Published online before print November 11, 2004, doi: 10.1161/01.RES.0000150368.56660.4f
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
© 2004 American Heart Association, Inc.
UltraRapid Communications |
Liver X Receptor Agonists Suppress Vascular Smooth Muscle Cell Proliferation and Inhibit Neointima Formation in Balloon-Injured Rat Carotid Arteries
From the Division of Endocrinology, Diabetes, and Hypertension (F.B., Y.T., E.C., J.L., W.A.H., R.E.L.) and the Department of Pathology (M.C.F.), David Geffen School of Medicine, University of California, Los Angeles; Department of Medicine/Cardiology (F.B., E.F.), German Heart Institute, Berlin, Germany; Division of Vascular Surgery (O.L.), University Hospital, Uppsala, Sweden; Department of Pathology-Oncology (K.K.), Karolinska Institute, Stockholm, Sweden; Department of Molecular and Cellular Biology (K.I.N.), Kyushu University, Fukuoka, Japan; and Division of Endocrinology and Molecular Medicine (D.B.), University of Kentucky College of Medicine, Lexington. Present address for R.E.L. is Takeda Pharmaceuticals of North America, Lincolnshire, Ill.
Correspondence and requests for reprints to Dennis Bruemmer, MD, University of Kentucky College of Medicine, Department of Internal Medicine, Division of Endocrinology and Molecular Medicine, Wethington Health Sciences Bldg, Rm 575, 900 S Limestone St, Lexington, KY 40536-0200. E-mail Dennis.Bruemmer{at}uky.edu
 |
Abstract |
|---|
 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
The liver X receptors
and ß (LXR and LXRß) are important regulators of cholesterol homeostasis in liver and macrophages. Synthetic LXR ligands prevent the development of atherosclerosis in murine models; however, the potential functional relevance of LXRs in vascular smooth muscle cells (VSMCs) has not been investigated. In the present study, we demonstrate that LXRs are expressed and functional in primary human coronary artery VSMCs (CASMCs). LXR ligands inhibited mitogen-induced VSMC proliferation and G1
S phase progression of the cell cycle. Inhibition of G1 exit by LXR ligands was accompanied by a dose-dependent inhibition of retinoblastoma protein (Rb) phosphorylation, which functions as the key switch for G1S cell cycle progression. LXR ligands suppressed mitogen-induced degradation of the cyclin-dependent kinase inhibitor p27Kip1, attenuated cyclin D1 and cyclin A expression, and inhibited the expression of S phase-regulatory minichromosome maintenance protein 6. Stabilization of p27kip1 by LXR ligands was mediated by supressing the transcriptional activation of the S phase kinase–associated protein 2 (Skp2), an F-box protein that targets p27Kip1 for degradation. Inhibition of Rb phosphorylation and G1S cell cycle progression by LXR ligands was reversed in VSMCs overexpressing Skp2, indicating that Skp2 as an upstream regulator of p27Kip1 degradation plays a central role in LXR ligand–mediated inhibition of VSMC proliferation. Furthermore, adenovirus-mediated overexpression of the S phase transcription factor E2F, which is released after Rb phosphorylation, reversed the inhibitory effect of LXR ligands on VSMC proliferation and S phase gene expression, suggesting that the primary mechanisms by which LXR ligands inhibit VSMC proliferation occur upstream of Rb phosphorylation. Finally, neointima formation in a model of rat carotid artery balloon injury was significantly attenuated after treatment with the LXR ligand T1317 compared with vehicle-treated animals. These data demonstrate that LXR ligands inhibit VSMC proliferation and neointima formation after balloon injury and suggest that LXR ligands may constitute a novel therapy for proliferative vascular diseases. The full text of this article is available online at http://circres.ahajournals.org.
Key Words: vascular smooth muscle cell • liver X receptor • arterial injury
|
Introduction |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
Cardiovascular disease is the leading cause of mortality in industrialized nations, accounting for nearly 50% of all deaths.1 Vascular smooth muscle cell (VSMC) activation, migration, and proliferation in response to injury play not only a decisive role for development of atherosclerosis but are also the primary pathophysiologic mechanism resulting in the failure of procedures used to treat occlusive proliferative atherosclerotic diseases, such as postangioplasty restenosis, transplant vasculopathy, and vein bypass graft failure.2,3 Although much effort has been devoted to targeting VSMC activation and proliferation, effective therapy to prevent occlusive vascular remodeling has not been established. Approximately 30% to 50% of patients undergoing percutaneous coronary interventions will experience restenosis, and 20% of these patients will require additional interventions, including coronary artery bypass surgery, a procedure that itself is limited by graft failure because of luminal obstruction.4 With recognition of the essential involvement of VSMC activation and proliferation in occlusive cardiovascular disease, therapeutic approaches designed to prevent and treat atherosclerosis and to limit failure of interventional therapeutic approaches have become a focus of research and development.
In response to vascular injury endothelial cells, VSMCs and macrophages secrete cytokines and growth factors that perpetuate the vasculoproliferative response. Cytokines and growth factors share a final proliferative signaling pathway: the cell cycle.5 Cell cycle progression is dependent on the expression of cyclin-dependent kinases (CDKs), which form holoenzymes with their regulatory subunits: the cyclins.6 Progression through the cell cycle requires the activity of cyclin/CDK complexes to phosphorylate the retinoblastoma protein (Rb), which acts as a transition point dedicating the cell to S phase.7 Phosphorylation of Rb in the late G1 phase releases the S phase transcription factor E2F to induce gene expression required for DNA synthesis and progression through the cell cycle.8 The enzymatic activation of cyclin/CDK complexes, and therefore Rb phosphorylation, is in turn regulated by CDK inhibitors (CDKIs), such as p27Kip1 and p21Cip1.9 Mitogen-induced downregulation of p27Kip1 by the ubiquitin-mediated proteolytic pathway during the G1 phase is pivotal for activation of the cyclin/CDK holoenzymes.10–12 Recent studies have demonstrated that the S phase kinase-associated protein 2 (Skp2), which functions as the receptor component of the Skp1–Cullin-F-box (SCF) ubiquitin ligase complex, is essential for regulation of p27Kip1 degradation.13 Molecules that target CDKs and CDKIs represent a new class of therapeutic agents that influence vasculoproliferative diseases.14 Although the mechanisms have not been investigated, stabilization of p27Kip1 is the primary molecular target for a variety of recent therapeutic advances to limit VSMC proliferation in vascular disease, including rapamycin,15 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase inhibitors,16 and thiazolidinediones.17
The liver X receptors and ß (LXR and LXRß) are members of the nuclear hormone receptor superfamily and have been suggested recently as potential targets for novel therapeutic interventions in human cardiovascular disease.18,19 LXR and LXRß are ligand-activated transcription factors that heterodimerize with the retinoid X receptor (RXR).20,21 LXR is expressed primarily in liver, intestine, adipose tissue, and macrophages, whereas LXRß is expressed ubiquitously.22 Endogenous ligands for these nuclear receptors are oxidized derivatives of cholesterol.23 In addition, specific synthetic LXR agonists as potential therapeutics for dyslipidemia and atherosclerosis have been developed. LXRs have been shown to regulate several important genes in reverse cholesterol transport and lipid metabolism, including the ATP-binding cassette transporter A1 (ABCA1),24 cholesterol ester transfer protein,25 apolipoprotein E (apoE),26 lipoprotein lipase,27 and sterol regulatory element-binding protein-1c.28 Activation of LXR in macrophages induces ABCA1 expression and stimulates apoA-I–mediated cholesterol efflux.24 Consistent with their ability to activate the reverse cholesterol transport, LXR ligands increase high-density lipoprotein cholesterol in mice.28 In addition, it has been suggested that LXRs regulate immune processes and inhibit inflammatory gene expression in macrophages.29,30 Direct evidence for the potential utility of LXR activators to prevent atherosclerosis derives from intervention studies. Two LXR agonists, T1317 and GW3965, have been shown to prevent development of atherosclerosis in murine models.31,32 These studies demonstrating antiatherosclerotic effects of LXR agonists may be taken as evidence for the potential benefits of specific LXR agonists to human cardiovascular disease.
Because the role of LXR in VSMCs has not been characterized, we analyzed the expression and function of LXR in human CASMCs. In the present study, we outlined a previously unrecognized role for LXR ligands to suppress VSMC proliferation and neointima formation after balloon injury. The mechanism by which LXR ligands inhibit VSMC proliferation and cell cycle progression involves an inhibition of Rb phosphorylation mediated through an inhibition of Skp2-dependent downregulation of p27Kip1. These observations suggest that LXR ligands may serve as novel therapeutic approaches to prevent cardiovascular disease.
|
Materials and Methods |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
Materials
Primary human CASMCs and smooth muscle growth medium-2 (SmGM-2) were commercially obtained from Cambrex Bio Science Walkersville, Inc. Hybond enhanced chemiluminescence (ECL) nitrocellulose membrane and ECL Western blotting detection reagents were purchased from Amersham Life Science, and horseradish peroxidase–linked anti-rabbit and anti-mouse antibodies were obtained from Cell Signaling. Cycloheximide was purchased from Sigma-Aldrich. Antibodies were purchased from the following suppliers: antiphosphorylated Rb (Ser 807/811) from Cell Signaling; cyclin D1 and E2F-1 from Upstate; cyclin A (sc-751), CDK4 (sc-749), CDK6 (sc-7181), p27Kip1 (sc-528), and minichromosomal maintenance protein 6 (MCM6) (sc-9843) from Santa Cruz Biotechnology; p27Kip1 for immunohistochemistry from BD Biosciences; Skp2 from Zymed Laboratories, Inc.; and LXR
from Calbiochem (318–329) and Perseus Proteomics, Inc. (2ZPPZ0412).
Cell Culture
CASMCs were maintained and passaged in SmGM-2 containing 5% FBS, 2 ng/mL human basic fibroblast growth factor, 0.5 ng/mL human epidermal growth factor, 50 µg/mL gentamycin, and 5 µg/mL bovine insulin according to manufacturer instructions. Early passage (four to eight) cells were grown to 60% to 70% confluence and serum deprived (0.4% FBS, no growth factors) for 24 hours. Cells were treated with LXR ligands for 24 hours before stimulation with platelet-derived growth factor (PDGF)-BB (R&D Systems) and insulin (Sigma-Aldrich) at the final concentration of 20 ng/mL and 1 µmol/L, respectively. Cell viability was measured using a commercially available Annexin V fluorescein isothiocyanate assay (Oncogene Research Products) according to manufacturer instructions. Both LXR ligands exhibited no effect on cell viability (data not shown). Lipoprotein-deficient serum (LPDS) was purchased from Intracel Resources. The compound T1317 was commercially obtained (Sigma-Aldrich). GW3965 was kindly provided by Dr Peter Tontonoz (University of California, Los Angeles). Adenovirus encoding human E2F-1 (Adx-E2F), driven by the cytomegalovirus (CMV) promoter, was provided by Dr Robb W. MacLellan (University of California, Los Angeles).33 Adenovirus containing CMV-driven green fluorescent protein (Adx-GFP) was used as described previously.34
Reverse Transcription and Quantitative Real-Time Polymerase Chain Reaction
Total RNA was isolated using the RNeasy Mini Kit (Qiagen), and 400 ng of total RNA was reverse transcribed with random hexamers using the TaqMan Reverse Transcription Reagent Kit (Applied Biosystems) according to manufacturer instructions. Real-time quantitative polymerase chain reaction (PCR) assays were performed by using an ABI-PRISM 7700 system (Applied Biosystems) in a total volume of 25 µL, using a TaqMan PCR Core Reagent Kit (Applied Biosystems). PCR thermocycling parameters were 2 minutes at 50°C, 10 minutes at 95°C, and 40 cycles of 95°C for 15 seconds and 60°C for 1 minute. Each sample was analyzed in triplicate and normalized to values for GAPDH mRNA expression using Taqman Human glyceraldehyde-3-phosphate dehydrogenase (GAPDH) Control Reagent (Applied Biosystems). Primer and probe sequences were used as described previously by Laffitte et al.35
Western Blot Analysis
Cells were harvested at the indicated time points and sonicated in solubilization buffer (20 mmol/L Tris-HCl, pH 7.5, 150 mmol/L NaCl, 1 mmol/L EDTA, 1 mmol/L EGTA, 1% Triton X-100, 2.5 mmol/L sodium pyrophosphate, 1 mmol/L sodium vanadate, 10 µg/mL each aprotinin and leupeptin, and 1 mmol/L phenylmethylsulfonyl fluoride). Whole cell lysates were cleared by centrifugation, and protein concentrations were determined by Lowry assay (Bio-Rad). Nuclear extracts were isolated using the NuCLEAR extraction kit according to manufacturer instructions (Sigma-Aldrich). Cell lysates containing equal amounts of protein were resolved by SDS-PAGE. Protein was transferred to nitrocellulose membranes (Hybond; Amersham Pharmacia Biotech). After blocking in 20 mmol/L Tris-HCl, pH 7.6, containing 150 mmol/L NaCl, 0.1% Tween-20, and 5% (wt/vol) nonfat dry milk, blots were incubated with specific antibodies described above. Immunoreactive bands were visualized using horseradish peroxidase-conjugated secondary antibodies (1:2000 dilution; Cell Signaling). Immunoreactivity was visualized using a chemiluminescence detection system (ECL; Amersham Pharmacia Biotech). Quantification was performed by densitometry and normalization to ß-actin.
Cell Growth Assay and Flow Cytometry
CASMCs, plated at a density of 1.0x106 cells on 100-mm culture plates, were serum deprived and incubated with LXR ligands as indicated. At 72 hours after stimulation with PDGF (20 ng/mL) and insulin (1 µmol/L), cells were harvested and cell proliferation was measured by counting the cells in a hematocytometer. For cell cycle analysis, CASMCs were harvested 24 hours after PDGF/insulin treatment. DNA was stained with 100 µg/mL propidium iodide for 30 minutes at 4°C, and 1x106 cells were then analyzed with a FACScan (Becton Dickinson). Data were based on six different experiments from three different preparations of CASMCs.
cDNA Array Assay
To analyze LXR ligand–induced regulation of gene expression, we used a commercially available cDNA array corresponding to 96 cell cycle regulatory human genes and 12 housekeeping genes (Cell Cycle GEArray Q series version 1; SuperArray) according to manufacturer instructions. Briefly, quiescent CASMCs were pretreated with the LXR ligand and stimulated with PDGF/insulin at the final concentration of 20 ng/mL and 1 µmol/L, respectively. At 24 hours after stimulation, RNA was isolated, and 2 µg of total RNA were used to synthesize a biotin-16–dUTP-labeled probe using AmpoLabeling-LPR method (30 cycles). Probes were hybridized to array membranes and signals were detected using chemiluminescence according to manufacturer instructions.
RNA Isolation and Northern Blotting
Total RNA was isolated using TRIzol reagent (Life Technologies) according to manufacturer instructions. Fifteen micrograms of total RNA were denatured in formamide/formaldehyde and electrophoresed through 1% formaldehyde-containing agarose gels. After electrophoresis, RNA was transferred to nylon membranes (Hybond N+; Amersham Pharmacia Biotech) by capillary blotting and fixed by UV cross-linking. Hybridization was performed using PerfectHyb Plus hybridization buffer (Sigma-Aldrich) as directed. cDNA for MCM6 was kindly provided by Dr Hiroshi Nojima (Osaka University, Japan).36 Probes used in the hybridization were radiolabeled with [-32P] dCTP (ICN) using Rediprime II random prime labeling system (Amersham Pharmacia Biotech). Blots were cohybridized with GAPDH cDNA (Sigma-Aldrich) to assess equal loading of samples. Quantification was performed by densitometry and normalization to GAPDH.
Transient Transfection and Luciferase Assay
Transient transfections were performed in triplicate in 12-well plates or 60-mm dishes. Human CASMCs were transfected with an Skp2 promoter construct (2 µg per well), an Skp2 expression vector (5 µg per 60-mm dish), or an LXR/LXRß expression vector (5 µg per 60-mm dish) using LipofectAMINE 2000 (Invitrogen). Six hours after transfection, cells were serum deprived (0.4% FBS, no growth factors) in the presence of the LXR ligand T1317 or GW3965. Luciferase activity was assayed 24 hours after PDGF (20 ng/mL) and insulin (1 µmol/L) stimulation using a Dual Luciferase Reporter Assay System (Promega) according to manufacturer instructions. LXR transactivation assays were performed by transient transfection of 2 µg/well of the LXRE-Luc reporter construct driven by three LXRE copies (TK-LXRE). After transfection using LipofectAMINE 2000, cells were incubated in media containing 5% LPDS for 18 hours before stimulation with T1317 (5 µmol/L) or GW3965 (3 µmol/L) for an additional 24 hours. Transfection efficiency was adjusted by normalizing firefly luciferase activities to renilla luciferase activities generated by cotransfection with 10 ng pRL-CMV (Promega). All experiments were repeated at least three times with different cell preparations. The Skp2 luciferase reporter construct has been described previously.37 The luciferase reporter construct driven by three copies of the LXR response element (TK-LXRE) and the LXR and LXRß expression vectors were kindly provided by Dr Peter Tontonoz (University of California, Los Angeles). The Skp2 expression vector was generously provided by Dr Randy Y.C. Poon (Department of Biochemistry, The Hong Kong University of Science and Technology, Hong Kong).38
Adenoviral Infection of CASMCs
CASMCs were infected with 100 plaque-forming units (pfu)/cell Adx-E2F or Adx-GFP in SmGM-2 containing 5% FBS for 24 hours. After starvation for 24 hours in the presence of the LXR ligand T1317, cells were stimulated with PDGF (20 ng/mL) and insulin (1 µmol/L) for 12 hours for RNA analysis, for 24 hours for protein analysis, and for 72 hours for cell proliferation assays. Experiments were repeated at least three times with different cell preparations.
Animal Experiments
Sprague-Dawley rats (3 months of age, weighing 330 to 360 g; Charles River Laboratories) received daily intraperitoneal injections of T1317 (50 mg/kg per day in dimethyl sulfoxide [DMSO]) or vehicle (DMSO) starting 2 days before the injury. The left and right common carotid artery was injured with a 2 French embolectomy catheter as described by Clowes et al.39 For all surgical procedures, animals were anesthetized through intraperitoneal injection of 75 mg/kg body weight ketamine and 10 mg/kg body weight xylazine diluted in sterile water. Fourteen days after arterial injury, animals were euthanized and the vasculature was cleared of blood by perfusion with PBS at 100 mm Hg followed by perfusion fixation with 4% paraformaldehyde in PBS for 15 minutes before harvesting. The common carotid artery was carefully excised and fixed in 4% paraformaldehyde overnight at 4°C before processing for paraffin embedding. All animal protocols were approved by the University of California, Los Angeles Animal Research Committee and complied with all federal, state, and institutional regulations.
Immunohistochemistry
From each paraffin-embedded vessel beginning from the distal cut end, sections 
1 mm apart were stained with hematoxylin-eosin and trichrom-elastin for measurement of vessel wall areas. Paraffin embedding and immunohistochemistry were performed as described previously.40
Morphometric Analysis of Intima/Media Ratios
Arterial specimens were blindly analyzed. Five hematoxylin-eosin stained sections obtained from each artery were examined and the intimal area, medial area, and intima/media (I/M) ratio were calculated using Image-Pro Plus software.
Statistical Analysis
ANOVA and paired or unpaired t test were performed for statistical analysis as appropriate. P values <0.05 were considered statistically significant. Results are expressed as mean±SEM.
|
Results |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
LXRs Are Expressed in Human CASMCs
LXR
is known to be primarily expressed in liver, kidney, intestine, adipose tissue, and macrophages, whereas LXRß is ubiqitously expressed.20 To determine the expression of LXRs in human CASMCs, we used quantitative real-time RT-PCR (Figure 1A), Western immunoblotting (Figure 1B), and confocal immunofluorescence (Figure 1C). Consistent with previous findings by Antonio et al,41 LXR and LXRß mRNA was present in human CASMCs as determined by quantitative real-time RT-PCR. Treatment of CASMCs with the LXR ligands T1317 or GW3965 selectively stimulated LXR mRNA expression, confirming the presence of an autoregulation of the LXR receptor by its ligands in VSMCs, similarly as described previously in macrophages.35 Western blotting analysis using an LXR-specific antibody demonstrated the presence of LXR protein in nuclear extracts of CASMCs (Figure 1B). CASMCs were transfected with human LXR and LXRß expression vectors, and expression of recombinant proteins was used to demonstrate the specificity of the human LXR antibody. LXR immunofluorescence was observed primarily in the nucleus of CASMCs, whereas an IgG control antibody exhibited only background staining (Figure 1C). Consistent with the primary nuclear localization of LXR, only weak staining was observed throughout the cytoplasm.
|
LXR Is Functional and Transactivates LXREs in Human CASMCs
After ligand-induced activation, LXR forms heterodimers with its counterpartner RXR and binds to LXREs in the promoter of target genes to regulate gene transcription.22 To analyze LXR function to transactivate LXREs in human CASMCs, cells were transiently transfected with an LXRE-Luciferase reporter construct (TK-LXRE) driven by three copies of the LXRE. As demonstrated in Figure 2, LXR ligands significantly increased LXRE reporter activity compared with unstimulated cells (1.9±0.2-fold at 5 µmol/L T1317 and 2.3±0.1-fold at 3 µmol/L GW3965 versus unstimulated cells; P<0.05). These transactivation assays demonstrate that LXR is not only expressed in human CASMCs but indicate that LXRs are transcriptionally active in human CASMCs.
|
LXR Ligands Inhibit Mitogen-Induced CASMC Proliferation and G1S Phase Progression
CASMC proliferation plays a crucial role for the development of restenosis after coronary angioplasty and for the progression of fatty streaks to atherosclerotic plaques.42,43 To determine the effect of synthetic LXR ligands on mitogen-induced CASMC proliferation, quiescent cells were treated with T1317 or GW3965 and stimulated with PDGF (20 ng/mL) and insulin (1 µmol/L) for 72 hours. Both LXR ligands inhibited PDGF/insulin-stimulated CASMC proliferation in a dose-dependent manner (84.2±4.6 inhibition at 5 µmol/L T1317 and 100% inhibition at 3 µmol/L GW3965, respectively; P<0.05; Figure 3A).
|
The effect of LXR ligands on cell cycle progression was determined by flow cytometry. Subconfluent CASMCs accumulated in G0/G1 phase after serum deprivation for 24 hours (91.3±4.3% in G0/G1 phase and 6.8±1.9% in S phase; Figure 3B). Mitogenic stimulation with the combination of PDGF (20 ng/mL) and insulin (1 µmol/L) for 24 hours induced the progression into S phase and the population of G0/G1 cells decreased substantially (74.3±7.4%), with a concomitant 3.7-fold increase of CASMCs in S phase (25.1±2.3%). Treatment of cells with LXR ligands substantially inhibited this mitogen-induced G1S progression of human CASMCs (10.8±2.1% and 7.8±1.7% cells in S phase with 5 µmol/L T1317 and 3 µmol/L GW3965 versus PDGF/insulin, respectively; P<0.05). These findings indicate that LXR activation inhibits CASMC proliferation by preventing mitogen-induced G1S phase progression.
LXR Ligands Inhibit Phosphorylation of Rb
To elucidate the mechanism by which LXR ligands inhibit G1S progression, we examined their effect on Rb phosphorylation at specific phosphorylation sites, such as Ser807/811. Cyclin/CDK-dependent Rb phosphorylation is necessary for cells to exit G1 and enter S phase.44 Quiescent CASMCs exhibited low levels of phosphorylated Rb at Ser807/811 (Figure 4), which increased substantially after 24-hour stimulation with PDGF (20 ng/mL) and insulin (1 µmol/L). Treatment of cells with either T1317 or GW3965 resulted in a dose-dependent inhibition of Rb phosphorylation at Ser807/811 (55.2±3.5% and 80.4±7.3% inhibition at 5 µmol/L T1317 and 3 µmol/L GW3965 versus PDGF/insulin alone; P<0.05; Figure 4). These observations suggest that LXR ligands inhibit CASMC proliferation and cell cycle progression, at least in part, through an inhibition of Rb phosphorylation.
|
LXR Ligands Inhibit Mitogen-Induced Skp2 and MCM6 mRNA Expression
To identify cell cycle genes regulated by LXR ligands, we used a DNA gene array containing genes known to control cell proliferation. Serum-deprived CASMCs were stimulated with PDGF (20 ng/mL) and insulin (1 µmol/L) in the presence of the LXR ligand T1317 (5 µmol/L). RNA was isolated after 24 hours and subjected to DNA array analysis. Expression of Skp2, an important upstream regulator of Rb phosphorylation,13 was low in quiescent CASMCs and induced by mitogenic stimulation. Treatment of VSMCs with the LXR ligand completely inhibited mitogen-induced Skp2 mRNA expression, suggesting that Skp2 plays an important role for the growth-inhibitory effects of LXR ligands (Figure 5A and 5B). Similarly, mitogen-induced expression of MCM6, an essential regulator of the DNA replicative machinery downstream of Rb phosphorylation, was suppressed by the LXR ligand (Figure 5A and 5B).
|
LXR Activation Prevents Mitogen-Induced p27Kip1 Degradation and Skp2 Expression
Mitogen-induced downregulation of p27Kip1 during G1 phase is essential for Rb phosphorylation by cyclin/CDK holoenzymes.10,45,46 Therefore, we next investigated the effect of LXR ligands on p27Kip1 protein expression after stimulation of human CASMCs with PDGF/insulin. Quiescent CASMCs expressed high levels of p27Kip1, which decreased markedly after 24 hours of stimulation with PDGF (20 ng/mL) and insulin (1 µmol/L; 34.1±7.1% of quiescent cells; P<0.05). As depicted in Figure 6A, the LXR ligand T1317 potently prevented mitogen-induced downregulation of p27Kip1 in a dose-dependent manner (87.9±4.3% of quiescent cells at 5 µmol/L T1317; P<0.05 versus PDGF/insulin alone).
|
, untreated cells; 
, T1317 treated cells). C, LXR ligands inhibit mitogen-induced Skp2 promoter activity in CASMCs. Cells were transiently transfected with a Skp2 promoter construct (pGL2–318) and serum deprived (0.4% FBS/SmGM-2) for 24 hours before mitogenic stimulation with PDGF/insulin. At 24 hours after stimulation, luciferase activity was measured. Transfection efficiency was adjusted by normalizing firefly luciferase activity to renilla luciferase activity generated by cotransfection with 10 ng pRL-CMV. D, Overexpression of Skp2 abrogates LXR ligand–mediated inhibition of Rb phosphorylation and cell cycle progression. CASMCs were transiently transfected with an Skp2 expression vector followed by serum deprivation in the presence or absence of LXR ligands. At 24 hours after stimulation with PDGF/insulin, cell extracts (50 µg) were assayed for Skp2 and phospho-Rb by Western blot analysis, or DNA was stained with propidium iodide for analysis of cell cycle distribution. Experiments were performed in triplicate (*P<0.05 vs mitogen-stimulated cells; mean±SEM).Previous studies suggest a central role of the F-box protein Skp2 in p27Kip1 degradation in vivo and in vitro.13,47 On the basis of the findings derived from the cDNA array experiments, we next investigated whether LXR ligands suppress mitogen-induced Skp2 protein expression. Quiescent human CASMCs expressed low levels of Skp2 protein, which were significantly increased after 24 hours of mitogenic stimulation (2.1±0.1-fold versus unstimulated cells; P<0.05; Figure 6A). Treatment of cells with T1317 dose-dependently inhibited PDGF/insulin-induced Skp2 protein expression (100% inhibition at 5 µmol/L T1317; P<0.05; Figure 6A).
To further determine the mechanisms by which LXR ligands prevent mitogen-induced degradation of p27Kip1, we examined their effects on mRNA expression levels and protein stability. p27Kip1 mRNA levels did not change with mitogenic stimulation or treatment with LXR ligands (data not shown). To determine p27Kip1 protein turnover, CASMCs were treated with PDGF (20 ng/mL) and insulin (1 µmol/L) for 6 hours, and cycloheximide (20 µg/mL) was added to block the de novo protein synthesis. The rate of p27Kip1degradation was substantially decreased in cells cotreated with T1317 (5 µmol/L) (Figure 6B). These data indicate that LXR ligands regulate p27Kip1 mainly through post-transcriptional mechanisms by stabilizing mitogen-induced p27Kip1 protein degradation.
Protein levels of Skp2 have been shown to be regulated at the transcriptional and post-transcriptional levels.37,48 To further examine the effect of LXR ligands on Skp2 transcription, CASMCs were transiently transfected with a human Skp2 promoter construct containing a 318-bp promoter fragment (pGL2–318; Figure 6C). PDGF/insulin-induced Skp2 promoter activity (2.0±0.29-fold induction versus quiescent cells; P<0.05) was completely inhibited by both LXR ligands (100% inhibition at 5 µmol/L T1317 and 3 µmol/L GW3965). Together, these data indicate that LXR ligands inhibit cell cycle progression of human CASMCs, at least in part, by inhibiting Skp2 transcription and resulting in a stabilization of mitogen-induced p27Kip1 degradation. To further corroborate that Skp2 is an important target for the efficacy of LXR ligands to inhibit Rb phosphorylation and cell cycle progression, Skp2 was overexpressed in human CASMCs using an Skp2 expression vector (Figure 6D). Ectopic Skp2 overexpression completely prevented the inhibition of Rb phosphorylation and G1S phase progression by LXR ligands (Figure 6D). These results further support the concept that Skp2 plays a central role in LXR ligand–mediated inhibition of cell proliferation.
LXR Ligands Inhibit Mitogen-Induced G1 Cyclin D1 and A and S phase MCM6 Expression
To further elucidate additional mechanisms by which LXR ligands inhibit Rb phosphorylation, we examined their effect on the expression of CDKs and their cyclin partners, for which Rb is a major physiological substrate. Phosphorylation of Rb by CDK4 or CDK6 complexes during the G1 phase and CDK2 at the G1/S interphase releases E2F protein and promotes transcription of genes essential for transition to the S phase.44 Cyclin D1 and A were expressed at low levels in quiescent CASMCs and increased 5.4±0.1-fold (cyclin D1) or 7.6±0.1-fold (cyclin A) after 24-hour mitogenic stimulation (both P<0.05 versus unstimulated cells). T1317 dose-dependently inhibited PDGF (20 ng/mL), and insulin (1 µmol/L) stimulated cyclin A and D1 expression (70.0±4.3% and 69.4±5.7% inhibition versus mitogen-stimulated cells at 5 µmol/L T1317; P<0.05; Figure 7A). Quiescent CASMCs expressed CDK4 and CDK6 protein, which did not change after mitogenic stimulation or treatment with T1317. These data are concordant with previous studies, demonstrating that CDKs are regulated predominantly at the post-translational level.49
|
The S phase of the cell cycle and DNA replication requires the function of MCM gene products,50 which are regulated by the S phase transcription factor E2F. To determine whether inhibition of Rb phosphorylation at the G1S phase translates to an inhibition of downstream S phase gene expression, we analyzed the effect of LXR activation on MCM6 expression. Quiescent human CASMCs expressed low levels of MCM6 protein, which increased 2.2±0.13-fold after a 24-hour stimulation with PDGF/insulin (Figure 7B). T1317 inhibited mitogen-induced MCM6 protein expression in a dose-dependent manner (94.8±7.9% inhibition versus PDGF/insulin-stimulated cells at 5 µmol/L T1317; P<0.05; Figure 7B). In concert, these findings suggest that inhibition of CASMC proliferation and Rb phosphorylation results from blocking the expression of essential G1 cyclins necessary for activating cyclin/CDK complexes to phosphorylate Rb. Subsequently, decreased Rb phosphorylation results in decreased expression of E2F-regulated MCM expression, proteins pivotal for the DNA replicative machinery during the S phase.
Adenoviral Overexpression of E2F-1 Reverses the Inhibitory Effect of LXR Ligands on Mitogenic Induction of MCM6
To provide additional evidence that the primary mechanism by which LXR ligands inhibit CASMC proliferation occurs upstream of Rb phosphorylation, we used an adenoviral expression vector (Adx-E2F) to overexpress E2F independent of Rb phosphorylation and analyzed CASMC proliferation (Figure 8A). Cells infected with an adenovirus overexpressing green fluorescent protein (Adx-GFP) served as control to assess for potential virus-mediated effects. In cells infected with Adx-GFP, treatment with the LXR agonist T1317 resulted in a potent inhibition of mitogen-induced CASMC proliferation (98.2±8.2% inhibition versus PDGF/insulin; P<0.05). In marked contrast, overexpression of E2F was associated with a profound induction of VSMC proliferation and prevented the inhibitory effect of the LXR agonist on CASMC proliferation (9.8±0.8-fold induction versus quiescent Adx-GFP infected VSMC; P<0.05). Similarly, the S phase and E2F target MCM6 gene was induced in CASMCs overexpressing E2F and the LXR agonist T1317 exhibited no effect to suppress MCM6 mRNA and protein expression in CASMCs overexpressing E2F-1 (Figure 8B). In concert, these findings support our conclusion that the inhibition of CASMC proliferation by LXR ligands is primarily mediated through an inhibition of E2F release from Rb and that LXR agonist suppresses events in the G1 phase of the cell cycle required for E2F release and proliferation of CASMCs.
|
LXR Ligand T1317 Inhibits Neointima Formation After Balloon Angioplasty
Our in vitro experiments revealed that LXR agonists potently inhibit CASMC proliferation. To finally determine whether these effects were applicable in vivo to inhibit neointima formation after arterial injury, T1317 (50 mg/kg per day) was administered to male Sprague-Dawley rats two days before bilateral balloon injury of the common carotid artery and daily for 14 days after injury. Figure 9A and 9B depicts representative sections of hematoxylin-eosin– and trichrom-elastin–stained carotid arteries from vehicle- and T1317-treated animals. Morphometric analysis revealed that the intimal area and I /M ratio was significantly reduced in LXR ligand–treated animals compared with vehicle-treated rats (intimal area 60327±12423 µm2 versus 152790±19764 µm2; I/M ratio 0.58±0.17 versus 1.57±0.21; both P<0.05; Figure 9C). In contrast, the medial area did not change in LXR agonist–treated rats (105009±18480 µm2) compared with the control group (97849±10445 µm2). Furthermore, in rats treated with the LXR agonist, p27Kip1 immunoreactivity in neointimal tissues was substantially higher compared with vehicle-treated animals (Figure 9D). Together, these results demonstrate that LXR ligands inhibit neointima formation in a model of rat carotid artery balloon injury and further support the important role of p27Kip1 for LXR ligand–mediated inhibition of VSMC proliferation and neointima formation.
|
|
Discussion |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
VSMC proliferation in the arterial wall plays a pivotal role for the development of postangioplasty restenosis and atherosclerosis.14,42 Here we demonstrate that LXRs are expressed and functional in human CASMCs and that LXR ligands inhibit mitogen-induced VSMC proliferation. The mechanism by which LXR ligands suppress VSMC proliferation involves an inhibition of Rb phosphorylation, E2F release, and G1
S phase cell cycle progression. Inhibition of Rb phosphorylation and cell cycle progression by LXR ligands is mediated by suppressing the Skp2 component of the ubiquitin ligase that regulates p27Kip1 degradation. Finally, we demonstrate that the effects of LXR ligands to suppress VSMC proliferation are applicable in vivo because treatment of rats with an LXR agonist inhibits neointima formation after balloon angioplasty.
LXR was first identified in the liver,22 but recent studies have demonstrated that LXR is expressed in a variety of additional tissues, such as the kidney,51 intestine,51 adipose tissue,52 and macrophages.53 In contrast to the tissue-specific expression of LXR, LXRß has been shown to be expressed ubiquitously.20,22 Consistent with a recent report,41 quantitative real-time RT-PCR demonstrated the presence of LXR and LXRß mRNA in CASMCs. Treatment of CASMCs with LXR agonists resulted in a prominent induction of LXR mRNA expression, which is in accordance with the recently demonstrated autoregulation of the LXR receptor by its ligands in macrophages.35 Consistent with the primary nuclear localization, we observed that LXR protein was present in nuclear cell extracts and that immunofluorescence was primarily observed in the nucleus with less staining in the cytoplasm. Data derived from transient transfection experiments using an LXRE luciferase reporter construct further suggest that LXR is functional active and able to transactivate LXREs in CASMCs.
In response to injury of the integrity of the arterial wall, VSMC proliferation results in the formation of a neointima, which represents a key pathophysiological mechanism for development of occlusive vascular disease. In addition to the concept that these VSMCs derive from the adjacent media and migrate to the subendothelial layer,54 recent evidence indicates that bone marrow–derived progenitor cells may also contribute to arterial remodeling in postangioplasty restenosis.55,56 To determine whether ligand-induced LXR activation interferes with the proliferative response of VSMCs after mitogenic stimulation, we analyzed the effect of LXR agonists on mitogen-induced CASMC proliferation. LXR ligands inhibited CASMC proliferation at concentrations similar to those required for the regulation of classical LXR target genes, such as ABCA1 and ABCG1.31,57,58 Analysis of cell cycle distribution indicated that LXR ligands inhibit mitogen-induced cell cycle progression and G1S phase progression. Hyperphosphorylation of Rb by G1 cyclin/CDKs results in release of the transcription factor E2F and is required for cells to enter S phase.45 Consistent with their ability to inhibit G1S cell cycle transition, LXR ligands prevented mitogen-induced phosphorylation of Rb, further suggesting that these compounds inhibit processes in the G1 phase of the cell cycle necessary for Rb phosphorylation.
Degradation of p27Kip1 during the G1 phase in response to mitogens is important for maximal activation of G1 cyclin/CDK complexes to phosphorylate Rb and, thus, for cell cycle progression from G1 to S phase.59,60 LXR ligands prevented mitogen-induced p27Kip1 degradation and, therefore, this is likely a major mechanism by which LXR ligands inhibit Rb phosphorylation and CASMC proliferation. Among the multiple cell cycle-regulatory proteins, p27Kip1 has been demonstrated to play an essential role for regulation of VSMC proliferation. p27Kip1 expression is markedly reduced in the intima and media after angioplasty, consistent with an injury-induced proliferative response.61,62 In addition, p27Kip1 mediates VSMC migration and proliferation, and overexpression of p27Kip1 inhibits VSMC growth and neointima formation.63,64 Stabilization of p27Kip1 is a primary molecular target for a variety of recent therapeutic advances to limit VSMC proliferation, including rapamycin,15 HMG-CoA reductase inhibitors,16 and thiazolidinediones.17 Although these antiproliferative agents prevent mitogen-induced p27Kip1 degradation, it remains unclear whether there are additional upstream targets regulating p27Kip1 levels.
p27Kip1 protein levels during the cell cycle are predominantly regulated through post-translational mechanisms, including ubiquitination and proteasome-dependent degradation.65 The F-box protein Skp2 has been shown to be an essential component of the SCFSkp2 complex catalyzing the ubiquitination-dependent degradation of p27Kip1.47 Recent observations by Bond et al indicate that Skp2 is an important regulator of rat VSMC p27Kip1 levels, Rb phosphorylation, and subsequent cell proliferation.66 Using microarray analysis corroborated by Western blot analysis, we identified that mitogen-induced expression of Skp2 protein is suppressed by LXR ligands. Because cell cycle–dependent expression of Skp2 has been demonstrated to be regulated at the transcriptional and post-transcriptional levels, we analyzed the effect of LXR ligands on Skp2 transcriptional activation. Mitogen-induced Skp2 promoter activity was suppressed by LXR ligands, suggesting that the antiproliferative effect of LXR ligands correlates with stabilization of p27Kip1, and this is most likely attributable to an inhibition of Skp2 transcription. In further support of this concept, LXR ligands exhibited no inhibition of cell cycle progression and Rb phosphorylation in CASMCs overexpressing Skp2. Therefore, Skp2 appears to be an important target for the antiproliferative efficacy of LXR ligands in CASMCs.
To identify additional mechanisms by which LXR ligands inhibit mitogen-induced Rb phosphorylation, we examined their effects on the expression of G1 cyclins and CDKs, which form holoenzymes to phosphorylate Rb. LXR ligands attenuated mitogen-induced cyclin D1 and cyclin A expression. Consistent with the primary post-translational regulation of CDK4 and CDK6,49 protein expression of these CDKs was not affected by LXR ligands. Although inhibition of Skp2 has been reported to increase cyclin D1 protein levels through decreased nuclear ubiquitination,67,68 we observed a potent inhibition of mitogen-induced cyclin D1 expression by LXR ligands. Recent studies by Carrano et al69 observed additive effects of Skp2 and cyclin D1 to induce S phase entry and cell division. Consistent with this, our results may indicate that LXR ligand–mediated inhibition of Skp2 and cyclin D1 expression defines two distinct pathways. Potentially, LXR ligands could primarily affect cyclin D1 transcription, as has been demonstrated for ligands of other nuclear receptors, such as the peroxisome proliferator–activated receptor 
70 and the RXR.71 However, the precise mechanisms by which LXR ligands affect mitogen-induced cyclin D1 expression remain to be further elucidated.
Phosphorylation of Rb results in a conformational change that releases the sequestered S phase transcription factor E2F, enabling it to transactivate target genes encoding the machinery for DNA synthesis.45,72 Inhibition of E2F transactivation has been reported to inhibit VSMC proliferation in vivo, whereas overexpression of E2F induces S phase gene expression and quiescent cells to enter S phase.73,74 Previous studies from our group have shown that mitogen-induced MCM expression in VSMC involves hyperphosphorylation of Rb and subsequent E2F release.75,76 In accord with the central role of E2F in regulating S phase gene expression, LXR ligands suppressed mitogen-induced expression of MCM6, a classical S phase and E2F-regulated target gene.77,78 To provide additional evidence that the molecular mechanisms resulting in an inhibition of Rb phosphorylation and CASMC proliferation by LXR ligands occur upstream of Rb phosphorylation in the G1 phase, we used an adenoviral construct to overexpress E2F independently of Rb phosphorylation. In CASMCs overexpressing E2F, LXR ligands exerted no effects on cellular proliferation and S phase gene expression, supporting that the primary mechanism by which LXR ligands inhibit CASMC proliferation occurs upstream of Rb phosphorylation and E2F release in G1. In concert, these findings indicate that inhibition of Rb phosphorylation and E2F release defines a novel mechanism for transrepression of gene expression by LXR ligands and indicate that the primary mechanism for the inhibition of CASMC proliferation by LXR ligands occurs upstream of Rb phosphorylation.
Consistent with the effect of LXR ligands on CASMC proliferation in vitro, we observed that intraperitoneal application of T1317 in a rat carotid artery injury model resulted in a significant decrease of the I/M ratio compared with vehicle-treated animals. Inhibition of neointima formation by the LXR agonist was associated with increased p27Kip1 expression in vivo, which further supports a key role of p27Kip1 for the efficacy of LXR ligands to prevent neointimal VSMC proliferation. The demonstration that LXR ligands inhibit CASMC proliferation and development of the neointima may raise interest in its potential utility to inhibit human postangioplasty restenosis. The finding that LXR ligands inhibit CASMC proliferation may have important therapeutic implications because restenosis and atherosclerosis are complex processes governed by the interaction of a variety of growth factors. Indeed, LXR activation has been suggested recently as potential target for novel therapeutic interventions in human cardiovascular disease.18,19 Selective loss of macrophage LXR activity increased atherosclerotic lesion development, suggesting that LXR functions as endogenous inhibitor of atherogenesis.79 Moreover, two LXR agonists, T1317 and GW3965, have been shown to decrease atherosclerosis in murine models.31,32 Whereas the antiatherogenetic effects of LXR activation have been attributed to the modulation of metabolic and inflammatory gene expression, our findings add a previously unrecognized role of LXR ligands to inhibit CASMC proliferation. The antiproliferative activity of LXR ligands resulted from their inhibition of Rb phosphorylation by modulating the expression of several key cell cycle regulators that control G1S phase progression. Attenuation of Skp2-dependent degradation of p27Kip1 appears to be the major mechanism ultimately leading to inhibition of Rb phosphorylation, E2F release, and E2F-dependent transactivation of S phase gene expression necessary for progression through the S phase. Thus, inhibition of CASMC proliferation by LXR ligands might indeed not only contribute to the antiatherosclerotic effects of LXR ligands but may also provide a novel therapeutic approach to limit proliferative vascular disease, which constitutes the major complication of strategies used to treat occlusive atherosclerotic disease.
|
Acknowledgments |
|---|
This study was supported by National Institutes of Health grant HL 58328 to W.A.H. and by scientist development grant 0435239N from the American Heart Association to D.B. F.B. was supported by a research fellowship from Philip Morris USA Incorporated. Y.T. was supported by Japan Heart Foundation/Bayer Yakuhin Research Grant Abroad. K.K. was supported by the Deutsche Forschungsgemeinschaft (KA 1820/1-1). We thank Dr Randy Y. C. Poon for providing the Skp2 expression vector and Longsheng Hong for excellent technical assistance in immunohistochemical procedures.
|
Footnotes |
|---|
Original received June 8, 2004; resubmission received October 25, 2004; accepted November 1, 2004.
|
References |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
- Yusuf S, Reddy S, Ounpuu S, Anand S. Global burden of cardiovascular diseases: part I: general considerations, the epidemiologic transition, risk factors, and impact of urbanization. Circulation. 2001; 104: 2746–2753.
[Abstract/Free Full Text] - Schwartz SM, Liaw L. Growth control and morphogenesis in the development and pathology of arteries. J Cardiovasc Pharmacol. 1993; 21 (suppl 1): S31–S49.[Medline] [Order article via Infotrieve]
- Dzau VJ, Braun-Dullaeus RC, Sedding DG. Vascular proliferation and atherosclerosis: new perspectives and therapeutic strategies. Nat Med. 2002; 8: 1249–1256.[CrossRef][Medline] [Order article via Infotrieve]
- Lowe HC, Oesterle SN, Khachigian LM. Coronary in-stent restenosis: current status and future strategies. J Am Coll Cardiol. 2002; 39: 183–193.
[Abstract/Free Full Text] - Boehm M, Nabel EG. The cell cycle and cardiovascular diseases. Prog Cell Cycle Res. 2003; 5: 19–30.[Medline] [Order article via Infotrieve]
- Morgan DO. Principles of CDK regulation. Nature. 1995; 374: 131–134.[CrossRef][Medline] [Order article via Infotrieve]
- Harbour JW, Dean DC. Rb function in cell-cycle regulation and apoptosis. Nat Cell Biol. 2000; 2: E65–E67.[CrossRef][Medline] [Order article via Infotrieve]
- Weinberg RA. E2F and cell proliferation: a world turned upside down. Cell. 1996; 85: 457–459.[CrossRef][Medline] [Order article via Infotrieve]
- Sherr CJ, Roberts JM. CDK inhibitors: positive and negative regulators of G1-phase progression. Genes Dev. 1999; 13: 1501–1512.
[Free Full Text] - Rao GN. Differential regulation of p27kip1 levels and CDK activities by hypertrophic and hyperplastic agents in vascular smooth muscle cells. Biochim Biophys Acta. 1999; 1448: 525–532.[Medline] [Order article via Infotrieve]
- Pagano M, Tam SW, Theodoras AM, Beer-Romero P, Del Sal G, Chau V, Yew PR, Draetta GF, Rolfe M. Role of the ubiquitin-proteasome pathway in regulating abundance of the cyclin-dependent kinase inhibitor p27. Science. 1995; 269: 682–685.
[Abstract/Free Full Text] - Shirane M, Harumiya Y, Ishida N, Hirai A, Miyamoto C, Hatakeyama S, Nakayama K, Kitagawa M. Down-regulation of p27(Kip1) by two mechanisms, ubiquitin-mediated degradation and proteolytic processing. J Biol Chem. 1999; 274: 13886–13893.
[Abstract/Free Full Text] - Sutterluty H, Chatelain E, Marti A, Wirbelauer C, Senften M, Muller U, Krek W. p45SKP2 promotes p27Kip1 degradation and induces S phase in quiescent cells. Nat Cell Biol. 1999; 1: 207–214.[CrossRef][Medline] [Order article via Infotrieve]
- Braun-Dullaeus RC, Mann MJ, Dzau VJ. Cell cycle progression: new therapeutic target for vascular proliferative disease. Circulation. 1998; 98: 82–89.
[Abstract/Free Full Text] - Gallo R, Padurean A, Jayaraman T, Marx S, Roque M, Adelman S, Chesebro J, Fallon J, Fuster V, Marks A, Badimon JJ. Inhibition of intimal thickening after balloon angioplasty in porcine coronary arteries by targeting regulators of the cell cycle. Circulation. 1999; 99: 2164–2170.
[Abstract/Free Full Text] - Laufs U, Marra D, Node K, Liao JK. 3-Hydroxy-3-methylglutaryl-CoA reductase inhibitors attenuate vascular smooth muscle proliferation by preventing rho GTPase-induced down-regulation of p27(Kip1). J Biol Chem. 1999; 274: 21926–21931.
[Abstract/Free Full Text] - Wakino S, Kintscher U, Kim S, Yin F, Hsueh WA, Law RE. Peroxisome proliferator-activated receptor gamma ligands inhibit retinoblastoma phosphorylation and G1S transition in vascular smooth muscle cells. J Biol Chem. 2000; 275: 22435–22441.
[Abstract/Free Full Text] - Jaye M. LXR agonists for the treatment of atherosclerosis. Curr Opin Investig Drugs. 2003; 4: 1053–1058.[Medline] [Order article via Infotrieve]
- Joseph SB, Tontonoz P. LXRs: new therapeutic targets in atherosclerosis? Curr Opin Pharmacol. 2003; 3: 192–197.[CrossRef][Medline] [Order article via Infotrieve]
- Peet DJ, Janowski BA, Mangelsdorf DJ. The LXRs: a new class of oxysterol receptors. Curr Opin Genet Dev. 1998; 8: 571–575.[CrossRef][Medline] [Order article via Infotrieve]
- Tontonoz P, Mangelsdorf DJ. Liver X receptor signaling pathways in cardiovascular disease. Mol Endocrinol. 2003; 17: 985–993.
[Abstract/Free Full Text] - Willy PJ, Umesono K, Ong ES, Evans RM, Heyman RA, Mangelsdorf DJ. LXR, a nuclear receptor that defines a distinct retinoid response pathway. Genes Dev. 1995; 9: 1033–1045.
[Abstract/Free Full Text] - Janowski BA, Grogan MJ, Jones SA, Wisely GB, Kliewer SA, Corey EJ, Mangelsdorf DJ. Structural requirements of ligands for the oxysterol liver X receptors LXRalpha and LXRbeta. Proc Natl Acad Sci U S A. 1999; 96: 266–271.
[Abstract/Free Full Text] - Venkateswaran A, Laffitte BA, Joseph SB, Mak PA, Wilpitz DC, Edwards PA, Tontonoz P. Control of cellular cholesterol efflux by the nuclear oxysterol receptor LXR alpha. Proc Natl Acad Sci U S A. 2000; 97: 12097–12102.
[Abstract/Free Full Text] - Luo Y, Tall AR. Sterol upregulation of human CETP expression in vitro and in transgenic mice by an LXR element. J Clin Invest. 2000; 105: 513–520.[Medline] [Order article via Infotrieve]
- Laffitte BA, Repa JJ, Joseph SB, Wilpitz DC, Kast HR, Mangelsdorf DJ, Tontonoz P. LXRs control lipid-inducible expression of the apolipoprotein E gene in macrophages and adipocytes. Proc Natl Acad Sci U S A. 2001; 98: 507–512.
[Abstract/Free Full Text] - Zhang Y, Repa JJ, Gauthier K, Mangelsdorf DJ. Regulation of lipoprotein lipase by the oxysterol receptors, LXRalpha and LXRbeta. J Biol Chem. 2001; 276: 43018–43024.
[Ab