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Am J Physiol Heart Circ Physiol 292: H2721-H2728, 2007. First published February 23, 2007; doi:10.1152/ajpheart.01174.2006
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Anti-atherosclerotic effects of sirolimus on human vascular smooth muscle cells

Kun L. Ma, Xiong Z. Ruan, Stephen H. Powis, John F. Moorhead, and Zac Varghese

Centre for Nephrology, Royal Free and University College Medical School, University College London, London, United Kingdom

Submitted 26 November 2006 ; accepted in final form 17 February 2007


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Sirolimus is a potent immunosuppressive agent and has an anti-atherosclerotic effect through its anti-proliferative property. The present study was undertaken to investigate the effect of sirolimus on intracellular cholesterol homeostasis in human vascular smooth muscle cells (VSMCs) in the presence of inflammatory cytokine IL-1beta. We explored the effect of sirolimus on the lipid accumulation of VSMCs in the presence of IL-1beta, using Oil Red O staining and quantitative measurement of intracellular cholesterol. The effect of sirolimus on the gene and protein expression of lipoprotein receptors and ATP binding cassettes (ABCA1 and ABCG1) was examined by real-time PCR and Western blotting, respectively. Furthermore, the effect of sirolimus on cholesterol efflux from VSMCs in the presence or absence of IL-1beta was also investigated using [3H] cholesterol efflux. Finally, we examined the effect of sirolimus on the production of inflammatory cytokines in VSMCs using ELISA. Sirolimus reduced intracellular lipid accumulation in VSMCs mediated by IL-1beta possibly due to the reduction of expression of low-density lipoprotein (LDL) and very low-density lipoprotein (VLDL) receptors. Sirolimus increased cholesterol efflux from VSMCs and overrode the suppression of cholesterol efflux induced by IL-1beta. Sirolimus also increased ABCA1 and ABCG1 genes expression, even in the presence of IL-1beta. We further confirmed that sirolimus inhibited mRNA and protein expression of inflammatory cytokines IL-6, tumor necrosis factor-{alpha}, IL-8, and monocyte chemoattractant protein-1. Inhibition of lipid uptake together with increasing cholesterol efflux and the inhibition of inflammatory cytokines are all important aspects of the anti-atherosclerotic effects of sirolimus on VSMCs.

cholesterol homeostasis; foam cell; inflammatory cytokine

ATHEROSCLEROSIS is a multifactorial disease; disordered lipid metabolism and chronic inflammation are important contributory factors (16, 32). Vascular smooth muscle cells (VSMCs) play a key role during the pathogenesis of vascular lesion, although they are not always the main cell component of atherosclerotic lesions, especially in the early stages. In normal vessels, the majority of VSMCs reside in the media, where they are quiescent and possess a "contractile" phenotype characterized by the abundance of actin- and myosin-containing filaments (4, 33). In disease states, VSMCs reenter the cell cycle, proliferate, and migrate from media to intima (33). After vessel injury, intimal VSMCs have a synthetic phenotype, characterized by hyperplasia or hypertrophy and matrix protein accumulation in the intima and/or media with or without lipid deposition, resulting in thickening and stiffness of the arterial wall. Foam cells have traditionally been regarded as being derived from macrophages because they express macrophage markers. However, it is now known that VSMCs can also be converted into foam cells. Evidence for this includes 1) VSMCs in tissue culture can accumulate cholesterol (45); 2) some lesional foam cells traditionally classified as macrophages are actually arterial VSMCs that have significantly altered their phenotype (30); 3) significant numbers of foam cells are present in atherosclerotic lesions in mice deficient in genes responsible for monocyte recruitment (2), leukocyte adhesion (6), and macrophage proliferation and differentiation (40); and 4) in advanced lesions from both Watanabe Heritable Hyperlipidemic rabbits and fat-fed rabbits (31), simultaneous tritium-labeled methyl-thymidine autoradiography and immunostaining for cell type-specific markers revealed that ~30% of the labeled cells were macrophages and 45% smooth muscle cells.

Sirolimus is a potent immunosuppressive agent used for the prophylaxis of transplant rejection. The cellular action of sirolimus is mediated by binding to the FK506-binding protein (i.e., FKBP12) and blocking signals induced by mammalian target of Rapamycin and arresting cell cycle progression from G1 to S phase transition (29). Recent studies have demonstrated that sirolimus has a beneficial effect in preventing restenosis following angioplasty in animal models and in clinical trials where sirolimus-coated stents have been shown to inhibit intimal thickening in patients with coronary artery disease (9, 12, 20, 41). Furthermore, some studies showed that sirolimus reduces atherosclerotic lesion size in apoE-null mice, whether administered intraperitoneally (3, 7) or orally (28). Although Elloso et al. (7) have reported increased low-density lipoprotein (LDL) cholesterol and high-density lipoprotein (HDL) cholesterol in sirolimus-treated apoE-null mice, interestingly, Castro et al. (3) found no significant effect of sirolimus on circulating lipids, including LDL cholesterol, HDL cholesterol, very LDL cholesterol, and intermediate density lipoprotein cholesterol. On the other hand, sirolimus did not aggravate hypercholesterolemia in fat-fed apoE-null mice (3, 7, 25, 26) while increasing circulating triglyceride levels in the same animal model (25). These discrepancies suggest that the anti-atherosclerotic effect of sirolimus could be related to the change of local cholesterol metabolism in the tissues mediated through its anti-proliferative and anti-inflammatory effects. This study particularly addresses the question how the anti-inflammatory effects of sirolimus modify the lipid-mediated vascular injury through its effects on intracellular cholesterol homeostasis.

In most cells, intracellular lipid content is governed by tight regulation of cholesterol influx and efflux pathways. The intracellular level of cholesterol is controlled by the uptake and synthesis of cholesterol through feedback regulation. Recent studies strongly indicated that atherogenesis is initiated by the interplay between cholesterol and cellular secretion of cytokines (especially IL-6) and apolipoprotein-E within the arterial wall (14). Ruan et al. (34, 37, 38) also reported that inflammatory cytokines could cause lipid accumulation by increasing native LDL uptake via LDL receptor (LDLr) or reducing efflux of intracellular lipid via ATP-binding cassette transporter A1 (ABCA1) in human mesangial cells. Therefore, the present investigation was undertaken to evaluate whether sirolimus ameliorates the imbalance of intracellular cholesterol homeostasis in human VSMCs mediated by inflammatory mediators.


   MATERIALS AND METHODS
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 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Cell culture. The primary human VSMCs from coronary artery (TCS cell works, Buckinghamshire, UK) were cultured in a basal medium in T75 flasks (Falcon, UK) supplemented with 5% fetal bovine serum, insulin, human epidermal growth factor, human fibroblast growth factor, 2 mmol/l L-glutamine, 0.5 ml 25 mg/ml gentamicin, and 50 µg/ml amphotericin B. Cell cultures were maintained in a humidified 95% air-5% CO2 incubator at 37°C, and the medium was changed every 48 h. Cells were subcultured with 0.25% trypsin-0.01% EDTA when cells were grown to subconfluence. The cells and all reagents for cell culture were obtained from TCS Cell Works. Recombinant interleukin-1 (IL-1beta, 1.0–3.3 x 108 U/mg) was obtained from R&D Systems (Europe, Abingdon, UK). Sirolimus (914.19 mol wt, code AY-22989-39) was supplied by Wyeth Pharmaceutics. All experiments were carried out in serum-free DMEM/F12 (1:1) medium containing 0.2% fatty acid-free BSA (Sigma, Poole, Dorset, UK).

Lipoprotein preparation. Plasma was collected from healthy human volunteers, and LDL was isolated by sequential ultracentrifugation as described in our previous publication (36). The study protocol was approved by the Institutional Review Board of Royal Free and Medical School, University College London and adhered to the tenets of the Declaration of Helsinki for experiments involving human samples. Antioxidants (100 µmol/l of EDTA and 20 µmol/l BHT) were added into the fresh LDL to prevent oxidation. The extent of lipid peroxidation of the LDL was estimated as the concentration of thiobarbituric acid reactive substances as described previously, and the results were expressed as nmoles of malondialdehyde per milligram LDL (nmol MDA/mg LDL). The levels of thiobarbituric acid reactive substances in native LDL used in the study were <0.1 nmol MDA/mg LDL.

Morphological examination. VSMCs were plated in chamber slides for tissue culture (Becton Dickinson Labware) and incubated in serum-free experimental medium with native LDL in the absence or presence of cytokines and cytokine plus sirolimus. After 24 h incubation, the cells were washed three times in phosphate-buffered saline (PBS), fixed for 30 min with 5% formalin solution in PBS, stained with Oil Red O for 30 min, and counterstained with hematoxylin for another 5 min. Finally, the cells were examined by light microscopy.

Quantitative measurement of intracellular free cholesterol-cholesterol ester. VSMCs were plated in six-well plates (Nunc, Naperville, IL). The cells then were incubated in serum-free DMEM/F12 (1:1) medium with 200 µg/ml of native LDL or 5 µg/ml of IL-1beta plus native LDL (200 µg/ml) in the absence or presence of 10 or 100 ng/ml sirolimus for 24 h. The total and free cholesterol were analyzed using the method described by Gamble et al. (10). In brief, the cells were collected and washed twice with PBS; lipids were extracted by addition of 1 ml chloroform-methanol (2:1) to the cell pellet. After sonification, the samples were centrifuged, and the lipid phase was collected. The samples were dried in vacuum and then dissolved in 2-propanol containing 10% Triton X-100. Cholesterol ester was converted to free cholesterol by cholesterol ester hydrolase for determination of total cholesterol. Cholesterol oxidase was employed to generate H2O2 from free cholesterol, and peroxidase was used to catalyze the reaction of H2O2 with 4-amino-antipyrine and phenol to yield a stable rose-color product. The concentration of total and free cholesterol per well was analyzed using a standard curve and normalized by total cell protein. The concentration of cholesterol ester was calculated using total cholesterol minus free cholesterol.

Cholesterol loading and efflux. VSMCs were loaded with 30 µg/ml cholesterol, 1 µg/ml 25-hydroxycholesterol, and 1 µCi/well [1{alpha}, 2{alpha}(n)-3H]cholesterol (Amersham, Little Chalfont, Bucks, UK) in serum-free medium for 48 h to equilibrate cellular cholesterol pools. After 48 h, fresh serum-free medium containing sirolimus (10 ng/ml or 100 ng/ml) was added in the presence or absence of the inflammatory cytokine IL-1beta (5 ng/ml) for another 24 h. After this incubation period, cells were washed three times in PBS, and ApoA1-mediated cholesterol efflux studies were immediately performed by adding fresh serum-free medium with or without 15 µg/ml ApoA1 (Calbiochem, Nottingham, UK) for 6 h. At the end of this incubation, the supernatant was collected and centrifuged at 13,000 rpm for 10 min to remove debris. Cells were lysed with 0.5 ml of 0.1 N NaOH. The radioactivity in both the supernatant and cellular lipid was measured by scintillation counting. ApoA1-induced [3H]cholesterol efflux was calculated by subtracting the radioactivity in supernatants without ApoA1 from the counts in supernatants containing ApoA1. The data were normalized by total [3H]cholesterol radioactivity (including in supernatant and cell) and were expressed as a percentage of control.

Quantification of specific transcripts by real-time RT-PCR. Total RNA was isolated from cultured VSMCs using the guanidinium-phenol-chloroform method. Total RNA (500 ng) was used as a template for RT-PCR. The RT reaction was set up using a kit from Applied Biosystems (Warrington, Cheshire, UK). After cDNA synthesis by RT, cDNA was split for the separate amplification for target genes using specific primers designed by Taqman Primer Express Software V2.0 as shown in Table 1. Real-time PCR was performed in an ABI 7000 using SYBR Green PCR kit according to the manufacturer's protocol (Applied Biosystems). After the PCR, a dissociation curve (melting curve) was constructed in the range of 60°C to 95°C. Relative amount of mRNA was calculated using the comparative threshold cycle (Ct) method ({Delta}{Delta}Ct method). The amplification efficiencies of the target and reference were shown to be approximately equal with a slope of log input amount to Ct < 0.1. Controls consisting of H2O or samples that were not reversely transcribed were negative for target and reference.


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  		Table 1. TaqMan primers for real-time PCR

 
Western blot analysis. Identical amounts of total protein from cultured VSMCs extracts were denatured and then subjected to electrophoresis on a 5% stacking and 8% separating SDS polyacrylamide gel. Electrophoretic transfer to nitrocellulose was accomplished at 100 V, 350 mA for 1 h in 25 mmol/l Tris, pH 8.3, 192 mmol/l glycine, 0.1% SDS, and 20% methanol. The membrane was then blocked with 2% blocker (Amersham Bioscience) for 1 h at room temperature and subjected to Western blotting analysis using chicken anti-human LDLr polyclonal antibody (Abcam, Cambridge, UK), rabbit anti-human VLDL receptor (VLDLr) polyclonal antibody(Santa Cruz Biotechnology) with horseradish peroxidase-labeled secondary antibodies (Abcam). Actin was also examined using a rabbit anti-actin antibody (Sigma, Poole, Dorset, UK) with a second antibody of goat anti-rabbit horseradish peroxidase-linked IgG (New England Biolabs, Herts, UK). Finally, the signals were detected using ECL advanced system (Amersham Bioscience).

Determination of IL-6, TNF-{alpha}, IL-8, and MCP-1 release. VSMCs in six-well plates were grown to confluence and then cultivated in serum-free medium containing 0.2% BSA (fatty acid free) for 24 h before the experiment. After addition of the stimuli, cells were incubated for 24 h, and then the supernatants of conditioned medium were collected and frozen at –70°C. Assays for IL-6, tumor necrosis factor (TNF)-{alpha}, IL-8, and monocyte chemoattractant protein-1 (MCP-1) were performed with an Enzyme-Linked Immunosorbent Assay Kit (R&D Systems) according to the manufacturer's instructions. The results were normalized with the concentration of total cell protein per well.

Data analysis. In all experiments, groups of data were evaluated for significance by one-way analysis of variance (ANOVA). Data were considered significant if the P value < 0.05.


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
Using Oil Red O staining, we checked the morphological change of VSMC loaded with native LDL in the presence or absence of inflammatory cytokine. We found that IL-1beta significantly increased lipid droplets accumulation of VSMC (Fig. 1B). However, sirolimus reduced lipid droplet accumulation of VSMC caused by IL-1beta (Fig. 1D). Furthermore, quantitative intracellular cholesterol analysis confirmed that sirolimus reduced cholesterol ester accumulation induced by IL-1beta in VSMCs, and sirolimus provided a protective role in decreasing lipid accumulation mediated by inflammatory cytokine in VSMCs (Fig. 1E).


Figure 1
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  		Fig. 1. Visualization of low-density lipoprotein (LDL) uptake and lipid droplets in vascular smooth muscle cells (VSMCs) after sirolimus treatment. VSMCs were incubated for 24 h in serum-free medium containing 200 µg/ml of native LDL in the absence (A) or presence of 5 ng/ml of IL-1beta (B) or 10 ng/ml of sirolimus (C) or 5 ng/ml of IL-1beta plus 10 ng/ml sirolimus (D). The cells were examined for lipid inclusions by Oil Red O (ORO) staining. The results are typical of those observed in four separate experiments (x400). E: effects of sirolimus on intracellular free cholesterol and cholesterol ester changes in LDL-loaded VSMCs. VSMCs were cultured for 24 h in serum-free medium containing 200 µg/ml of native LDL in the absence (control; Ctr) or presence of 5 ng/ml of IL-1beta or 10 ng/ml of sirolimus or 5 ng/ml of IL-1beta plus 10 ng/ml sirolimus. Intracellular free cholesterol and cholesterol ester were assayed as described in MATERIALS AND METHODS. Values are means ± SD of duplicate wells from 4 experiments. *P < 0.05 vs. control. **P < 0.01 vs. control. ***P < 0.01 vs. IL-1beta.

 
To investigate the potential mechanisms of these phenomena, we investigated effects of sirolimus on the gene expression of lipoprotein receptors that mediate cholesterol uptake. Sirolimus significantly reduced VLDLr and LDLr gene expression in VSMCs, even in the presence of IL-1beta (Fig. 2A). We also checked gene expression of type A scavenger receptor CD36; however, we found no significant changes (results not shown). The influence of sirolimus on the protein expression of VLDLr and LDLr was examined by Western blotting. Sirolimus reduced VLDLr and LDLr protein expression, especially in the presence of IL-1beta, which is consistent with the mRNA results (Fig. 2, B and C).


Figure 2
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  		Fig. 2. Effect of sirolimus on the mRNA and protein expression of lipoprotein receptors in VSMCs. VSMCs were incubated in serum-free medium (control) or serum-free medium with 10–100 ng/ml of sirolimus (Sir10, Sir100) or 5 ng/ml of IL-1beta (IL-1beta) or 5 ng/ml of IL-1beta plus 10–100 ng/ml of sirolimus (IL-1beta + Sir10, IL-1beta + Sir100) for 24 h. The mRNA expression of very LDL receptor (VLDLr) and LDL receptor (LDLr) was determined following the {Delta}{Delta} threshold cycle (Ct) protocol for real time RT-PCR as described in MATERIALS AND METHODS. beta-Actin served as the housekeeper gene. Results represent the means ± SD from 4 experiments (A). The protein levels of VLDLr and LDLr were examined by Western blotting (B). Histogram represent means ± SD of the densitometric scans of the VLDLr and LDLr protein bands from four experiments, normalized by comparison with actin, and expressed as a percentage of control and protein (C). *P <0.05 vs. control, **P < 0.01 vs. control, ***P < 0.01 vs. IL-1beta group.

 
We also examined the effects of sirolimus on intracellular cholesterol efflux under inflammatory stress. We found that IL-1beta (5 ng/ml) reduces ApoA1-mediated cholesterol efflux from lipid-loaded VSMCs. However, sirolimus (10 ng/ml, 100 ng/ml) can restore the suppression induced by inflammation (Fig. 3). Since ABCA1 and ABCG1 are the major players in mediating intracellular cholesterol efflux, we evaluated the effect of sirolimus on ABCA1 and ABCG1 mRNA expression. We found that sirolimus increased both ABCA1 and ABCG1 gene expression in lipid-loaded VSMCs even in the presence of IL-1beta, which significantly reduced ABCA1 mRNA steady-state level (Fig. 4, A and B). These results suggest that sirolimus restores the suppression of ABCA1 and ABCG1 pathways induced by IL-1beta.


Figure 3
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  		Fig. 3. Effect of sirolimus on the intracellular cholesterol efflux of VSMCs loaded with lipid. VSMCs were preloaded with cholesterol as described in MATERIALS AND METHODS. The cholesterol-loaded cells were incubated in serum-free medium (control) or serum-free medium with 10 or 100 ng/ml of sirolimus or 5 ng/ml of IL-1beta, or IL-1beta (5 ng/ml) plus sirolimus (10 ng/ml) or IL-1beta (5 ng/ml) plus sirolimus (100 ng/ml) for 24 h. The cholesterol efflux was measured. Data represent the means ± SD of 4 independent experiments. *P < 0.01 vs. control, **P < 0.01 vs. IL-1beta group.

 

Figure 4
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  		Fig. 4. Effect of airolimus on the mRNA expression of ATP binding cassettes [ABCA1 (A) and ABCG1 (B)] in lipid-loaded VSMCs. VSMCs were loaded with 30 µg/ml cholesterol and 1 µg/ml 25-hydroxy-cholesterol in serum-free medium for 48 h. After that, VSMCs were incubated in serum-free medium (control) or serum-free medium with 10–100 ng/ml of sirolimus (Sir10, Sir 100) or 5 ng/ml of IL-1beta (IL-1beta), or 5 ng/ml of IL-1beta plus 10–100 ng/ml of sirolimus (IL-1beta + Sir10, IL-1beta + Sir100) for 24 h. The mRNA expression of ABCA1 and ABCG1 was determined following the {Delta}{Delta}Ct protocol for real-time RT-PCR as described in MATERIALS AND METHODS. beta-Actin served as the housekeeper gene. Results represent the means ± SD from 4 experiments. *P < 0.001 vs. control, **P < 0.001 vs. IL-1beta group.

 
Interestingly, sirolimus also inhibited both mRNA (Fig. 5, A and B) and protein (Fig. 6, A–C) expression of inflammatory cytokines in non-lipid-loaded and lipid-loaded VSMCs, suggesting that sirolimus also decreases lipid accumulation by inhibiting inflammatory mediators. All of our results suggest that sirolimus may play an important role in the maintenance of intracellular cholesterol homeostasis of VSMCs by several different mechanisms.


Figure 5
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  		Fig. 5. Effect of sirolimus on the gene expression of inflammatory cytokines in VSMCs without (A) or with lipid loading (B). VSMCs were preloaded with cholesterol as described in MATERIALS AND METHODS. Both normal and cholesterol-loaded VSMCs were incubated in serum-free medium (control) or serum-free medium with different concentrations (10–100 ng/ml) of sirolimus (Sir10 and Sir100) or 5 ng/ml of IL-1beta (IL-1beta) or 5 ng/ml of IL-1beta plus different concentrations of sirolimus (IL-1beta + Sir10, IL-1 beta+ Sir100) for 24 h. The mRNA of IL-6, tumor necrosis factor (TNF)-{alpha}, IL-8, and monocyte chemoattractant protein-1 (MCP-1) were determined following the {Delta}{Delta}Ct protocol for real-time RT-PCR as described in MATERIALS AND METHODS. beta-Actin served as the housekeeper gene. Results represent the means ± SD from 4 experiments. *P <0.01 vs. control, **P < 0.01 vs. IL-1beta group.

 

Figure 6
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  		Fig. 6. Effect of sirolimus on the protein productions of inflammatory cytokines in VSMCs without or with lipid loading. VSMCs were preloaded with cholesterol as described in MATERIALS AND METHODS. Both normal and cholesterol-loaded VSMCs were incubated in serum-free medium (control) or serum-free medium with different concentrations (10–100 ng/ml) of sirolimus (Sir10 and Sir100) or 5 ng/ml of IL-1beta (IL-1beta) or 5 ng/ml of IL-1beta plus different concentrations of sirolimus (IL-1beta + Sir10, IL-1beta + Sir100) for 24 h. Supernatants were collected and assayed for IL-6 (A), IL-8 (B), and MCP-1 (C) as described in MATERIALS AND METHODS. Results are expressed as means ± SD of 4 independent experiments. *P < 0.05 vs. control, **P < 0.001 vs. control, ***P < 0.05 vs. IL-1beta group, ****P < 0.001 vs. IL-1beta group.

 

   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
Activation of immune cells, excessive VSMC proliferation, and migration within the arterial wall are thought to play important role in neointimal thickening in both experimental animals models and humans (11). Studies have demonstrated that sirolimus attenuated neointimal thickening in some animal models of alloimmune and mechanical injury (3, 5). Moreover, sirolimus reduced human coronary in-stent restenosis (15, 39). Sirolimus also can directly inhibit the VSMC proliferation and migration that contribute to atherosclerosis (29). Remarkably, sirolimus significantly reduced aortic cholesterol content (1) and atherosclerotic lesion size (3, 7, 28) in fat-fed apoE-null mice. Despite the demonstrated atheroprotective effect of sirolimus in ApoE-null mice, caution must be exercised regarding its possible clinical use in patients experiencing atherosclerosis, since several studies have shown that sirolimus can exacerbate preexisting hypercholesterolemia and hypertriglyceridemia in humans (13, 19, 21). In this study, we examined the effect of sirolimus on intracellular cholesterol homeostasis in the presence of inflammatory cytokine IL-1beta and determined whether this compound reduces intracellular lipid accumulation and foam cell formation in the human coronary artery smooth muscle cell, one of the main cells in atherosclerotic lesions.

IL-1beta is a prototypic proinflammatory cytokine that plays an important role in vascular inflammation and the pathogenesis of atherosclerosis. IL-1beta can be synthesized by macrophages, endothelial cells, and VSMCs, and the levels IL-1beta in the vessel wall are also elevated in atherosclerosis (8, 17, 24). It is well-known that IL-1beta induces the production of cytokines and chemokines and increases the expression of adhesion molecules on endothelial cells, thus leading to the recruitment of inflammatory cells. In addition, IL-1beta contributes to the development of tissue damage by stimulating cell proliferation and the release of matrix metalloproteases (18) leading to destabilization/and finally rupture of vulnerable atherosclerotic plaques. At a clinical level, circulating cytokines have a prognostic role since they are useful markers for predicting future coronary events in patients with advanced atherosclerosis and in patients after acute coronary syndromes (42). More recently, it has been demonstrated that overexpression of sIL-1Ra, a natural inhibitor of IL-1, can inhibit the development of atherosclerotic lesions in ApoE–/– mice. By crossing ApoE–/– mice with IL-1Ra–/– mice, this doubly deficient model exhibited a severe form of aortic inflammation with massive infiltration of macrophages in the adventitia, lipid accumulation in macrophages, and marked destruction of the elastic lamina. However, ApoE–/– IL-1Ra+/+ developed atherosclerosis, whereas ApoE+/+ IL-1Ra –/– had no signs of atherosclerosis or vascular inflammation (18).

We demonstrated that sirolimus reduced lipid droplets accumulation and cholesterol ester content in VSMCs caused by IL-1beta. We have demonstrated previously that IL-1beta induces cholesterol esterification by increasing acyl-CoA:cholesterol acyl-transferase (ACAT) activity in VSMCs (35). Taken together, these findings suggest that sirolimus may inhibit IL-1beta-induced cholesterol esterification.

We have also demonstrated that IL-1beta increased VLDL receptor and LDL receptor mRNA and protein expression in VSMCs, suggesting that inflammatory cytokine IL-1beta disrupted cholesterol homeostasis by increasing receptor-mediated cholesterol uptake. However, sirolimus decreased intracellular lipid accumulation of VSMCs by reducing mRNA and protein expression of VLDLr and LDLr even in the presence of IL-1beta. Our results also suggest that sirolimus increases intracellular cholesterol efflux by upregulating ABCA1 and ABCG1 gene expression in the presence of IL-1beta. Importantly, sirolimus overrode the suppression of cholesterol efflux induced by IL-1beta. Thus sirolimus may ameliorate imbalance of intracellular cholesterol homeostasis in VSMCs mediated by inflammatory cytokines, in agreement with the anti-atherosclerotic effect of sirolimus in some animal models and clinical studies of human cardiovascular diseases.

Our results may also partly explain some dose-dependent adverse effects of sirolimus, especially hyperlipidemia with a high plasma VLDL and LDL concentration in transplant patients after sirolimus treatment (13, 21, 22). Sirolimus reduces lipids influx to peripheral cells such as VSMCs by inhibiting receptor-mediated uptake of LDL and VLDL, the carriers of cholesterol and triglyceride, respectively. We have also observed a reduction in VLDL and LDLr in hepatocyte cultures (not published). In addition, some studies have also reported that sirolimus alters the insulin signaling pathway by increasing adipose tissue lipase activity and/or decreasing lipoprotein lipase activity, resulting in increased hepatic synthesis of triglyceride, increased secretion of VLDL, and increased hypertriglyceridemia (23). However, sirolimus also significantly increased cholesterol efflux from the cells through the ABCA1 and ABCG1 pathways as demonstrated here, which may reduce intracellular cholesterol level and increase HDL formation, which protects against atherosclerosis. Our in vitro studies demonstrate that sirolimus reduces intracellular cholesterol concentration of VSMCs despite its hyperlipidemic potential.

Inflammation is a risk factor for atherosclerosis (32). We assumed that sirolimus inhibits inflammatory processes directly. Therefore, we examined the effect of sirolimus on the inflammatory cytokine excretion of VSMC by using IL-1beta as stimulator. Our results showed that sirolimus directly inhibited mRNA expression of inflammatory cytokines IL-6, TNF-{alpha}, IL-8, and MCP-1. The reduction of IL-6, IL-8, and MCP-1 protein levels was confirmed by ELISA assay (note: TNF-{alpha} level was too low to be measured in the supernatants of VSMC cultures). Some previous studies have also demonstrated effects of sirolimus on the expression and excretion of inflammatory cytokines and chemokines in mesangial cells and in a macrophage cell line (43), as well as in transplant models (27, 44).

It is clear that VSMCs in mature, normal blood vessels exhibit a differentiated, quiescent, contractile morphology, but injury induces a phenotypic modulation toward a proliferative, dedifferentiated, migratory phenotype with upregulated extracellular matrix protein synthesis (synthetic phenotype), which contributes to intimal hyperplasia. Martin et al. (17a) have demonstrated that sirolimus treatment induces differentiation in cultured synthetic phenotype VSMCs from multiple species. VSMCs treated with sirolimus assumed a contractile morphology, quantitatively reflected by a 67% decrease in cell area. Total protein and collagen synthesis were also inhibited by sirolimus. Sirolimus induced expression of the VSMCs differentiation marker contractile proteins smooth muscle (SM) {alpha}-actin, calponin, and SM-myosin heavy chain (SM-MHC), as observed by immunoblotting and immunohistochemistry. The interaction among mammalian target of Rapamycin pathway, cell phenotype, and cholesterol hemostasis need to be investigated.

In summary, our results suggest that inflammatory cytokines can cause lipid accumulation by disrupting cholesterol homeostasis in VSMCs. Sirolimus prevents this lipid accumulation by 1) inhibiting gene and protein expression of VLDLr and LDLr; 2) upregulating ABCA1 and ABCG1 gene expression thereby overriding the reduction in cholesterol efflux induced by IL-1beta; and 3) reducing inflammatory cytokine production, which may provide an additional mechanism for the anti-atherosclerotic effect of this compound.


   GRANTS
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
This work was supported by the Royal Free Hospital Special Trustees Grant 115 through Z. Varghese.


   ACKNOWLEDGMENTS
 
We thank Wyeth Pharmaceutics for providing sirolimus for the experiments.


   FOOTNOTES
 

Address for reprint requests and other correspondence: Z. Varghese, Centre for Nephrology, Royal Free & Univ. College Medical School, Univ. College London, Royal Free campus, Rowland Hill St., London NW3 2PF, UK (e-mail: z.varghese{at}medsch.ucl.ac.uk)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


   REFERENCES
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 

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