche Forschungsgemeinschaft (Sa568/2-1). The excellent technical assistance of Petra Epping is greatly appreciated.
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References |
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1. Schwartz SM, Reidy M. Common mechanisms of proliferation of smooth muscle in atherosclerosis and hypertension. Hum Pathol. 1987; 18 : 240 –247.[Medline] [Order article via Infotrieve]
2. Schwartz SM, Campbell GR, Campbell JH. Replication of smooth muscle cells in vascular disease.
Circ Res. 1986;
58
: 427
–444.
3. Ross R. The pathogenesis of atherosclerosis-an update. N Engl J Med. 1986; 314 : 488 –500.[Medline] [Order article via Infotrieve]
4. Ross R, Raines EW, Bowen-Pope DF. The biology of platelet-derived growth factor. Cell. 1986; 46 : 155 –169.[Medline] [Order article via Infotrieve]
5. Gennis RB. Biomembranes, Molecular Structure and Function. New York, NY: Springer-Verlag; 1989 : 167 –198.
6. Shinitzky M, Barenholz Y. Fluidity parameters of lipid regions determined by fluorescence polarization. Biochim Biophys Acta. 1978; 515 : 367 –394.[Medline] [Order article via Infotrieve]
7. Gleason MM, Medow MM, Tulenko TN. Excess membrane cholesterol alters calcium movements, cytosolic calcium levels, and membrane fluidity in arterial smooth muscle cells.
Circ Res. 1991;
69
: 216
–227.
8. Bialecki RA, Tulenko TN, Colucci WS. Cholesterol enrichment increases basal and agonists-stimulated calcium influx in rat vascular smooth muscle cells. J Clin Invest. 1991; 88 : 1894 –1900.[Medline] [Order article via Infotrieve]
9. Sen L, Bialecki RA, Smith E, Smith TW, Colucci WS. Cholesterol increases the L-type voltage-sensitive calcium channel current in arterial smooth muscle cells.
Circ Res. 1992;
71
: 1008
–1014.
10. Renaud JF, Schmid A, Romey G, Nano J-L, Lazdunski M. Mevinolin, an inhibitor of cholesterol biosynthesis, drastically depressed Ca2+ channel activity and uncouples excitation from contraction in cardiac cells in culture.
Proc Natl Acad Sci U S A. 1986;
83
: 8007
–8011.
11. Kutryk MJ, Pierce GN. Stimulation of sodium-calcium exchange by cholesterol incorporation into isolated cardiac sarcolemmal vesicles.
J Biol Chem. 1988;
263
: 13167
–13172.
12. Berk BC, Brock TA, Gimbrone MA Jr, Alexander RW. Early agonist-mediated ionic events in cultured vascular smooth muscle cells.
J Biol Chem. 1987;
262
: 5065
–5072.
13. Sachinidis A, Locher R, Vetter W, Tatje D, Hoppe J. Different effects of PDGF-isoforms on rat vascular smooth muscle cells.
J Biol Chem. 1990;
265
: 10238
–10243.
14. Sardet C, Franchi A, Pouyssegur J. Molecular cloning, primary structure, and expression of the human growth factor-activatable Na+/H+ antiporter. Cell. 1989; 56 : 271 –280.[Medline] [Order article via Infotrieve]
15. Hepler PK. The role of calcium in cell division. Cell Calcium. 1994; 16 : 322 –330.[Medline] [Order article via Infotrieve]
16. Rozengurt E. Early signals in the mitogenic response.
Science. 1986;
234
: 161
–166.
17. Chamley JH, Campbell GR, Ross R. The smooth muscle cell in culture. Physiol Rev. 1979; 39 : 1 –61.
18. Sachinidis A, Flesch M, Ko Y, Schr
r K, Bhm M, Düsing R, Vetter H. Thromboxane A2 and vascular smooth muscle cell proliferation.
Hypertension. 1995;
26
: 771
–780.
19. Cooper RA, Leslie MH, Fischkoff S, Shinitzky M, Schattil SJ. Factors influencing the lipid composition and fluidity of red membranes in vitro: production of red cells possessing more than two cholesterols per phospholipid. Biochemistry. 1978; 17 : 327 –331.[Medline] [Order article via Infotrieve]
20. New RRC. Liposomes, a Practical Approach. New York, NY: IRL Press at Oxford University Press; 1990: 105 –107.
21. Findlay JBC, Evans WH. Biological Membranes, a Practical Approach. New York, NY: IRL Press at Oxford University Press; 1990 : 104 –105.
22. Redgrave TG, Robert DCK, West CE. Separation of plasma lipoproteins by density-gradient ultracentrifugation. Anal Biochem. 1975; 65 : 42 –49.[Medline] [Order article via Infotrieve]
23. Ko Y, Totzke G, Seewald S, Schmitz U, Schiermeyer B, Meyer zu Brickwedde K, Vetter H, Sachinidis A. Native low-density lipoprotein (LDL) induces the expression of the early-growth response gene-1 in human umbilical arterial endothelial cells. Eur J Cell Biol. 1995; 68 : 306 –312.[Medline] [Order article via Infotrieve]
24. Bradford M. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem. 1976; 72 : 248 –254.[Medline] [Order article via Infotrieve]
25. Wieland H, Seidel D, Advances in the analysis of plasmalipoproteins. Innere Medizin. 1978; 7 : 290 –300.
26. Grynkiewicz G, Poenie M, Tsien RY. A new generation of Ca2+ indicators with greatly improved fluorescence properties.
J Biol Chem. 1985;
260
: 3440
–3450.
27. Brockbank K, England PJ. A rapid method for preparation of sarcolemmal vesicles from rat aorta, and the stimulation of calcium uptake into vesicles by cyclic AMP-dependent protein kinase. FEBS Lett. 1980; 122 : 67 –71.[Medline] [Order article via Infotrieve]
28. Edwards MJ, Maguire HM. Purification and properties of rat heart 5' nucleotidase.
Mol Pharmacol. 1970;
6
: 641
–647.
29. Green DE, Mii S, Kohout PM. Studies on the terminal electron transport system, I: succinic dehydrogenase.
J Biol Chem. 1955;
217
: 551
–567.
30. Medow MS, Segal S. Age related changes in fluidity of rat renal brushborder membrane vesicles. Biochem Biophys Res Commun. 1987; 142 : 849 –856.[Medline] [Order article via Infotrieve]
31. Strickberger SA, Russek LN, Phair RD. Evidence for increased aortic plasma membrane calcium transport caused by experimental atherosclerosis in rabbits.
Circ Res. 1988;
62
: 75
–80.
32. Phair RD. Cellular calcium and atherosclerosis. Cell Calcium. 1988; 9 : 275 –284.[Medline] [Order article via Infotrieve]
33. Kaplan DR, Morrison DK, Wong G, McCormic F, Williams L. PDGF ß-receptor stimulates tyrosine phosphorylation of GAP and association of GAP with a signaling complex. Cell. 1990; 61 : 125 –133.[Medline] [Order article via Infotrieve]
34. Rozengurt E. Early signals in the mitogenic response.
Science. 1986;
234
: 161
–166.
35. Ma Y-H, Reusch HP, Wilson E., Escobedo JA, Fantl WJ, Williams LT, Ives HE. Activation of Na+/H+ exchange by platelet-derived growth factor involves phosphatidylinositol 3'-kinase and phospholipase C.
J Biol Chem. 1994;
269
: 30734
–30739.
36. Diliberto PA, Gordon G, Herman B. Platelet-derived factor (PDGF) receptor activation modulates the calcium mobilizing activity of the PDGF ß receptor in Balb/c3T3 fibroblasts.
J Biol Chem. 1991;
266
: 12612
–12617.
37. Kondo T, Konishi F, Inui H, Inagami T. Differing signal transductions elicited by three isoforms of platelet-derived growth factor in vascular smooth muscle cells.
J Biol Chem. 1993;
268
: 4458
–4464.
38. Roe MW, Hepler JR, Harden TK, Herman B. Platelet-derived growth factor and angiotensin II cause increases in cytosolic free calcium by different mechanisms in vascular smooth muscle cells. J Cell Physiol. 1989; 139 : 100 –108.[Medline] [Order article via Infotrieve]
39. Block L, Emmons LR, Vogt E, Sachinidis A, Vetter W, Hoppe J. Ca2+-channel blockers inhibit the action of recombinant platelet-derived growth factor in vascular smooth muscle cells.
Proc Natl Acad Sci U S A. 1989;
86
: 2388
–2392.
40. Berk BC, Alexander RW, Brock TA, Gimbrone MA, Webb RC. Vasoconstriction: a new activity for platelet-derived growth factor.
Science. 1986;
232
: 87
–90.
41. Sachinidis A, Locher R, Hoppe J, Vetter W. The platelet-derived growth factor isomeres, PDGF-AA, PDGF-AB and PDGF-BB, induce contraction of vascular smooth muscle cells by different intracellular mechanisms. FEBS Lett. 1990; 275 : 95 –98.[Medline] [Order article via Infotrieve]
42. Sage SO, Jobson TM, Rink TJ. Agonist-evoked changes in cytosolic calcium concentration in human platelets: studies in physiological bicarbonate.
J Physiol Lond. 1990;
420
: 31
–45.
43. Kimura M, Nakamura K, Fenton JW, Andersen TT, Reeves JP, Aviv A, Role of external Na+ and cytosolic pH in agonist-evoked cytosolic Ca2+ in response in human platelets. Am J Physiol. 1994; 267 : C1543 –C1552.[Medline] [Order article via Infotrieve]
44. Fless GM, Kirchhausen R, Fischer-Dzoga K, Wissler RW, Scanu AM. Serum low density lipoproteins with mitogenic effect on cultured aortic smooth muscle cells. Atherosclerosis. 1982; 41 : 171 –183.[Medline] [Order article via Infotrieve]
45. Sachinidis A, Mengden T, Locher R, Brunner C, Vetter W. Novel cellular activities for low-density lipoprotein in vascular smooth muscle cells.
Hypertension. 1990;
15
: 704
–711.
46. Scott-Burden T, Resink TJ, Hahn AW, Baur U, Box RJ, Buehler FR. Induction of growth-related metabolism in human vascular smooth muscle cells by low density lipoprotein.
J Biol Chem. 1989;
264
: 12582
–12589.
47. Sachinidis A, Ko Y, Wieczorek A, Weisser B, Locher R, Vetter W, Vetter H. Lipoproteins induce expression of the early growth response gene-1 in vascular smooth muscle cells from rat. Biochem Biophys Res Commun. 1993; 192 : 794 –799.[Medline] [Order article via Infotrieve]
48. Nilsson J, Sjlund M, Palmberg L, Von Euler AM, Jonzon B, Thyberg J. The calcium antagonist nifedipine inhibits arterial smooth muscle cell proliferation.
Atherosclerosis. 1985;
58; 109
–122.[Medline]
[Order article via Infotrieve]
49. Ko Y, Totzke G, Graack G, Heidgen F, Meyer zu Brickwedde K, Düsing R, Vetter H, Sachinidis A. Action of dihydropyridine calcium channel blockers on early-growth response gene expression and cell growth in vascular smooth muscle cells. J Hypertens. 1993; 11 : 1171 –1178.[Medline] [Order article via Infotrieve]
50. Shinitzky M. An efficient method for modulation of cholesterol level in cell membrane. FEBS Lett. 1978; 85 : 317 –320.[Medline] [Order article via Infotrieve]
51. Haley NJ, Shio H, Fowler S. Characterization of lipid-laden aortic cells from cholesterol-fed rabbits, I: resolution of aortic cell populations by metrizamide density gradient centrifugation. Lab Invest. 1977; 37 : 278 –296.
52. Thomson GR. A Handbook of Hyperlipidaemia. London, UK: Current Science Ltd; 1986: 24.
53. Smith EB, Slater RS. Relationship between low-density lipoprotein in aortic intima and serum-lipid levels. Lancet. 1972; 1 : 463.[Medline] [Order article via Infotrieve]
54. Castelli WP, Anderson K. A population at risk: prevalence of high cholesterol levels in hypertensive patients in the Framingham Study. Am J Med. 1986; 80 (suppl 2A): 23 –32.[Medline] [Order article via Infotrieve]
55. Martin MJ, Hulley SB, Browner WS, Kuller LH, Wentworth D. Serum cholesterol, blood pressure, and mortality: implications from a cohort of 361662 men. Lancet. 1986; 2 : 933 –936.[Medline] [Order article via Infotrieve]
56. Daly MM. Effect of hypertension on the lipid composition of rat aortic intima-media.
Circ Res. 1972;
31
: 410
–416.
57. Robertson AL, Khairallah PA. Arterial endothelial permeability and vascular diseases, the ‘trap door’ effect. Exp Mol Pathol. 1973; 18 : 241 –260.[Medline] [Order article via Infotrieve]
58. Vasile E, Simionescu M, Simionescu N. Visualization of the binding, endocytosis, and transcytosis of low-density lipoprotein in arterial endothelium in situ.
J Cell Biol. 1983;
96
: 1677
–1689.
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