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Correspondence to: Johan Thyberg, Dept. of Cell and Molecular Biology, Karolinska Institutet, Box 285, S-17177 Stockholm, Sweden. E-mail: johan.thyberg@cmb.ki.se
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Summary |
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Vascular smooth muscle cells (SMCs) grown in primary culture are converted from a contractile to a synthetic phenotype. This includes a marked morphological reorganization, with loss of myofilaments and formation of a large ERGolgi complex. In addition, the number of cell surface caveolae is distinctly reduced and the handling of lipoprotein-derived cholesterol changed. Here we used filipin as a marker to study the distribution of cholesterol in SMCs by electron microscopy. In contractile cells, filipinsterol complexes were preferentially found in caveolae and adjacent ER cisternae (present in both leaflets of the membranes). After exposure to LDL or cholesterol, labeling with filipin was increased both in membrane organelles and in the cytoplasm. In contrast, treatment with mevinolin (a cholesterol synthesis inhibitor) or ß-cyclodextrin (a molecule that extracts cholesterol from cells) decreased the reaction with filipin but did not affect the close relation between the ER and the cell surface. In synthetic cells, filipinsterol complexes were diffusely spread in the plasma membrane and the strongest cytoplasmic reaction was noted in endosomes/lysosomes, both under normal conditions and after incubation with LDL or cholesterol. On the basis of the present findings, we propose a mechanism for direct exchange of cholesterol between the plasma membrane and the ER and more active in contractile than in synthetic SMCs. (J Histochem Cytochem 50:185195, 2002)
Key Words: caveolae, cholesterol, smooth muscle cells, differentiation, filipin
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Introduction |
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Caveolae are small, flask-shaped invaginations of the plasma membrane with important functions in signal transduction (
We have previously reported that there is a phenotype-dependent variation in the number of caveolae in vascular SMCs both in vivo and in vitro. The differentiated contractile SMCs that are found in the normal arterial media and during the first 12 days of primary culture demonstrate a severalfold higher density of plasma membrane caveolae than the fibroblast-like synthetic SMCs that appear in the forming neointima after vascular injury and after 35 days of primary culture (
In the present investigation, electron microscopic and cytochemical techniques were used to further explore the differences in cholesterol distribution and transport between contractile and synthetic SMCs. A major purpose was to find ultrastructural support for the idea that caveolae are involved in selective uptake and discharge of cholesterol at the cell surface. For this, enzymatically isolated SMCs were seeded on a substrate of fibronectin in serum-free medium and incubated in primary culture for various times, either without further additions or in the presence of cholesterol (as a part of LDL or bound to cyclodextrin). In some experiments the cells were also treated with the 3-hydroxy-3-methylglutaryl co-enzyme A (HMG CoA) reductase inhibitor mevinolin to suppress endogenous cholesterol synthesis (
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Materials and Methods |
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Materials
Ham's medium F12, newborn calf serum (NCS), and collagenase were obtained from Gibco BRL (Paisley, Scotland), and culture plastics from Nunc (Roskilde, Denmark). The culture medium was supplemented with 10 mM Hepes/10 mM Tes (pH 7.3), 50 µg/ml L-ascorbic acid, and 50 µg/ml gentamycin sulfate (medium F12). Bovine serum albumin (BSA), bovine plasma fibronectin, human low-density lipoprotein (LDL), water-soluble cholesterol (encapsulated into the hydrophobic cavity of methyl-ß-cyclodextrin), mevinolin, mevalonic acid lactone, 2-hydroxypropyl-ß-cyclodextrin, filipin complex, and dimethyl sulfoxide (DMSO) were from Sigma (St Louis, MO). To prepare culture substrates, fibronectin was diluted to 10 µg/ml in Dulbecco's PBS, pH 7.3, and adsorbed to the bottom of plastic dishes for 1520 hr at 20C. Before seeding of cells, the dishes were rinsed twice with PBS and incubated with medium F12/0.1% BSA for 30 min to block unspecific binding sites. Mevinolin and mevalonic acid lactone were dissolved in 95% ethanol at 5 and 200 mM and added to the cultures in final concentrations of 5 or 500 µM, respectively (controls were given equivalent amounts of ethanol). Cyclodextrin was dissolved in culture medium at a concentration of 3% directly before use and sterile-filtered (0.2 µm).
Cell Culture
SMCs were isolated from the aortic media of 350400-g male SpragueDawley rats by digestion with 0.1% collagenase in medium F12/0.1% BSA (
Electron Microscopy
The cells were fixed in 3% glutaraldehyde in 0.1 M sodium cacodylateHCl buffer (pH 7.3) with 0.05 M sucrose for at least 2 hr, followed by rinsing in buffer for 12 hr. They were subsequently exposed to 0.2 mg/ml filipin in 0.1 M cacodylate buffer (pH 7.3) for 15 hr at 20C (
Quantitation of FilipinSterol Complexes
To evaluate the effects of the experimental treatments on the cholesterol content of the plasma membrane, approximate mid-sagittal sections through the central parts of the cells (adjacent to the nucleus) were photographed at a final magnification of x50,00070,000. Within the samples so obtained, the number of caveolae on the cell surface was counted under an illuminated magnifier and the length of the plasma membrane within the plane of section was measured using a digital instrument (Calculated Industries; Yorba Linda, CA). In addition, the number of filipinsterol complexes was counted separately in plasma membrane domains with and without caveolae.
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Results |
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Changes in Filipin Staining During Phenotypic Modification of SMCs in Primary Culture
In agreement with earlier descriptions, the newly isolated rat aortic SMCs retained a morphology characteristic of a differentiated contractile phenotype for at least 12 days of culture on a substrate of fibronectin in serum-free medium. During this time, a large part of the cytoplasm was occupied by myofilaments and secretory organelles, such as the ER and Golgi, were small in size (Fig 1A). Thereafter, a marked reorganization of the cells took place, with a decrease in the fractional volume of myofilaments and a corresponding growth in size of the ER and Golgi. In parallel, there was also a distinct reduction in the number of plasma membrane caveolae (Fig 1B). In the majority of the SMCs, this transition from a contractile to a synthetic phenotype was completed after 45 days of culture (see
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The reactivity with filipin varied dependent on both the phenotypic state of the cells and the experimental treatments applied here. On the whole, filipinsterol complexes were found on the cell surface, in vesicles of endosomal or lysosomal nature, in the ER (including the nuclear envelope), and in Golgi-associated vesicles. In contrast, no or only weak staining was detected in stacked Golgi cisternae and mitochondria. Both sides of the membranes were positive, but the outer part of the plasma membrane and the luminal part of the endosome/lysosome and the ER membranes showed the most prominent labeling. In contractile SMCs, the reactivity of the plasma membrane was concentrated in but not restricted to the regions where caveolae opened on the cell surface (see
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Quantitative estimations revealed that the number of caveolae per unit length of plasma membrane was 8090% lower in synthetic than in contractile SMCs (without special treatments). However, the density of filipinsterol complexes in caveolar membranes was the same in the two cell types. In contrast, non-caveolar membranes displayed a higher content of such complexes in synthetic than in contractile cells (Table 1 and Table 2; p<0.05). No complexes of the type described above were seen in cells not treated with filipin during the EM preparation (see Fig 1A and Fig 1B).
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Effects of Experimental Treatments on Filipin Staining of SMCs in Primary Culture
The cells were subjected to various experimental treatments to either increase or decrease their cholesterol content. After incubation with 100 µg/ml LDL for 210 hr, the number of caveoalae increased about threefold in synthetic cells but reached only about one third of the level maintained in contractile cells (Table 1). At the same time, the number of filipinsterol complexes was augmented both on the plasma membrane and in intracellular compartments. In synthetic cells, the surface labeling was enhanced both in caveolar and non-caveolar regions, whereas contractile cells showed a clear elevation only in the latter regions (Table 1). Especially in contractile SMCs, strong staining was also observed in ER cisternae and signs of a close spatial relation between portions of the ER and the cell surface were abundant (Fig 4A). In synthetic cells, there was a further increase in the number of endosomes/lysosomes and filipinsterol complexes were found in both the membrane and the lumen of these organelles, and in the adjacent cytoplasm (Fig 4B). A few cells in an intermediate phenotype displayed many myelin-like deposits (without evident filipin labeling) associated with the Golgi complex (Fig 5A). These structures were 50100 nm in diameter and located either free in the cytoplasm or within the lumen of Golgi vesicles or cisternae (Fig 5B). A modest increase in the number of cytoplasmic lipid droplets was also noted, mainly in synthetic cells. Exposure of the cultures to a lower concentration of LDL (1020 µg/ml) or cholesterol-loaded ß-cyclodextrin (2 µg/ml) throughout 5 days of culture gave rise to changes similar to those just described (not shown).
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Treatment of the cells with 5 µM mevinolin for 5 days partially inhibited the shift from a contractile to a synthetic phenotype and induced apoptosis in about 20% of the cells, as judged by nuclear condensation and disintegration of cytoplasmic organelles (unpublished observations). Caveolae were reduced in number in the phenotypically modified cells, but not as much as normally seen during the shift into a synthetic state (i.e., in the absence of the drug). The filipinsterol complexes were also fewer (Table 2) and the plasma membrane had a smoother contour than in the controls (Fig 6A and Fig 6B). Likewise, no or weak staining with filipin was noted in endosomes/lysosomes, ER cisternae, and Golgi-associated vesicles. In spite of this, a close spatial relation between caveolae and ER-like cisternae was still possible to detect. Simultaneous treatment with 500 µM mevalonic acid lactone counteracted the effects of mevinolin on the structural rebuilding of the cells as well as the reaction with filipin (Table 2). On the other hand, addition of LDL (20 µg/ml) or cholesterol-loaded ß-cyclodextrin (2 µg/ml) to the culture medium did not eliminate the inhibitory effect of mevinolin on the change in cell phenotype (unpublished observations). The number of filipinsterol complexes on the cell surface (Table 2) and in compartments such as endosomes/lysosomes and the ER was, however, equal to or even higher than in control cells kept in normal medium (Fig 7A and Fig 7B).
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Exposure of the SMCs to 3% ß-cyclodextrin (without cholesterol) for 6 hr caused an almost total loss of caveolae and no or only very few filipinsterol complexes were observed (Fig 8A and Fig 8B). Otherwise, the morphology of the cells was surprisingly well preserved, but an increase in the number of apoptotic cells was noted. If the treatment with ß-cyclodextrin was restricted to 2 hr, the effect on caveolae and filipin staining was less complete but still highly significant (Table 3). In contrast, no signs of cell damage were evident after this time.
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Discussion |
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Cholesterol is an essential component in the membranes of mammalian cells and an intricate system has been developed to regulate the cellular levels of this lipid. In part, cholesterol reaches the cell via the circulation, packaged in LDL particles. These are ingested by receptor-mediated endocytosis and transferred to lysosomes, where free cholesterol is generated by enzymatic digestion. After transport out of the lysosomes, the cholesterol molecules are used for membrane biogenesis in the ER/Golgi complex and sub-sequently carried to the cell surface. If supplied in excess, they may also be converted into cholesteryl esters and stored in cytoplasmic lipid droplets. Cholesterol is further synthesized de novo by most cells, using enzymes residing in the ER (
During the past several years it has also become apparent that cholesterol is unevenly distributed in the membranes of cells, being most abundant in microdomains referred to as detergent-resistant membranes or rafts, and in cell surface caveolae (
The findings of the present electron microscopic and cytochemical investigation using filipin as a probe indicate that cholesterol is preferentially associated with caveolae on the surface of contractile SMCs and is more randomly dispersed over the surface of synthetic SMCs. Among the cytoplasmic organelles, the most distinct labeling was found in ER cisternae (including the nuclear envelope), Golgi-associated vesicles, and endosomes/lysosomes. In contrast, no or only few filipinsterol complexes were found in stacked Golgi cisternae and mitochondria. With regard to the Golgi complex, this is in agreement with recent observations by fluorescence microscopy showing a dot-like perinuclear pattern in SMCs exposed to filipin, distinct from the reticulate pattern seen after immunostaining for mannosidase II, a marker enzyme of medial Golgi cisternae (
In the literature, partly different views have been expressed concerning the role of the Golgi complex in cholesterol transport. First, cholesterol synthesized in the ER has been found to reach the cell surface also when protein secretion is inhibited, implying a path that bypasses the Golgi complex (
Especially in the contractile SMCs, a close spatial relationship was noted between caveolae and ER-like cisternae or cytoplasmic vesicles. In these regions, filipinsterol complexes appeared both on the extracellular/luminal and the cytoplasmic sides of the membranes, and sometimes also in the intervening free cytoplasm. Interestingly, such a link between caveolar and intracellular membrane compartments was observed not only in control cells but also in cells exposed to LDL or water-soluble cholesterol (increased number of filipinsterol complexes) and in cells deprived of cholesterol by treatment with the HMG CoA reductase inhibitor mevinolin or ß-cyclodextrin (reduced number of filipinsterol complexes). It is proposed that the above-mentioned contacts serve in the translocation of cholesterol and other lipid molecules between the plasma membrane and intracellular membranes. Such a process could make it possible to pass newly synthesized cholesterol from the ER to the plasma membrane without the involvement of the Golgi complex and without the need for vesicle budding and fusion. After selective transfer of cholesterol from extracellular lipoproteins to the plasma membrane, it could likewise represent a means for movement of cholesterol from the cell surface to the ER and other cytoplasmic membrane systems.
Summing up, the findings of this and other recent studies from our laboratory indicate that the number of caveolae and the distribution/handling of cholesterol in vascular SMCs differ in a phenotype-dependent manner (
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Acknowledgments |
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Supported by the Swedish Medical Research Council, the Swedish Heart Lung Foundation, the King Gustaf V 80th Birthday Fund, and the Karolinska Institutet.
The author thanks Karin Blomgren and Birgitta Björkroth for expert technical assistance.
Received for publication November 14, 2000; accepted August 29, 2001.
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