Effects of sirolimus on mesangial cell cholesterol homeostasis: a novel mechanism for its action against lipid-mediated injury in renal allo
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《美国生理学杂志》
Centre for Nephrology, Royal Free and University College Medical School, London, United Kingdom
ABSTRACT
Lipoprotein abnormalities are present in a high proportion of renal transplant patients. It is accepted that dyslipidemia is associated with atherosclerosis and in the progression of renal disease. Lipid abnormalities may also play a significant role in the development of chronic allograft nephropathy. Sirolimus was found to have an antiatherosclerotic effect in the apolipoprotein E-knockout mice model of hyperlipidemia through its antiproliferative effects. As lipid-mediated renal injury is important in the pathogenesis of glomerulosclerosis which shares common pathogenic mechanisms with atherosclerosis, in this study we have tested the hypothesis that sirolimus prevents lipid-mediated renal injury through the modulation of cholesterol homeostasis of mesangial cells and its anti-inflammatory effects on macrophages. We demonstrated that sirolimus reduced lipid accumulation, as measured by oil red O staining in human mesangial cells (HMCs). Using real-time PCR, we screened the mRNA expression of lipoprotein receptors. Sirolimus significantly suppressed LDL and VLDL receptors and CD36 gene expression. It also increased cholesterol efflux from HMCs by increasing peroxisome proliferator-activated receptor- (PPAR), PPAR, liver X receptor-, and ATP binding cassette A1 (ABCA1) gene expression. Sirolimus overrode the suppression of cholesterol efflux and ABCA1 gene expression induced by the inflammatory cytokine IL-1. Furthermore, sirolimus significantly inhibited inflammatory cytokines IL-6 and TNF- production in macrophages. These data suggest that sirolimus may prevent cellular cholesterol accumulation even in the presence of hyperlipidemia and inflammation, by regulating both cholesterol homeostasis and inflammatory responses.
human mesangial cells; inflammatory cytokine; foam cell
CHRONIC ALLOGRAFT NEPHROPATHY (CAN) is the most important cause of renal allograft loss after the first posttransplant year. Clinically, it is characterized by a slow decline of renal function with an increase in plasma creatinine, proteinuria, hypertension, and hyperlipidemia. It is becoming apparent that both alloantigen-dependent and -independent factors may combine to activate the cellular and molecular mediators of tissue injury, repair, and remodeling. The cellular components comprise vascular smooth muscle cells and inflammatory cells, including T cells, monocytes, and macrophages. A characteristic histopathological manifestation of CAN is the graft vascular lesion known as transplant-associated arteriosclerosis. This lesion is characterized by a diffuse concentric intimal thickening of graft arteries. There is a high prevalence of lipoprotein abnormalities in renal transplant patients, which is associated with atherosclerosis and a decline in renal function. Dyslipidemia may also play a significant role in the development CAN (29). In addition, death with a functioning graft is now the commonest cause of allograft loss, accounting for 42% of allograft losses (17), to which cardiovascular disease is the biggest contributory factor. Several pretransplant cardiovascular risk factors such as hyperlipidemia, hypertension, and diabetes have been found to correlate with the high incidence of cardiovascular disease events in this population. Although it was thought that dyslipidemia can be a necessary and sufficient cause for premature atherosclerosis, a large body of evidence has identified the importance of inflammation in modifying lipid-mediated vascular and renal injury (3).
Atherosclerosis is now recognized as a chronic inflammatory disease (4). Inflammation, lipid accumulation, and foam cell formation are characteristic features of the earliest lesion of atherosclerosis, the so-called fatty streak involving vascular smooth muscle cells and macrophages. Lipid-mediated renal injury, causing the progression of renal disease, was first suggested by our group (15). We proposed the term "glomerular atherosclerosis" on the basis that atherosclerosis and glomerulosclerosis share common pathogenic mechanisms. Others confirmed our findings that lipid-mediated renal injury is an important component of glomerulosclerosis (11). In addition, glomerular foam cells are seen in renal allografts (30). In most cells, intracellular cholesterol accumulation is prevented by tight regulation of influx and efflux pathways (3). Conventionally, foam cells are thought to be derived predominantly from macrophages. However, other cell types, such as smooth muscle cells and human mesangial cells (HMCs) can be converted into foam cells. Many lipoprotein receptors, such as the low-density lipoprotein receptor (LDLr), the type A scavenger receptor (Scr), and the very-low-density lipoprotein receptor (VLDLr) mediate cholesterol uptake. Unlike the LDLr, rising intracellular cholesterol concentration does not suppress Scr, thus providing a mechanism for unregulated cholesterol uptake. We demonstrated that HMCs express an inducible form of Scr by which cells can acquire lipids and convert to foam cells in developing glomerulosclerosis (23). The LDLr is the primary receptor for binding and internalization of plasma-derived LDL and regulates plasma LDL concentration (8). Brown and Goldstein (1) observed that intracellular cholesterol concentration tightly regulates LDLr activity through a feedback system (1), which protects cells from native LDL accumulation. Therefore, native LDL is ineffective in generating lipid-rich foam cells under physiological conditions. However, our previous studies indicated that inflammatory mediators caused dysregulation of LDLr expression (25). Because intracellular lipid content is governed by both influx and efflux, we have also shown that inflammatory cytokine IL-1 reduced cholesterol efflux, a process mediated by the protein ATP binding cassette A1 (ABCA1) (20).
Several lines of evidence suggest that sirolimus may attenuate atherosclerosis. In clinical application, the sirolimus-eluting coronary stent has been demonstrated to significantly decrease restenosis rates (18). Elloso et al. (6) demonstrated a more specific antiatherosclerotic effect of sirolimus in the apolipoprotein (apo) E knockout mouse model of hyperlipidemia. In a more recent study, it has been shown that sirolimus elicits a marked reduction of aortic atherosclerosis in apo E-null mice fed a high-fat diet, despite sustained hypercholesterolemia. Furthermore, this attenuation of atherosclerosis was thought to be through a p27 kip1-independent pathway, resulting in the reduced expression of positive cell cycle regulators and MCP-1 within the arterial wall (5). It is well established that mesangial cells are closely related to vascular smooth muscle cells and have both LDL and Scrs (23, 25). This study was designed to determine whether sirolimus could prevent lipid accumulation in mesangial cells and to investigate the effect of sirolimus on cellular cholesterol homeostasis and inflammation.
MATERIALS AND METHODS
Cell culture. A stable line of HMCs that had been immortalized by transfection with T-SV40 and H-ras oncogenes was used in all experiments (kindly supplied by Dr. J. D. Sraer, Hpital Tenon, Paris, France). These cells have been fully characterized and retain most of the morphological and physiological features of nontransfected mesangial cells (24, 25, 27). The cells were cultured in growth medium comprising RPMI 1640 supplemented with 5% fetal calf serum (FCS), 2 mmol/l glutamine, 100 U/ml penicillin, 100 μg/ml streptomycin, 2.5 μg/ml amphotericin, 5 μg/ml insulin, 5 μg/ml human transferrin, and 5 ng/ml sodium selenite. Experiments were carried out in serum-free RPMI 1640 medium containing 0.2% BSA (Sigma, Poole, Dorset, UK). All reagents for cell culture were obtained from GIBCO BRL (Paisley, UK). Recombinant interleukin-1 (IL-1; 1.0–3.3 x 108 U/mg) was obtained from R&D Systems (Abingdon, UK). Sirolimus (rapamycin) was supplied by Wyeth Pharmaceutics.
Preparation of lipoprotein. LDL was isolated by sequential ultracentrifugation as described previously (21) and stored at 4°C in the presence of antioxidants [100 μmol/l of EDTA and 20 μmol/l of butylated hydroxytoluene (BHT)].
Morphological examination and quantification of lipid accumulation. HMCs were plated in chamber slides for tissue culture (Nunc, Naperville, IL) and incubated in serum-free RPMI 1640 medium with 200 μg/ml of native LDL or 5 μg/ml of IL-1 plus native LDL in the absence or presence of 10 or 100 ng/ml sirolimus. After 24-h incubation, the cells were washed three times with 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. HMCs were plated in 12-well plates (Nunc). The cells then were incubated in serum-free RPMI 1640 medium with 200 μg/ml of native LDL or 5 μg/ml of IL-1 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. (7). In brief, the cells were collected and washed twice with PBS; lipids were extracted by the addition of 1 ml chloroform/methanol (2:1) to the cell pellet. After sonification, the sample was centrifuged and the lipid phase was collected. Then, 0.5 ml of 0.9% NaCl was added to the liquid and the lipid layer in the bottom of tube was carefully collected. The sample was then dried in vacuum and samples were dissolved in 95% ethanol. 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 p-hydroxyphenylacetic acid to yield a stable fluorescent product. The concentration of total and free cholesterol per well was analyzed using a standard curve and normalized by measuring the concentration of total cell protein using the Lowry protein assay. The concentration of cholesterol ester was calculated using total cholesterol minus free cholesterol.
RT-PCR. Total RNA was isolated from HMCs treated with sirolimus as described later and used as a template for RT-PCR using an RNA PCR kit from ABI (Applied Biosystems, Warrington, Cheshire, UK). The RT reaction was set up in a 20-μl mixture containing 50 mmol/l KCl, 10 mmol/l Tris·HCl, 5 mmol/l MgCl2, 1 mmol/l of each dNTP, 2.5 μmol/l random hexamers, 20 U RNAsin, and 50 U of Moloney murine leukemia virus reverse transcriptase. Incubations were performed in a DNA Thermal Cycler (PerkinElmer 9700) for 10 min at room temperature, followed by 30 min at 42°C and 5 min at 99°C. After cDNA synthesis by RT, real-time quantitative PCR was performed on a TaqMan ABI 7000 Sequence Detection System (Applied Biosystems, Foster City, CA) using TaqMan universal PCR Master Mix (Applied Biosystems, Branchburg, NJ) with specific primers of target genes (Table 1). Thermal cycler conditions contained holds for 2 min at 50°C and 10 min at 95°C, followed by 40 cycles of 20 s at 95°C, 20 s at 55°C, and 30 s at 72°C. The relative amount of mRNA was calculated using comparative CT method (CT). -Actin served as the reference housekeeping gene. 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.
View this table:
Cholesterol loading and cholesterol efflux assay. The human THP-1 cell line was incubated with 160 nmol/l of PMA for 5 days to differentiate cells into macrophages. The differentiated THP-1 cell and HMCs were incubated with medium that contained RPMI 1640, 30 μg/ml of cholesterol, 1 μg/ml of 25-hydroxycholesterol (25-HC, Sigma), and 1 μl of [3H]cholesterol (1 μCi/μl, Amersham, Little Chalfont, Bucks, UK). After 48 h, fresh serum-free medium containing sirolimus was added in the presence or absence of IL-1 (5 ng/ml) for a further 24 h. After this incubation period, cells were washed twice in PBS and apo A1-mediated cholesterol efflux studies were immediately performed by adding fresh serum-free RPMI 1640 medium with or without 15 μg/ml of apo A1 (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. The radioactivity in both the supernatant and cellular lipid was measured by scintillation counting. Apo A1-induced [3H]cholesterol efflux was calculated by subtracting the radioactivity in supernatants without apo A from the counts in supernatants containing apo A. The data were normalized by determining total [3H]cholesterol radioactivity (including radioactivity in supernatants and cells) and were expressed as a percentage of control.
Proinflammatory cytokine production. We studied proinflammatory cytokine production in THP-1 cell lines. THP-1 cells were differentiated by incubating with PMA (125 nmol/l) for 5 days. The cells then were incubated in serum-free medium containing different concentration of sirolimus in the presence or absence of 10 μg/ml LPS for 24 h. Supernatants were collected and assayed for IL-6 and TNF- using protocols supplied by the manufacturer (R&D Systems) and normalized to cell protein concentrations.
Data analysis. In all experiments, data were evaluated for significance by one-way ANOVA using Minitab software. Data were considered significant at P < 0.05.
RESULTS
Our study demonstrated that at concentrations of 10–100 ng/ml sirolimus reduced lipid droplet accumulation in HMCs in the presence of the inflammatory cytokine IL-1 (Fig. 1). Quantitative intracellular cholesterol analysis confirmed that sirolimus reduced cholesterol ester accumulation induced by IL-1 in HMCs, suggesting that sirolimus may inhibit IL-1-induced cholesterol esterification (Fig. 2). To examine the effect of sirolimus on the gene expression involved in lipid uptake, we screened the mRNA expression of lipoprotein receptors under the influence of IL-1 and sirolimus. Sirolimus suppressed LDLr, VLDLr, and CD36 gene expression and prevented induction of LDLr, VLDLr genes induced by IL-1 (Fig. 3). Because intracellular lipid content is governed by both influx and efflux mechanisms, the balance between lipid uptake by lipoprotein receptors and cholesterol efflux mechanisms is important. We investigated the effect of sirolimus on cholesterol efflux in both HMCs and THP-1 macrophages. Sirolimus overrode the suppression of cholesterol efflux induced by IL-1 (Fig. 4). Furthermore, we examined the PPAR-LXR-ABCA1 pathway, which has previously been demonstrated to control ABCA1 transcription. Sirolimus increased ABCA1, LXR, and PPAR gene expression and overrode the suppression of those genes induced by IL-1 in HMCs (Fig. 5). Production of inflammatory mediators by macrophages is thought to be an important factor in the glomerular atherosclerotic process; we therefore investigated the effect of sirolimus on anti-inflammation in culture of the human THP-1 cell line. Sirolimus significantly inhibited the production of the proinflammatory cytokine TNF- and IL-6 induced by LPS in THP-1 cells (Figs. 6 and 7).
DISCUSSION
Sirolimus is a macrocyclic natural product that possesses potent immunosuppressive activity (26). Like other immunosuppressive drugs, sirolimus inhibits T cell proliferation (4). It expresses its immunosuppressive and antiproliferative activities through inhibition of the kinase activity of the mammalian target of rapamycin (mTOR) and regulation of the cyclin-dependent kinase inhibitor p27kipl (4, 9, 13, 16). The migration and proliferation of SMC in vessel wall are critical events in the progression of atherosclerosis (19). One of mechanisms of the antiatherosclerotic effect of sirolimus is through inhibition of cell proliferation (14). However, it has also been shown that the beneficial effects of sirolimus on the vascular wall were independent of inhibition of cell proliferation mediated by the p27kip1 pathway (5). Because the cellular cholesterol content of the macrophages in apo E knockout mice treated with sirolimus appears to be much lower than that of control animals, as evidenced by both morphology and oil red O staining (6), sirolimus may affect local lipid homeostasis within the vascular wall and kidney.
In the present study, we have investigated the effect of sirolimus on cholesterol homeostasis in HMCs. The generally accepted mechanism of foam cell formation involves uptake of modified (usually oxidized) LDL via the Scr family (2). CD36 has been identified as a type B Scr that may be the main receptor for modified LDL uptake in HMCs. However, sirolimus reduced CD36 gene expression, which may reduce modified LDL accumulation in cells. LDLr is the main lipoprotein receptor in HMCs. Unmodified lipoprotein is internalized via the LDLr. The expression of LDLr is tightly regulated by intracellular cholesterol concentration, thereby protecting cells from lipid accumulation (8). We have previously demonstrated that inflammatory mediators could disrupt LDLr feedback regulation and make native LDL accumulation occur in HMCs, suggesting that native LDL may be pathogenic under inflammatory stress (25). In this study, we have demonstrated that sirolimus inhibited LDLr gene expression, which significantly reduced native LDL accumulation induced by IL-1. Sirolimus also reduced VLDLr expression in HMCs. These results suggest that sirolimus may decrease lipid uptake by reducing LDLr, VLDLr, and CD36 gene expression under inflammatory stress.
Because intracellular lipid content is governed by both influx and efflux mechanisms, the balance between lipid uptake through lipoprotein receptors as described above and cholesterol efflux is important. We have previously demonstrated that the inflammatory cytokine IL-1 inhibits cholesterol efflux by inhibiting PPAR-LXRa-ABCA1 pathways (20). The present results show that sirolimus significantly increased PPAR, PPAR, LXR, and ABCA1 gene expression and also enhanced ABCA1-mediated cholesterol efflux in the presence of IL-1. This suggests that sirolimus may prevent lipid accumulation by both reducing cholesterol uptake and increasing cholesterol efflux pathways. This may explain why sirolimus treatment was associated with a 30%, dose-dependent, elevation in HDL-cholesterol reported by Elloso et al. (6). Interestingly, the ability of sirolimus to increase cellular cholesterol efflux is also observed in cholesterol-loaded macrophages in this study.
Inflammation acts as a partner with hypercholesterolemia during the development of atherosclerosis and glomerulosclerosis (22, 28). It is a feature of many chronic progressive renal diseases and is evidenced histologically by the accumulation of macrophages, cholesterol, and cholesteryl esters in sclerotic glomeruli (10). It has been recognized that inflammatory cells infiltrate the glomeruli, and macrophages play an important pathogenic role in the formation of foam cells. Therefore, we examined whether sirolimus has anti-inflammatory effects in a cultured macrophage cell line. Our results showed that sirolimus significantly inhibited the production of the inflammatory cytokines TNF- and IL-6 in THP-1 cells. Although clinical studies in renal transplant patients have shown that sirolimus-treated patients suffered more frequently from hyperlipidemia, these patients had better renal function compared with patients maintained on calcineurin-inhibiting drugs, which may also be a reflection of its anti-inflammatory effect in modifying a lipid-mediated renal injury (12).
In summary, sirolimus prevented lipid accumulation in HMCs by reducing lipid uptake and increasing cholesterol efflux. In addition, it also inhibited proinflammatory cytokine (TNF- and IL-6) production. These results may provide additional explanations for the quantitative reduction in atherosclerosis plaque formation that has been observed in sirolimus-treated apo E knockout mice. Therefore, in addition to its antiproliferative effects, sirolimus may prevent atherosclerosis and CAN by dual actions on cholesterol homeostasis and inhibition of inflammation.
GRANTS
This work was supported by Royal Free Hospital Special Trustees Grant 115 and by Wyeth Pharmaceutics.
DISCLOSURES
None of the authors has a financial interest in Wyeth Pharmaceutics.
ACKNOWLEDGMENTS
We thank Professor J. D. Sraer (Hpital Tenon, Paris, France) for the kind gift of the immortalized human mesangial cell line and Wyeth Pharmaceutics for providing sirolimus for the experiments. We also thank Professor Fu Zhou for technical help with the intracellular cholesterol assay.
FOOTNOTES
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.
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ABSTRACT
Lipoprotein abnormalities are present in a high proportion of renal transplant patients. It is accepted that dyslipidemia is associated with atherosclerosis and in the progression of renal disease. Lipid abnormalities may also play a significant role in the development of chronic allograft nephropathy. Sirolimus was found to have an antiatherosclerotic effect in the apolipoprotein E-knockout mice model of hyperlipidemia through its antiproliferative effects. As lipid-mediated renal injury is important in the pathogenesis of glomerulosclerosis which shares common pathogenic mechanisms with atherosclerosis, in this study we have tested the hypothesis that sirolimus prevents lipid-mediated renal injury through the modulation of cholesterol homeostasis of mesangial cells and its anti-inflammatory effects on macrophages. We demonstrated that sirolimus reduced lipid accumulation, as measured by oil red O staining in human mesangial cells (HMCs). Using real-time PCR, we screened the mRNA expression of lipoprotein receptors. Sirolimus significantly suppressed LDL and VLDL receptors and CD36 gene expression. It also increased cholesterol efflux from HMCs by increasing peroxisome proliferator-activated receptor- (PPAR), PPAR, liver X receptor-, and ATP binding cassette A1 (ABCA1) gene expression. Sirolimus overrode the suppression of cholesterol efflux and ABCA1 gene expression induced by the inflammatory cytokine IL-1. Furthermore, sirolimus significantly inhibited inflammatory cytokines IL-6 and TNF- production in macrophages. These data suggest that sirolimus may prevent cellular cholesterol accumulation even in the presence of hyperlipidemia and inflammation, by regulating both cholesterol homeostasis and inflammatory responses.
human mesangial cells; inflammatory cytokine; foam cell
CHRONIC ALLOGRAFT NEPHROPATHY (CAN) is the most important cause of renal allograft loss after the first posttransplant year. Clinically, it is characterized by a slow decline of renal function with an increase in plasma creatinine, proteinuria, hypertension, and hyperlipidemia. It is becoming apparent that both alloantigen-dependent and -independent factors may combine to activate the cellular and molecular mediators of tissue injury, repair, and remodeling. The cellular components comprise vascular smooth muscle cells and inflammatory cells, including T cells, monocytes, and macrophages. A characteristic histopathological manifestation of CAN is the graft vascular lesion known as transplant-associated arteriosclerosis. This lesion is characterized by a diffuse concentric intimal thickening of graft arteries. There is a high prevalence of lipoprotein abnormalities in renal transplant patients, which is associated with atherosclerosis and a decline in renal function. Dyslipidemia may also play a significant role in the development CAN (29). In addition, death with a functioning graft is now the commonest cause of allograft loss, accounting for 42% of allograft losses (17), to which cardiovascular disease is the biggest contributory factor. Several pretransplant cardiovascular risk factors such as hyperlipidemia, hypertension, and diabetes have been found to correlate with the high incidence of cardiovascular disease events in this population. Although it was thought that dyslipidemia can be a necessary and sufficient cause for premature atherosclerosis, a large body of evidence has identified the importance of inflammation in modifying lipid-mediated vascular and renal injury (3).
Atherosclerosis is now recognized as a chronic inflammatory disease (4). Inflammation, lipid accumulation, and foam cell formation are characteristic features of the earliest lesion of atherosclerosis, the so-called fatty streak involving vascular smooth muscle cells and macrophages. Lipid-mediated renal injury, causing the progression of renal disease, was first suggested by our group (15). We proposed the term "glomerular atherosclerosis" on the basis that atherosclerosis and glomerulosclerosis share common pathogenic mechanisms. Others confirmed our findings that lipid-mediated renal injury is an important component of glomerulosclerosis (11). In addition, glomerular foam cells are seen in renal allografts (30). In most cells, intracellular cholesterol accumulation is prevented by tight regulation of influx and efflux pathways (3). Conventionally, foam cells are thought to be derived predominantly from macrophages. However, other cell types, such as smooth muscle cells and human mesangial cells (HMCs) can be converted into foam cells. Many lipoprotein receptors, such as the low-density lipoprotein receptor (LDLr), the type A scavenger receptor (Scr), and the very-low-density lipoprotein receptor (VLDLr) mediate cholesterol uptake. Unlike the LDLr, rising intracellular cholesterol concentration does not suppress Scr, thus providing a mechanism for unregulated cholesterol uptake. We demonstrated that HMCs express an inducible form of Scr by which cells can acquire lipids and convert to foam cells in developing glomerulosclerosis (23). The LDLr is the primary receptor for binding and internalization of plasma-derived LDL and regulates plasma LDL concentration (8). Brown and Goldstein (1) observed that intracellular cholesterol concentration tightly regulates LDLr activity through a feedback system (1), which protects cells from native LDL accumulation. Therefore, native LDL is ineffective in generating lipid-rich foam cells under physiological conditions. However, our previous studies indicated that inflammatory mediators caused dysregulation of LDLr expression (25). Because intracellular lipid content is governed by both influx and efflux, we have also shown that inflammatory cytokine IL-1 reduced cholesterol efflux, a process mediated by the protein ATP binding cassette A1 (ABCA1) (20).
Several lines of evidence suggest that sirolimus may attenuate atherosclerosis. In clinical application, the sirolimus-eluting coronary stent has been demonstrated to significantly decrease restenosis rates (18). Elloso et al. (6) demonstrated a more specific antiatherosclerotic effect of sirolimus in the apolipoprotein (apo) E knockout mouse model of hyperlipidemia. In a more recent study, it has been shown that sirolimus elicits a marked reduction of aortic atherosclerosis in apo E-null mice fed a high-fat diet, despite sustained hypercholesterolemia. Furthermore, this attenuation of atherosclerosis was thought to be through a p27 kip1-independent pathway, resulting in the reduced expression of positive cell cycle regulators and MCP-1 within the arterial wall (5). It is well established that mesangial cells are closely related to vascular smooth muscle cells and have both LDL and Scrs (23, 25). This study was designed to determine whether sirolimus could prevent lipid accumulation in mesangial cells and to investigate the effect of sirolimus on cellular cholesterol homeostasis and inflammation.
MATERIALS AND METHODS
Cell culture. A stable line of HMCs that had been immortalized by transfection with T-SV40 and H-ras oncogenes was used in all experiments (kindly supplied by Dr. J. D. Sraer, Hpital Tenon, Paris, France). These cells have been fully characterized and retain most of the morphological and physiological features of nontransfected mesangial cells (24, 25, 27). The cells were cultured in growth medium comprising RPMI 1640 supplemented with 5% fetal calf serum (FCS), 2 mmol/l glutamine, 100 U/ml penicillin, 100 μg/ml streptomycin, 2.5 μg/ml amphotericin, 5 μg/ml insulin, 5 μg/ml human transferrin, and 5 ng/ml sodium selenite. Experiments were carried out in serum-free RPMI 1640 medium containing 0.2% BSA (Sigma, Poole, Dorset, UK). All reagents for cell culture were obtained from GIBCO BRL (Paisley, UK). Recombinant interleukin-1 (IL-1; 1.0–3.3 x 108 U/mg) was obtained from R&D Systems (Abingdon, UK). Sirolimus (rapamycin) was supplied by Wyeth Pharmaceutics.
Preparation of lipoprotein. LDL was isolated by sequential ultracentrifugation as described previously (21) and stored at 4°C in the presence of antioxidants [100 μmol/l of EDTA and 20 μmol/l of butylated hydroxytoluene (BHT)].
Morphological examination and quantification of lipid accumulation. HMCs were plated in chamber slides for tissue culture (Nunc, Naperville, IL) and incubated in serum-free RPMI 1640 medium with 200 μg/ml of native LDL or 5 μg/ml of IL-1 plus native LDL in the absence or presence of 10 or 100 ng/ml sirolimus. After 24-h incubation, the cells were washed three times with 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. HMCs were plated in 12-well plates (Nunc). The cells then were incubated in serum-free RPMI 1640 medium with 200 μg/ml of native LDL or 5 μg/ml of IL-1 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. (7). In brief, the cells were collected and washed twice with PBS; lipids were extracted by the addition of 1 ml chloroform/methanol (2:1) to the cell pellet. After sonification, the sample was centrifuged and the lipid phase was collected. Then, 0.5 ml of 0.9% NaCl was added to the liquid and the lipid layer in the bottom of tube was carefully collected. The sample was then dried in vacuum and samples were dissolved in 95% ethanol. 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 p-hydroxyphenylacetic acid to yield a stable fluorescent product. The concentration of total and free cholesterol per well was analyzed using a standard curve and normalized by measuring the concentration of total cell protein using the Lowry protein assay. The concentration of cholesterol ester was calculated using total cholesterol minus free cholesterol.
RT-PCR. Total RNA was isolated from HMCs treated with sirolimus as described later and used as a template for RT-PCR using an RNA PCR kit from ABI (Applied Biosystems, Warrington, Cheshire, UK). The RT reaction was set up in a 20-μl mixture containing 50 mmol/l KCl, 10 mmol/l Tris·HCl, 5 mmol/l MgCl2, 1 mmol/l of each dNTP, 2.5 μmol/l random hexamers, 20 U RNAsin, and 50 U of Moloney murine leukemia virus reverse transcriptase. Incubations were performed in a DNA Thermal Cycler (PerkinElmer 9700) for 10 min at room temperature, followed by 30 min at 42°C and 5 min at 99°C. After cDNA synthesis by RT, real-time quantitative PCR was performed on a TaqMan ABI 7000 Sequence Detection System (Applied Biosystems, Foster City, CA) using TaqMan universal PCR Master Mix (Applied Biosystems, Branchburg, NJ) with specific primers of target genes (Table 1). Thermal cycler conditions contained holds for 2 min at 50°C and 10 min at 95°C, followed by 40 cycles of 20 s at 95°C, 20 s at 55°C, and 30 s at 72°C. The relative amount of mRNA was calculated using comparative CT method (CT). -Actin served as the reference housekeeping gene. 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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Cholesterol loading and cholesterol efflux assay. The human THP-1 cell line was incubated with 160 nmol/l of PMA for 5 days to differentiate cells into macrophages. The differentiated THP-1 cell and HMCs were incubated with medium that contained RPMI 1640, 30 μg/ml of cholesterol, 1 μg/ml of 25-hydroxycholesterol (25-HC, Sigma), and 1 μl of [3H]cholesterol (1 μCi/μl, Amersham, Little Chalfont, Bucks, UK). After 48 h, fresh serum-free medium containing sirolimus was added in the presence or absence of IL-1 (5 ng/ml) for a further 24 h. After this incubation period, cells were washed twice in PBS and apo A1-mediated cholesterol efflux studies were immediately performed by adding fresh serum-free RPMI 1640 medium with or without 15 μg/ml of apo A1 (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. The radioactivity in both the supernatant and cellular lipid was measured by scintillation counting. Apo A1-induced [3H]cholesterol efflux was calculated by subtracting the radioactivity in supernatants without apo A from the counts in supernatants containing apo A. The data were normalized by determining total [3H]cholesterol radioactivity (including radioactivity in supernatants and cells) and were expressed as a percentage of control.
Proinflammatory cytokine production. We studied proinflammatory cytokine production in THP-1 cell lines. THP-1 cells were differentiated by incubating with PMA (125 nmol/l) for 5 days. The cells then were incubated in serum-free medium containing different concentration of sirolimus in the presence or absence of 10 μg/ml LPS for 24 h. Supernatants were collected and assayed for IL-6 and TNF- using protocols supplied by the manufacturer (R&D Systems) and normalized to cell protein concentrations.
Data analysis. In all experiments, data were evaluated for significance by one-way ANOVA using Minitab software. Data were considered significant at P < 0.05.
RESULTS
Our study demonstrated that at concentrations of 10–100 ng/ml sirolimus reduced lipid droplet accumulation in HMCs in the presence of the inflammatory cytokine IL-1 (Fig. 1). Quantitative intracellular cholesterol analysis confirmed that sirolimus reduced cholesterol ester accumulation induced by IL-1 in HMCs, suggesting that sirolimus may inhibit IL-1-induced cholesterol esterification (Fig. 2). To examine the effect of sirolimus on the gene expression involved in lipid uptake, we screened the mRNA expression of lipoprotein receptors under the influence of IL-1 and sirolimus. Sirolimus suppressed LDLr, VLDLr, and CD36 gene expression and prevented induction of LDLr, VLDLr genes induced by IL-1 (Fig. 3). Because intracellular lipid content is governed by both influx and efflux mechanisms, the balance between lipid uptake by lipoprotein receptors and cholesterol efflux mechanisms is important. We investigated the effect of sirolimus on cholesterol efflux in both HMCs and THP-1 macrophages. Sirolimus overrode the suppression of cholesterol efflux induced by IL-1 (Fig. 4). Furthermore, we examined the PPAR-LXR-ABCA1 pathway, which has previously been demonstrated to control ABCA1 transcription. Sirolimus increased ABCA1, LXR, and PPAR gene expression and overrode the suppression of those genes induced by IL-1 in HMCs (Fig. 5). Production of inflammatory mediators by macrophages is thought to be an important factor in the glomerular atherosclerotic process; we therefore investigated the effect of sirolimus on anti-inflammation in culture of the human THP-1 cell line. Sirolimus significantly inhibited the production of the proinflammatory cytokine TNF- and IL-6 induced by LPS in THP-1 cells (Figs. 6 and 7).
DISCUSSION
Sirolimus is a macrocyclic natural product that possesses potent immunosuppressive activity (26). Like other immunosuppressive drugs, sirolimus inhibits T cell proliferation (4). It expresses its immunosuppressive and antiproliferative activities through inhibition of the kinase activity of the mammalian target of rapamycin (mTOR) and regulation of the cyclin-dependent kinase inhibitor p27kipl (4, 9, 13, 16). The migration and proliferation of SMC in vessel wall are critical events in the progression of atherosclerosis (19). One of mechanisms of the antiatherosclerotic effect of sirolimus is through inhibition of cell proliferation (14). However, it has also been shown that the beneficial effects of sirolimus on the vascular wall were independent of inhibition of cell proliferation mediated by the p27kip1 pathway (5). Because the cellular cholesterol content of the macrophages in apo E knockout mice treated with sirolimus appears to be much lower than that of control animals, as evidenced by both morphology and oil red O staining (6), sirolimus may affect local lipid homeostasis within the vascular wall and kidney.
In the present study, we have investigated the effect of sirolimus on cholesterol homeostasis in HMCs. The generally accepted mechanism of foam cell formation involves uptake of modified (usually oxidized) LDL via the Scr family (2). CD36 has been identified as a type B Scr that may be the main receptor for modified LDL uptake in HMCs. However, sirolimus reduced CD36 gene expression, which may reduce modified LDL accumulation in cells. LDLr is the main lipoprotein receptor in HMCs. Unmodified lipoprotein is internalized via the LDLr. The expression of LDLr is tightly regulated by intracellular cholesterol concentration, thereby protecting cells from lipid accumulation (8). We have previously demonstrated that inflammatory mediators could disrupt LDLr feedback regulation and make native LDL accumulation occur in HMCs, suggesting that native LDL may be pathogenic under inflammatory stress (25). In this study, we have demonstrated that sirolimus inhibited LDLr gene expression, which significantly reduced native LDL accumulation induced by IL-1. Sirolimus also reduced VLDLr expression in HMCs. These results suggest that sirolimus may decrease lipid uptake by reducing LDLr, VLDLr, and CD36 gene expression under inflammatory stress.
Because intracellular lipid content is governed by both influx and efflux mechanisms, the balance between lipid uptake through lipoprotein receptors as described above and cholesterol efflux is important. We have previously demonstrated that the inflammatory cytokine IL-1 inhibits cholesterol efflux by inhibiting PPAR-LXRa-ABCA1 pathways (20). The present results show that sirolimus significantly increased PPAR, PPAR, LXR, and ABCA1 gene expression and also enhanced ABCA1-mediated cholesterol efflux in the presence of IL-1. This suggests that sirolimus may prevent lipid accumulation by both reducing cholesterol uptake and increasing cholesterol efflux pathways. This may explain why sirolimus treatment was associated with a 30%, dose-dependent, elevation in HDL-cholesterol reported by Elloso et al. (6). Interestingly, the ability of sirolimus to increase cellular cholesterol efflux is also observed in cholesterol-loaded macrophages in this study.
Inflammation acts as a partner with hypercholesterolemia during the development of atherosclerosis and glomerulosclerosis (22, 28). It is a feature of many chronic progressive renal diseases and is evidenced histologically by the accumulation of macrophages, cholesterol, and cholesteryl esters in sclerotic glomeruli (10). It has been recognized that inflammatory cells infiltrate the glomeruli, and macrophages play an important pathogenic role in the formation of foam cells. Therefore, we examined whether sirolimus has anti-inflammatory effects in a cultured macrophage cell line. Our results showed that sirolimus significantly inhibited the production of the inflammatory cytokines TNF- and IL-6 in THP-1 cells. Although clinical studies in renal transplant patients have shown that sirolimus-treated patients suffered more frequently from hyperlipidemia, these patients had better renal function compared with patients maintained on calcineurin-inhibiting drugs, which may also be a reflection of its anti-inflammatory effect in modifying a lipid-mediated renal injury (12).
In summary, sirolimus prevented lipid accumulation in HMCs by reducing lipid uptake and increasing cholesterol efflux. In addition, it also inhibited proinflammatory cytokine (TNF- and IL-6) production. These results may provide additional explanations for the quantitative reduction in atherosclerosis plaque formation that has been observed in sirolimus-treated apo E knockout mice. Therefore, in addition to its antiproliferative effects, sirolimus may prevent atherosclerosis and CAN by dual actions on cholesterol homeostasis and inhibition of inflammation.
GRANTS
This work was supported by Royal Free Hospital Special Trustees Grant 115 and by Wyeth Pharmaceutics.
DISCLOSURES
None of the authors has a financial interest in Wyeth Pharmaceutics.
ACKNOWLEDGMENTS
We thank Professor J. D. Sraer (Hpital Tenon, Paris, France) for the kind gift of the immortalized human mesangial cell line and Wyeth Pharmaceutics for providing sirolimus for the experiments. We also thank Professor Fu Zhou for technical help with the intracellular cholesterol assay.
FOOTNOTES
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.
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