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Cardiovascular Research 2005 68(1):128-135; doi:10.1016/j.cardiores.2005.05.004
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Copyright © 2005, European Society of Cardiology

Decreased caveolin-1 in atheroma: Loss of antiproliferative control of vascular smooth muscle cells in atherosclerosis

Carsten Schwenckea,1, Alexander Schmeissera,1, Christina Waltera, Rolf Wachtera, Sven Pannacha, Brigitta Wecka, Ruediger C. Braun-Dullaeusa, Michael Kasperb and Ruth H. Strassera,*

aMedical Clinic II, Department of Cardiology, University of Technology Dresden, Fetscherstr. 76, 01307, Dresden, Germany
bDepartment of Anatomy, University of Technology Dresden, Dresden, Germany

* Corresponding author. Tel.: +49 351 4501704; fax: +49 351 4501702. Email address: Ruth.Strasser{at}mailbox.tu-dresden.de

Received 20 November 2004; revised 15 April 2005; accepted 3 May 2005


   Abstract
Top
 Abstract
1. Introduction
 2. Methods
 3. Results and discussion
 Acknowledgments
 References
 
Objective: Proliferation of vascular smooth muscle cells (VSMC) is involved in the pathogenesis of primary atherosclerosis and restenosis after angioplasty. On the background of the antiproliferative activities of caveolin-1, the present study focused on the expression of caveolin-1 in proliferating VSMC of human atheroma.

Methods: VSMC were isolated from wild-type (Wt) and caveolin-1 knockout mice (Cav-/-). Proliferation of Wt-VSMC after supplementation of serum or Cav-/-VSMC after adenoviral overexpression of caveolin-1 was documented by either Western blot analysis of the cyclin-dependent kinase (Cdk) inhibitor p27kip1 and the proliferating cell nuclear antigen (PCNA) or BrdU incorporation. Using immunohistochemistry the proliferation of VSMC derived from atheroma of human carotid vessels as well as the expression of caveolin-1 in these cells were investigated ex vivo.

Results: Supplementation of serum to Wt-VSMC resulted in an augmented cell cycle entry and a concomitant decrease of caveolin-1 expression. Inversely, adenoviral overexpression of caveolin-1 in Cav-/-VSMC inhibited cellular proliferation. Corresponding to these in vitro data, the expression of caveolin-1 was significantly decreased in proliferating VSMC of human atheroma.

Conclusion: The proliferation of VSMC in vitro and in human atheroma is associated with a decrease of caveolin-1 expression. These data suggest that the loss of antiproliferative control by caveolin-1 plays a pivotal role in VSMC proliferation in atherosclerosis.

KEYWORDS Atherosclerosis; Caveolae; Signal transduction; Smooth muscle


   1. Introduction
Top
 Abstract
 1. Introduction
2. Methods
 3. Results and discussion
 Acknowledgments
 References
 
Caveolae, 50–100 nm plasmalemmal vesicles, represent subcompartments of the plasma membrane that exist most abundantly in terminally differentiated cells like adipocytes, endothelial cells and myocytes (reviewed in Ref. [1]). Caveolin, a 21–24 kDa integral membrane protein, is a major protein component of caveolae. Multiple members of the caveolin gene family have been identified (caveolin-1-{alpha} and -β, caveolin-2, and -3) that differ in tissue distribution [1]. Although caveolae play a major role in vesicular and cholesterol trafficking, the recent identification of various signaling molecules in caveolae and their functional interaction with caveolin suggest that they may also participate in transmembrane signaling [1]. Several groups have shown a compartmentation of various signaling molecules in caveolae. These include heterotrimeric G proteins [2], adenylyl cyclase [3] and PKC isoforms [4], certain receptor-tyrosine kinases (including EGF-R and PDGF-R) [5,6], H-Ras [7] and eNOS [8]. Moreover, an inhibition of their enzymatic activity by a short cytosolic domain derived from the N-terminal region of caveolin-1, called the caveolin scaffolding domain, has been demonstrated [9]. In effect, caveolin-1 seems to act as a scaffolding protein, able to negatively regulate the activity of signaling molecules.

Interestingly, many of the signaling molecules that either interact with or are inhibited by caveolin-1 are involved in the mediation of mitogenic signals to the nucleus. Therefore, it is in line that caveolin-1 mRNA and protein expression are reduced during cell transformation by activated oncogenes such as v-abl and H-ras [10] and conversely that the recombinant expression of caveolin-1 in v-abl and H-ras transfomed NIH-3T3 fibroblasts inhibits both the p42/44 MAP kinase and the basal transcriptional activation of a mitogen-sensitive promoter [11,12]. In support of these data, the antisense-mediated down-regulation of caveolin-1 in NIH-3T3 fibroblasts leads to a hyper-activation of the p42/44 MAP kinase pathway [13]. Taken together, these data lead to the hypothesis that caveolin-1 inhibits cellular proliferation. Consequently the loss of caveolin-1 is a hallmark of dedifferentiation and proliferation.

Cellular proliferation is involved in the pathogenesis of vascular proliferative diseases such as primary atherosclerosis and restenosis after angioplasty [14]. Whereas early events in atherogenesis are characterized by an altered endothelial function and by the recruitment of mononuclear leucocytes to the intima, the progression of atheroma involves the proliferation of VSMC, their migration from the underlying media to the intima and their production of extracellular matrix macromolecules [14,15].

Based on the recently postulated role of caveolin-1 in the modulation of cell cycle progression, the foci of the present study were 1) to establish the causal relationship of caveolin-1 in the proliferation of VSMC using adenoviral overexpression and 2) to investigate the possible pathophysiologic role of caveolin-1 in human atherosclerosis.


   2. Methods
Top
 Abstract
 1. Introduction
 2. Methods
3. Results and discussion
 Acknowledgments
 References
 
The authors declare that the investigation conforms with the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health and that the investigation conforms with the principles outlined in the Declaration of Helsinki.

2.1 Materials
Dulbecco's modified Eagle's medium (DMEM), DMEM/HAM's F12 and fetal bovine serum (FBS) was from GIBCO BRL (Karlsruhe, Germany). The following primary antibodies were used: mouse monoclonal anti-caveolin-1 (clone 2297), mouse monoclonal anti-caveolin-1 (clone C060), rabbit polyclonal anti-caveolin (PharMingen, Heidelberg, Germany), mouse monoclonal anti-smooth muscle {alpha}-actin ({alpha}-sm actin) (clone 1A4) (Coulter-Immunotech, Hamburg, Germany), mouse monoclonal anti-PCNA (sc-56), mouse monoclonal anti-p27Kip1 (F8) (Santa Cruz Biotechnology, Heidelberg, Germany), mouse monoclonal anti-β-actin (Sigma, Deisenhofen, Germany), and rabbit anti-cleaved caspase 3 (Asp175) (Cell Signaling, Frankfurt/M., Germany). Secondary anti-mouse and anti-rabbit antibodies were purchased from Amersham (Freiburg, Germany).

2.2 Cell culture
Primary VSMC were isolated from the aorta of wild-type mice and caveolin knockout mice by enzymatic dissociation [16] and maintained in DMEM/HAM's F12 supplemented with 20% fetal bovine serum (FBS), penicillin (100 U/ml), and streptomycin (100 mg/ml) in a humidified 95% air/5% CO2 incubator. Caveolin-1 knockout mice (Cav-/-) and wild-type mice (C57BL/6 mice) were a generous gift of Dr. Marek Drab (Institute of Pharmacology, Technical University of Dresden). Expression of {alpha}-sm actin was demonstrated by immunohistochemical staining with a monoclonal antibody directed against {alpha}-sm actin.

Human embryonic kidney (HEK) 293 cells were purchased from ATCC (Manassas, USA) and cultivated in DMEM with 4,5 g/l glucose, 10% FBS, penicillin (100 U/ml), and streptomycin (100 mg/ml) in a humidified 95% air/5% CO2 incubator.

2.3 Construction of caveolin-1 adenovirus
The recombinant adenovirus was prepared with the Adeno-x expressionsystem from BD Bioscience (Heidelberg, Germany) according to the manufacturer's instruction. In brief, the full open-reading frame of mouse caveolin-1 was cloned from mouse aorta total RNA by polymerase chain reaction using appropriate primers containing NheI and KpnI restriction sites at 5' and 3'. cDNA was digested with NheI and KpnI (MBI Fermentas, St. Leon-Rot, Germany) and ligated in sense orientation at the cloning site of the pshuttle vector using a ligation kit from MBI Fermentas. The construct was sequenced by AGOWA (Berlin, Germany). After amplification in E. coli (INV{alpha}F', Invitrogen, Karlsruhe, Germany) the expression cassette was excised from pshuttle vector and ligated to E1- and E3-delated Adeno-x viral DNA. The caveolin-1 containing Adeno-x viral DNA (Ax-Cav-1) was amplified in E. coli (Stbl2, Invitrogen). The recombinant Adeno-x viral DNA was packaged into infectious adenovirus by transfecting HEK 293 cells. An adenovirus without caveolin-1 insert was used as a control (Ax-{Delta}).

2.4 Cell proliferation assay
Caveolin-1 knockout mouse (Cav-/-) VSMC were plated into 96-well plates at ~70% confluence and infected with either Ax-Cav-1 or Ax-{Delta} at 6.8 x 107 ifU/ml. Time of infection and serum conditions differed between experimental settings. BrdU incorporation into Cav-/-VSMC was assessed with BrdU cell proliferation ELISA from Roche (Mannheim, Germany).

2.5 Transmission electron microscopy
Caveolin-1 knockout mouse (Cav-/-) VSMC infected with either Ax-Cav-1 or Ax-{Delta} were fixed with 2.5% glutaraldehyde in 0.1 M cacodylarte buffer and embedded in Epon according to standard protocols. Sections were photographed at a magnification of x 16,700.

2.6 Immunoblotting
Thirty micrograms of cellular lysates were separated by SDS-poly-acryl-amide gel electrophoresis (SDS-PAGE) and transferred to Fluoro Trans® W membranes (Pall, Dreieich, Germany). Membranes were blocked in 5% nonfat dry milk and subjected to immunoblotting using mouse monoclonal anti-caveolin-1 (clone 2297), PCNA (sc-56), p27Kip1 (F8) or cleaved caspase 3 (Asp 175). Bound primary antibodies were visualized using Amersham (Freiburg, Germany) chemiluminescence Western blotting detection reagents. Equal protein loading was examined by protein staining with ponceau S.

2.7 Statistical analysis
Statistical analysis was performed using analysis of variance and Student–Newman–Keuls test for significance.

2.8 Immunohistochemistry
Specimens from human atheroma were obtained from a total of twenty patients with symptomatic carotid artery disease (history of stroke or transient ischemic attack) undergoing carotid endarterectomy. Control carotid arteries were derived from healthy accident victims during autopsy. Consecutive paraffin sections (5 µm) of routinely fixed (4% buffered formaldehyde) samples of carotid arteries were used for the avidin–biotin complex (ABC) immunoperoxidase technique and two-colour immunofluorescence. Sections were deparaffinized and pretreated with microwave radiation [17], washed in phosphate-buffered saline (PBS), and then incubated with normal horse serum followed by incubation with the primary antibody. All steps were performed at room temperature. For immunodetection a commercially available ABC-technique (mouse kit 6102; Vectastain Elite Kit, Serva, Heidelberg, Germany) was used according to the manufacturer's instructions. Visualization of peroxidase localization was performed using 3,3'-diaminobenzidine as substrate.


   3. Results and discussion
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results and discussion
Acknowledgments
 References
 
3.1 Effect of VSMC proliferation on caveolin-1 expression
Primary VSMC were isolated from mouse aorta by enzymatic dissociation [16] and the expression of smooth muscle {alpha}-actin ({alpha}-sm actin) was demonstrated by staining with an {alpha}-sm actin antibody (data not shown). Based on the potential role of caveolin-1 in cell cycle progression we investigated the protein expression of caveolin-1 in proliferating VSMC. In order to induce quiescence, serum was withdrawn from VSMC for 2 days. As shown in Fig. 1A, the subsequent supplementation of 10% FBS induced a significant decrease of caveolin-1 expression in a time-dependent manner. Proliferation of VSMC after serum-stimulation was documented by an increase of PCNA (Fig. 1B), a nuclear protein important for G1-phase progression [18]. However, the expression of β-actin serving as housekeeping protein remained unchanged under these conditions. Cell cycle entry and progression depends on the regulated interaction of cyclin-dependent kinases (Cdks) with their activators, the cyclins and Cdk inhibitors [19]. The Cdk inhibitor p27kip1 plays an important role in cell cycle regulation as its high levels, present in quiescent cells, decline upon mitogen induction. In line with the above-mentioned increase of PCNA expression after serum-stimulation, we could additionally demonstrate cell cycle entry of VSMC by a significant decrease of p27kip1 (Fig. 1C). This in vitro model indicates a regulatory role of caveolin-1 in VSMC proliferation.


Figure 1
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  		Fig. 1 Effect of VSMC proliferation on caveolin-1 expression. A. VSMC were serum-starved for 48 h. Cells were harvested 8, 12, 16, 20, 24 and 32 h after supplementation of 10% FBS. Thirty micrograms of cellular lysates were subjected to SDS-PAGE and Western blot analysis with a monoclonal caveolin-1 antibody. Caveolin-1 was detected on SDS-PAGE as double bands (caveolin-1-{alpha} and -β). Equal protein loading was examined by protein staining with ponceau S. The intensity of caveolin-1 bands was normalized to protein loading. The lower part shows a whole series of experiments (n = 8, *p ≥ 0.05, **p ≥ 0.005). The upper part of the panel shows a representative Western blot out of eight experiments. These data indicate a significant decrease of caveolin-1 in VSMC after serum supplementation. B. Cell cycle entry of VSMC after serum supplementation was verified by Western blot analysis of PCNA expression. The lower part shows a whole series of experiments (n = 8, *p ≥ 0.05, **p ≥ 0.005). The upper part of the panel shows a representative Western blot out of eight experiments. For equal protein loading Western blot analysis of β-actin was performed. C. Western blot analysis of the Cdk inhibitor p27kip1 after serum stimulation. A whole series of experiments (n = 8, *p ≥ 0.05, **p ≥ 0.005) and a representative Western blot are shown.

 
A previous report showed that serum stimulation causes a decrease of caveolin-1 protein levels in human coronary artery smooth muscle cells [20]. In accordance with these data Thyberg et al. [21,22] demonstrated by immunofluorescence and immunoelectron microscopy a decrease of caveolin-1 protein expression and plasma membrane-associated caveolae in proliferating VSMC.

3.2 Overexpression of caveolin-1 inhibits proliferation of VSMC
To establish a causal relationship between caveolin-1 expression and cell cycle progression we overexpressed caveolin-1 in VSMC, isolated from caveolin-1 knockout mice (Cav-1-/-). For these studies Cav-1-/-VSMC were infected with an adenovirus-expressing caveolin-1 (Ax-Cav-1) or a similar vector without cDNA insert (Ax-{Delta}). Western blot analysis demonstrated that caveolin-1 is completely absent in Cav-1-/-VSMC. However, infection with Ax-Cav-1 induced an overexpression of caveolin-1 in Cav-1-/-VSMC (Fig. 2A). Transmission electron microscopy was performed to visualize plasmalemmal caveolae. While Cav-1-/-VSMC are conspicuously devoid of caveolae, these cells have numerous uniformly sized caveolae after adenoviral overexpression of caveolin-1 (Fig. 2B).


Figure 2
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  		Fig. 2 Caveolin-1 protein expression and caveolae formation after adenoviral overexpression. A. Western blot analysis showing caveolin-1 protein expression in wild-type VSMC (Wild-type) and VSMC from caveolin-1 knockout mice infected with an adenovirus expressing caveolin-1 (Ax-Cav-1) or a similar vector without cDNA insert (Ax-{Delta}). B. The transmission electron micrographs shown are imaged at a magnification of x 16,700 (for ease of view, images are further magnified to x 33,400). VSMC isolated from caveolin-1 knockout mice (Cav-1-/-) are devoid of caveolae. Caveolin-1 overexpression in caveolin-1 deficient VSMC (Ax-Cav-1) leads to a wide formation of caveolae.

 
To define changes in cell cycle progression of adenovirally infected Cav-1-/-VSMC, a standard protocol was used to determine the incorporation of BrdU into the DNA of proliferating VSMC. BrdU incorporation was significantly reduced after infection of Cav-1-/-VSMC with Ax-Cav-1 compared with Ax-{Delta} infected cells (Fig. 3), demonstrating that the expression of caveolin-1 directly regulates cellular proliferation. Western blot analysis of Ax-Cav-1- and Ax-{Delta}-infected VSMC with an antibody directed against cleaved caspase 3 excluded that the decreased proliferation of Cav-1-/-VSMC after caveolin-1 overexpression was caused by apoptosis (data not shown).


Figure 3
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  		Fig. 3 Adenoviral overexpression of caveolin-1 inhibits proliferation of vascular smooth muscle cells. VSMC derived from caveolin-1 knockout mice were infected with an adenovirus expressing caveolin-1 (Ax-Cav-1) or a similar vector without cDNA insert (Ax-{Delta}) under serum-free conditions for 24 h followed by incubation in medium containing 20% FBS for aditional 24 h. BrdU incorporation into caveolin-deficient VSMC was assessed with BrdU cell proliferation ELISA. A whole series of experiments (n = 6, **p ≥ 0.005) is shown.

 
3.3 Caveolin-1 expression in human atherosclerosis
The expression of caveolin-1 in VSMC was further investigated in human tissue. Therefore, specimens from human atheroma were obtained from a total of twenty patients with symptomatic carotid artery disease undergoing carotid endarterectomy. As controls, caveolin-1 was detected in VSMC of healthy carotid arteries by immunohistochemical staining (Fig. 4A). The abundance of VSMC in atheroma was confirmed by staining with {alpha}-sm actin. It has to be emphasized that VSMC of healthy carotid arteries were lacking PCNA immunoreactivity. These data demonstrate the contractile phenotype of VSMC. In striking contrast to control vessels, caveolin-1 was markedly decreased in sections derived from human atheroma (Fig. 4B). However, VSMC were still detectable in these sections, as confirmed by {alpha}-sm actin-immunostaining. PCNA immunoreactivity verified the proliferation of VSMC in human atherosclerosis. Interestingly, in particular VSMC in the margin of the atheromatous lesion showed an impressive PCNA staining (Fig. 4B). During the course of these experiments, it was noted that caveolin-1 was not completely absent from human atheroma. As shown in Fig. 4C, caveolin-1 was still detectable in the endothelium of vasa vasorum.


Figure 4
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  		Fig. 4 Expression of caveolin-1 in VSMC of human atheroma. Consecutive paraffin sections from human atheroma were obtained from patients with carotid artery disease undergoing endarterectomy. Representative data out of twenty patients are shown. A. Immunoperoxidase staining of caveolin-1 (Caveolin-1), {alpha}-sm actin ({alpha}-SM Actin) and PCNA (PCNA) in control vessels (Control). A magnification of 1:300 is demonstrated. Control vessels show a strong immunostaining for both caveolin-1 and {alpha}-sm actin whereas they are devoid of PCNA immunostaining. B. Immunostaining of caveolin-1 (Caveolin-1), {alpha}-sm actin ({alpha}-SM Actin) and PCNA (PCNA) in human atheroma (Atheroma). Caveolin-1 is markedly decreased in VSMC of atheroma. VSMC show PCNA immunostaining predominantly in the margin of the atherosclerotic lesion. C. Immunostaining of caveolin-1 in human atheroma. Caveolin-1 is detectable exclusively in the endothelium of vasa vasorum (as indicated by arrows).

 
These data indicate that the proliferation of VSMC in human atherosclerosis is accompanied by a decrease of caveolin-1 expression, as shown before in vitro. In addition, this newly characterized decline of caveolin-1 expression in proliferating smooth muscle cells of human atheroma strongly links the loss of the antiproliferative control mediated by caveolin-1 to the progression of human atherosclerosis. Using a rabbit arterial injury model Peterson et al. demonstrated an attenuation of caveolin-1 expression in phenotypically altered VSMC [23]. Three weeks after balloon-injury immunostaining for caveolin-1 showed an intense staining throughout the media, however, less caveolin-1 immunoreactivity within the neointima was evident when compared with the media. Our data are in good agreement with a study, showing a decreased caveolin-1 mRNA expression in atherosclerotic lesions [24]. However, to our knowledge this is the first study directly demonstrating by immunohistochemistry a decreased expression of caveolin-1 in proliferating VSMC in human atheroma.

Besides caveolae function in vesicular and cholesterol trafficking, several observations provide evidence that caveolin-1 plays a pivotal role in the regulation of cell cycle progression. Functionally, it appears that caveolin-1 inhibits mitogenic signaling events. For example, caveolin-1 has been shown to interact with and suppress the kinase activity of several members of the Ras-p42/44 MAP kinase cascade, including MEK and ERK [25]. In accordance with these data the recombinant expression of caveolin-1 in transformed NIH 3T3 fibroblasts or cell lines derived from human cancers suppresses their transformed phenotype [26]. The recombinant overexpression of caveolin-1 in NIH 3T3 fibroblasts as well as the expression of caveolin-1 as a transgene induced cells to exit the S phase of the cell cycle with a concomitant increase in the G0/G1 population and a reduction in cellular proliferation [27]. In addition to repression of the Ras-p42/44 MAP kinase pathway, caveolin-1 has been found to transcriptionally repress cyclin D1 expression [28]. Conversely, the antisense-mediated down-regulation of caveolin-1 in NIH 3T3 cells caused a hyper-activation of the p42/44 MAP kinase pathway and consecutively an increased cellular proliferation [13].

These in vitro data have been further substantiated by the generation of mice that lack the caveolin-1 gene (Cav-1-/-) [29,30]. This caveolin-1 knockout revealed in vivo an abnormal hypercellular lung phenotype with thickened alveolar septa and an increase of lung endothelial cells, cell types and tissue that normally express high levels of caveolin-1 [29,30]. Isolation of both primary embryonic fibroblasts and cardiac fibroblasts demonstrated that the loss of caveolin-1 in these cell types leads to an elevated cellular proliferation, which correlated with the hyperactivation of the p42/44 MAP kinase cascade [29,31]. Additionally, we could demonstrate most recently an increased percentage of Cav-1-/-VSMC in the S-phase, as compared to their caveolin-1 expressing wild-type counterparts [32]. Cav-1-/-mice also show vascular defects, such as increased neointimal hyperplasia in response to vascular injury [33]. This phenotype appears to be due to hyperactivation of the p42/44 MAP kinase cascade and upregulation of cyclin D1 in Cav-1-/-VSMC. Taken together, these data suggest that caveolin-1 can act as an antiatherogenic molecule in vascular smooth muscle cells.

Caveolae and caveolin-1 are involved in regulating several signal transduction pathways that play a pathogenetic role in atherosclerosis and restenosis after coronary angioplasty. It is noteworthy that caveolae and caveolin-1 are present in almost every cell type that has been implicated in the development of an atheroma. These include endothelial cells, macrophages, and VSMC. Depending on the cell type examined, the pathogenetic role of caveolin-1 might be either proatherogenic or antiatherogenic. Frank et al. [34] recently reported that the loss of caveolin-1 in ApoE/Cav-1 double-knockout mice conferred protection against atheroma formation. In this double-knockout animal model the loss of caveolin-1 resulted in the down-regulation of endothelium-derived proatherogenic molecules, namely, CD36 and VCAM-1. These findings suggest that caveolin-1 deficiency may lead to reduced transcytosis of lipoproteins across endothelial cells. Another important function of caveolin-1 in endothelial cells is its ability to negatively regulate eNOS activity [35]. The phenotypical characterization of the Cav-1-/-mouse showed indeed an increased endothelial NO production [29]. eNOS is involved in early steps that lead to the development of an atheroma. This initiation of the atherosclerotic lesion is characterized by the adhesion of leukocytes to the surface of endothelial cells, mediated by the expression of adhesion molecules like VCAM-1. However, there is up to date no evidence of a down-regulation of caveolin-1 in endothelial cells in non-knockout models. Furthermore, we now provide evidence of a stable caveolin-1 expression in ECs of human atherosclerotic plaques. Instead, reduced caveolin expression was found in VSMC pointing towards caveolin's role for the progression of an atheroma which involves the accumulation and proliferation of VSMC [14,15].

In the present study we demonstrated that caveolin-1 deficiency resulted in increased proliferation of primary VSMC. Conversely, overexpression of caveolin-1 in Cav-1-/-VSMC inhibited cellular proliferation. However, the salient finding of the present study is the attenuation of caveolin-1 expression in proliferating VSMC of human atheroma. Our report extends earlier observations demonstrating the pivotal role of caveolin-1 in cell cycle regulation. Taken together, these findings suggest that caveolin-1 may act as an antiatherogenic molecule in vascular smooth muscle cells.


   Acknowledgments
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results and discussion
 Acknowledgments
References
 
The authors thank Mrs. S. Bramke for skillful immunohistochemical assistance and Mrs. P. Barthel for expert Western blot analysis.


   Notes
 
1 C. Schwencke and A. Schmeisser contributed equally to this work. Back

Time for primary review 22 days


   References
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results and discussion
 Acknowledgments
 References
 

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