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FJ
EXPRESS SUMMARY ARTICLE The Full-length version of this article is also available, published online February 19, 2003 as doi:10.1096/fj.02-0396fje. |
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,
,
* Department of Biomedical Science and Biotechnology, University of Cagliari, Cagliari, Italy; and Departments of
Experimental Pathology,
National Institute for the Research on Cancer, Genova-Biotechnology Unit in the Department of Experimental Pathology, and
Interdepartmental Center for the Research on Cancer, University of Bologna, Bologna, Italy
2Correspondence: Department of Biomedical Science and Biotechnology, University of Cagliari, Via Porcell, 4, 09124, Cagliari, Italy. E-mail: sdessi{at}vaxca1.unica.it
SPECIFIC AIMS
Abnormal vascular smooth muscle cell (VSMC) proliferation is considered a key event in a number of human diseases, which go by the name "vascular proliferative diseases," and the role exerted by growth factors, their receptors, and signal transduction in allowing or preventing/inhibiting this proliferation received consistent attention in the last years.
Conversely, it is known that the progression of VSMC into cell cycle is accompanied by signals other that transduction, i.e., profound and substantial biochemical changes in cholesterol metabolism and in intracellular cholesterol trafficking. This prompted us to evaluate and compare the regulation of VSMC growth in vitro with that of cholesterol trafficking, with the aim of identifying a downstream control point of many, if not all, mitogenic stimuli and eventually identifying new targets for future drug development in the field of vascular proliferative diseases.
PRINCIPAL FINDINGS
The findings presented here suggest a plausible link between two key events in the progression of vascular proliferative diseases, providing some evidence that the cholesterol ester pathway may have a functional role in the progression of vascular lesions, i.e., in the signal transduction pathways governing the VSMC cycle at the G1/S transition (Fig. 1
).
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1. Time-dependent changes in the cholesterol ester pathway during cell replication of VSMC
Time-dependent changes in the cholesterol esterification pathway have been examined during the proliferation of VSMC. Quiescent VSMCs were stimulated to growth by adding 10% fetal calf serum (FCS), and the incorporation of 3H-thymidine into DNA was analyzed at various times after stimulation. DNA synthesis began to increase 24 h after serum stimulation, reached a peak at 36 h, and thereafter decreased. Changes in the 3H-thymidine incorporation were preceded by similar changes in the 14C-oleate incorporated into cholesterol esters, which began to increase at 12 h, reached maximal levels at 18–24 h, and thereafter declined. The mRNA of cyclin D1 together with those of acylCoA: cholesterol acyltransferase (ACAT) and multidrug resistance 1 (MDR1) was expressed at increased levels at 12–18 h and then decreased. In contrast, the mRNA level of caveolin-1 was reduced 12–24 h after serum stimulation, compared with quiescent cells. By densitometric analysis of the Western blot, serum caused a rapid increase in the basal extracellular-regulated kinase (ERK)1/2–mitogen-activated protein kinase (MAPK) expression, whereas the caveolin-1 protein level was reduced.
2. Effect of inhibitors of cholesterol esterification on the proliferation of VSMC
To explore the hypothesized mechanism by which cholesterol esterification could influence the VSMC proliferation, we performed cell-cycle analyses in the presence or absence of two different inhibitors of cholesterol esterification (Sandoz 58–035, 5 µM; progesterone, 1 µM). A significant inhibition of 14C-oleate incorporated into cholesterol levels was evident in the VSMCs treated by each inhibitor as early as 12 h after serum stimulation. This effect was followed by a significant reduction of 3H-thymidine incorporation into DNA, evident after a delay of 
12 h with respect to it and reflecting changes in cell number, as confirmed in parallel experiments based on cell count. Under our experimental conditions, neither Sandoz 58–035 or progesterone caused accumulation of intracellular cholesterol or promoted changes in cell shape or spreading nor did each of them induce apoptotic bodies, as evaluated by microscopic inspection of the culture.
3. Effect of cholesterol esterification inhibitors on ACAT, MDR1, and caveolin-1 expression
As shown in Figure 2
A, the treatment of serum-stimulated VSMC with Sandoz 58–035 or progesterone induced a down-regulation of the MDR1, ACAT, and cyclin D1 mRNA expression, evident 12 h after serum stimulation and at maximal effect after 18 h of serum stimulation. In contrast, caveolin-1 mRNA levels were up-regulated by the inhibitors, the effect being evident as early as 12 h after VSMC serum stimulation. Changes in caveolin-1 protein abundance were also evident at 6 and 12 h in the treated cells (Fig. 2B
).
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4. Effect of cholesterol esterification inhibitors on the cell cycle
To further verify the effect of the inhibition of cholesterol esterification by Sandoz 58–035 and progesterone on cell growth, we determined the cell-cycle distribution of the treated VSMC by flow cytometry. Whereas in the absence of serum stimulation, 83% of the VSMC remained in the growth-arrested phase of the cell cycle (G0/G1), 50% of them proceeded to the S phase after 24 h from serum stimulation (Fig. 3
A). In contrast, 77% and 71% of the cells treated with Sandoz 58–035 and progesterone, respectively, were blocked in the G0/G1 phase after 24 h from serum stimulation, indicating a scarce progression of them to the S and G2/M phases (Fig. 3A
).
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A strong and rapid inhibition of the mitogenic signaling ERK1/2 pathway was caused by the treatment of VSMC with inhibitor of cholesterol esterification (Fig. 3B
)
CONCLUSIONS AND SIGNIFICANCE
Considerable evidence has been accumulated in recent years by us and others, indicating that cholesterol and other isoprenoids produced throughout the cholesterol biosynthetic pathway play a key role in eukaryotic normal and neoplastic cell proliferation. An accumulation of cholesterol esters was evident in all the proliferating tissues analyzed, together with a drastic reduction of high-density lipoprotein cholesterol in the plasma compartment, so indicating the necessity of an endogenous source of newly synthesized cholesterol for membrane biogenesis and of esterified cholesterol for cell proliferation.
Increased cholesterol esterification by ACAT in the endoplasmic reticulum (ER) and enhanced proliferation of VSMC are considered hallmark events in the pathogenesis of vascular diseases; however, a functional link between them is as yet poorly elucidated.
In this study, we provide some evidence that cholesterol esterification may have a role in the signal transduction pathways governing the VSMC cycle at the G1/S transition. Our results also demonstrate that the rate of VSMC proliferation positively correlates with MDR1 mRNA levels, whereas it negatively correlates with caveolin-1 expression. MDR1 is known to be involved in controlling cholesterol trafficking from the plasma membrane to the ER, whereas caveolin-1, acting in an opposite direction to MDR1, transports cholesterol from the ER to the plasma membrane and mediates the efflux of free cholesterol derived from de novo synthesis or from low-density lipoproteins (LDLs). All these data suggest that cholesterol esterification may have a role in regulating the growth rate of VSMC and that MDR1 and caveolin-1 may contribute to this regulation by modulating, in an opposite manner, the availability of cholesterol substrate in the ER, which is a major determinant of ACAT activity.
Recent data indicate a direct role for caveolin-1 in the interaction, with a number of caveolae-associated signaling molecules such as the Ras-MAPK cascade to modulate or suppress their enzymatic activities, suggesting for caveolin-1, a function as a negative regulator of many different classes of signaling molecules. In addition, studies in cholesterol-depleted cells demonstrate that a reduced cholesterol level in caveolae is by itself a signal to activate pathways leading to cell division, suggesting that the tumor-suppressing effect of caveolin-1 may be a result of its ability to maintain and/or reconstitute the necessary concentration of cholesterol in caveolae.
In view of the postulated roles for caveolin-1 in cholesterol transport and in negative regulation of signal transduction, our results point to the possibility that cholesterol esterification may influence VSMC cycle progression by reducing the caveolin-1 expression. This is in line with the observation that proliferating VSMC showed lower expression of caveolin-1 and that caveolin-1 levels returned to control when VSMC growth was reduced by the treatment with inhibitors of cholesterol esterification.
Future investigations should improve our knowledge of the proliferative mechanisms in VSMC and of the relevance of the cholesterol esterification pathway in the progression of vascular proliferative diseases. In addition, therapeutic strategies for preventing/treating proliferative vascular disorders, including the use of cholesterol esterification inhibitors, should improve concurrent advances in understanding VSMC function/proliferation.
FOOTNOTES
1 To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.02-0396fje; to cite this article, use FASEB J. (February 19, 2003) 10.1096/fj.02-0396fje 
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