- Open Access
Discovery and implementation of transcriptional biomarkers of synthetic LXR agonists in peripheral blood cells
- Elizabeth A DiBlasio-Smith1,
- Maya Arai1,
- Elaine M Quinet2,
- Mark J Evans2,
- Tad Kornaga1,
- Michael D Basso2,
- Liang Chen2,
- Irene Feingold3,
- Anita R Halpern2,
- Qiang-Yuan Liu2,
- Ponnal Nambi2,
- Dawn Savio2,
- Shuguang Wang2,
- William M Mounts1,
- Jennifer A Isler4,
- Anna M Slager4,
- Michael E Burczynski4,
- Andrew J Dorner1 and
- Edward R LaVallie1Email author
© DiBlasio-Smith et al; licensee BioMed Central Ltd. 2008
- Received: 05 August 2008
- Accepted: 16 October 2008
- Published: 16 October 2008
LXRs (Liver X Receptor α and β) are nuclear receptors that act as ligand-activated transcription factors. LXR activation causes upregulation of genes involved in reverse cholesterol transport (RCT), including ABCA1 and ABCG1 transporters, in macrophage and intestine. Anti-atherosclerotic effects of synthetic LXR agonists in murine models suggest clinical utility for such compounds.
Blood markers of LXR agonist exposure/activity were sought to support clinical development of novel synthetic LXR modulators.
Transcript levels of LXR target genes ABCA1 and ABCG1 were measured using quantitative reverse transcriptase/polymerase chain reaction assays (qRT-PCR) in peripheral blood from mice and rats (following a single oral dose) and monkeys (following 7 daily oral doses) of synthetic LXR agonists. LXRα, LXRβ, ABCA1, and ABCG1 mRNA were measured by qRT-PCR in human peripheral blood mononuclear cells (PBMC), monocytes, T- and B-cells treated ex vivo with WAY-252623 (LXR-623), and protein levels in human PBMC were measured by Western blotting. ABCA1/G1 transcript levels in whole-blood RNA were measured using analytically validated assays in human subjects participating in a Phase 1 SAD (Single Ascending Dose) clinical study of LXR-623.
A single oral dose of LXR agonists induced ABCA1 and ABCG1 transcription in rodent peripheral blood in a dose- and time-dependent manner. Induction of gene expression in rat peripheral blood correlated with spleen expression, suggesting LXR gene regulation in blood has the potential to function as a marker of tissue gene regulation. Transcriptional response to LXR agonist was confirmed in primates, where peripheral blood ABCA1 and ABCG1 levels increased in a dose-dependent manner following oral treatment with LXR-623. Human PBMC, monocytes, T- and B cells all expressed both LXRα and LXRβ, and all cell types significantly increased ABCA1 and ABCG1 expression upon ex vivo LXR-623 treatment. Peripheral blood from a representative human subject receiving a single oral dose of LXR-623 showed significant time-dependent increases in ABCA1 and ABCG1 transcription.
Peripheral blood cells express LXRα and LXRβ, and respond to LXR agonist treatment by time- and dose-dependently inducing LXR target genes. Transcript levels of LXR target genes in peripheral blood are relevant and useful biological indicators for clinical development of synthetic LXR modulators.
- Peripheral Blood Mononuclear Cell
- Peripheral Blood Cell
- Reverse Cholesterol Transport
- Human Peripheral Blood Mononuclear Cell
- ABCG1 Expression
The liver X receptors (LXRα and LXRβ, also known as NR1H3 and NR1H2, respectively) belong to the nuclear hormone receptor family of ligand-activated transcription factors. LXRs are involved in controlling the expression of a spectrum of genes that regulate cholesterol biosynthesis and export in the liver as well as cholesterol efflux from peripheral tissues [1–3]. In this way, LXRs act as cholesterol sensors in the body. As such, the naturally occurring, activating ligands for LXRs in vivo include specific oxidized cholesterol metabolites such as 24 (S),25-epoxycholesterol, 22 (R)-, 24 (S)-, and 27-hydroxycholesterol . When these ligands bind to LXRs, they displace co-repressors and allow the ligand-bound LXR (which forms an obligate heterodimer with retinoid X receptor (RXR), the receptor for 9-cis-retinoic acid) to regulate the expression of target genes by binding to specific promoter response elements (LXREs) in target genes of LXR action [5–8]. In the liver, LXRs regulate the expression of genes that control cholesterol metabolism and homeostasis, such as cholesterol 7α-hydroxylase (in mice), which controls the cholesterol/bile acid synthetic pathway, and sterol regulatory element-binding protein-1c, a key transcription factor that regulates expression of genes important in fatty acid biosynthesis [9, 10]. The role for each LXR isoform in these processes has been elucidated by studies of pan-LXRα/β agonists in LXRα KO mice [11, 12]. LXRα and β have also been shown to be expressed in macrophage, where they play an important role in regulating cholesterol efflux from macrophage in atherosclerotic lesions [13–15]. In macrophage, LXR activation results in the induction of several genes. Among these induced genes are those encoding the ATP-binding cassette proteins, such as ABCA1 and ABCG1, which are plasma membrane-associated transport proteins that are responsible for mediating cholesterol efflux as the initial step of the "reverse cholesterol transport" (RCT) process thereby controlling cholesterol mobilization from lipid-laden macrophages [16, 17]. This "effluxed" cholesterol is subsequently transferred to plasma acceptor proteins such as high-density lipoprotein (HDL), which then delivers excess cholesterol to the liver  for eventual excretion. The action of LXR activation in the liver stimulates bile acid production and excretion of this cholesterol. In addition, LXRs are expressed in the intestine where they limit dietary cholesterol uptake by regulating the expression of ABC family members ABCA1 and ABCG5/ABCG8 that reside on the apical surface of enterocytes and act as efflux pumps moving cholesterol out of absorptive cells into the intestinal lumen .
Since LXRs are important regulators of reverse cholesterol transport in macrophages, we and others have developed synthetic LXR agonists that have been shown to be capable of stimulating macrophages in atherosclerotic plaques to efflux the scavenged cholesterol and limiting plaque progression [19–23]. This attribute is of particular disease relevance because lipid accumulation in these cells, through the uptake of oxLDL/LDL, is believed to be of fundamental importance to the etiology and pathogenesis of atherogenesis and atherosclerosis and other chronic inflammatory diseases [24–28]. We have recently developed a novel LXR agonist LXR-623 that has been shown to be anti-atherogenic in mouse models of atherosclerosis (manuscript in preparation).
To assist in the clinical development of LXR-623, we sought to identify peripheral blood biomarkers of LXR agonist exposure and activity. Initial biomarker discovery experiments in rodents revealed that peripheral blood cells respond to orally dosed LXR-623 by substantially increasing the transcriptional level of ABCA1 and ABCG1 in a dose-dependent manner. These data were confirmed in primate studies, where it was shown that peripheral blood cell expression of ABCA1 and ABCG1 mRNA was significantly increased in a dose-dependent manner by LXR-623 following 7 days of dosing. These findings were extended to human cells by treating PBMC from normal human donors ex vivo with LXR-623, which showed that ABCA1 and ABCG1 expression was similarly regulated in human peripheral blood cells. Furthermore, despite the assumption that monocytes (the circulating macrophage-precursor cell type in PBMC) are the only LXR agonist-responsive cell type in PBMC, it was shown that T- and B-cells (in addition to monocytes) also express LXRα and LXRβ and respond to LXR agonist treatment by upregulating ABCA1 and ABCG1 gene expression. Based upon these findings, external standard based qRT-PCR assays were developed to measure copy numbers of ABCA1 and ABCG1 transcripts in whole blood cell RNA from human subjects in a Phase 1 SAD (Single Ascending Dose) clinical study of LXR-623. In a representative subject both ABCA1 and ABCG1 transcripts were rapidly upregulated with similar temporal profiles following a single dose of LXR-623. We conclude that the pharmacodynamic effects of synthetic LXR agonist compounds can be measured in vivo by monitoring the expression of selected LXR target genes in peripheral blood cells. This approach should prove useful for future clinical development of the present compound and other candidate LXR agonist compounds.
All cell culture reagents were obtained from Gibco-Invitrogen (Carlsbad, CA). LXR agonists T0901317 [N-(2,2,2,-trifluoro-ethyl)-N-[4-(2,2,2,-trifluoro-1-hydroxy-1-trifluoromethyl-ethyl)-phenyl]-benzenesulfonamide] [8, 22] and GW3965 [3-(3-(2-chloro-3-trifluoromethylbenzyl-2,2 diphenylethylamino)propoxy)phenylacetic acid]  were prepared following standard chemical syntheses from published literature. LXR-623 was synthesized by the Wyeth Chemical and Screening Sciences group. Mouse Universal Reference Total RNA (catalog # 636657) and Human Universal Reference Total RNA (catalog # 636538) was purchased from Clontech (Mountain View, CA).
Mouse blood collection and RNA isolation
Blood (~300 uL) obtained from C57/Bl6 mice treated with LXR-623 agonist compound was immediately mixed with 1.3 mL of RNAlater (Ambion, Austin, TX), and frozen at -80°C until further processing to RNA. RNA was isolated from the thawed samples using the RiboPure Blood Kit (Ambion #1928) following the manufacturer's protocol. Quantitation of total RNA samples was performed using an Eppendorf BioPhotometer 6131. RNA quality was assessed using an Agilent BioAnalyzer with the RNA Nano-chip (Agilent Technologies, Santa Clara, CA).
Rat blood and tissue collection and RNA isolation
Male Long Evans rats (Charles River Labs) weighing approximately 300 g were administered a single gavage treatment of 1 ml 2% Tween 80/0.5% methylcellulose containing sufficient compound to deliver the indicated doses. At various times following dosing, the rats were anesthetized with isoflurane and peripheral blood was removed by cannulation of the abdominal aorta. Approximately 2.5 ml blood was collected into PAXgene Blood RNA Tubes (Qiagen, Valencia, CA; # 262115) and RNA was prepared according to the manufacturer's protocol. Spleens were removed and frozen in liquid nitrogen prior to processing for RNA isolation using the RNeasy Mini RNA Isolation Kit (Qiagen). Total RNA was quantified by RiboGreen (Invitrogen, Carlsbad, CA). For determination of drug levels, compounds were extracted from EDTA plasma into 1:1 acetonitrile:water and quantified by LC/MS/MS.
Non-human primate blood collection and RNA isolation
Cynomolgus monkeys were treated for 7 days with LXR agonist LXR-623 at either 15 mg/kg/day or 50 mg/kg/day PO. Serum and whole blood samples were collected at predose (day 0) and following dosing on day 7. Whole blood (2.5 ml) was collected into PAXgene Blood RNA Tubes (Qiagen catalog # 262115), incubated overnight at room temperature, frozen on dry ice and stored at -80°C. Isolation of RNA from PAXgene tubes was performed according to the manufacturer's protocol. Quantitation of total RNA samples was performed using an Eppendorf BioPhotometer 6131 (Eppendorf, Hamburg, Germany). RNA quality was assessed using an Agilent BioAnalyzer with the RNA Nano-chip (Agilent).
Human PBMC and purified blood cell collection and RNA isolation
Whole blood was collected in 8 mL CPT tubes (Becton-Dickinson, Franklin Lakes, NJ) from healthy donors and the CPT tubes were processed for the isolation of PMBCs according to the manufacturer's protocol. All PBMC preps from a single donor were pooled for cell counts and subsequent analysis. The cell number and cellular composition of each PBMC fraction was determined by Pentra C60+ automated cell counter (Horiba ABX, Montpelier, France). For ex vivo treatment with LXR agonist, the purified PBMC were resuspended in culture medium (RPMI + 10% fetal calf serum + 1% penicillin/streptomycin with 1% L-glutamine), transferred to 6-well (9.5 cm2 each) tissue culture dishes at approximately 5 × 106 cells per well, and 2 uM LXR-623 or vehicle (DMSO) were added. After 18 hours of culture, RNA isolation and qPCR analysis for LXRα, LXRβ, ABCA1, ABCG1, and PLTP was performed. At time of harvest, conditioned media was removed and centrifuged at 450 × g for 5 minutes to pellet any cells that were not adherent. The adherent cells remaining on the plate were lysed by the addition of 1.2 ml RLT lysis buffer (Qiagen) containing 150 mM 2-mercaptoethanol (Sigma, St. Louis, MO) to the plate, the lysed cells were scraped from the plate with a cell lifter, and the lysed cells in RLT buffer were transferred to the cell pellet from the centrifuged conditioned media. The cell pellet was resuspended by vortexing, and the total cell lysate was used for RNA isolation using the RNeasy Mini RNA Isolation Kit (Qiagen). Quantitation of total RNA samples was performed using an Eppendorf BioPhotometer 6131; RNA yields averaged 4.5 ug total RNA per culture well. RNA quality was assessed using an Agilent BioAnalyzer with the RNA Nano-chip (Agilent).
Fresh human PBMC, T cells, B cells, and monocytes from normal human donors were purchased from AllCells (Emeryville, CA). Each cell set was derived from the same donor for comparison of response within a donor. The cells were cultured, treated, and harvested as described above for the PBMC cultures.
Human whole blood collection and RNA isolation
ABCA1 and ABCG1 expression was evaluated in human clinical samples from a Wyeth-sponsored, single-center Phase 1 single ascending dose (SAD) clinical study (3201A1-100) of LXR-623 encompassing 40 healthy human subjects. Whole blood was collected into PAXgene tubes 2 hours prior to dosing and at time points of 2, 4, 12, 24, and 48 hours following oral administration of a single dose of LXR-623. RNA was purified from the PAXgene tubes as described above for the non-human primate samples. Sample RNA quality was assessed using an Agilent BioAnalyzer with the RNA Nano-chip (Agilent), using the RIN (RNA Integrity Number) algorithm  provided with the instrument software. For these samples, the mean RIN ranged from 4.1–8.8, with a mean RIN of 6.8.
Preparation and purification of cDNA
Purified RNA was converted to cDNA for subsequent qRT-PCR using the High Capacity cDNA Archive Kit (Applied Biosystems, Foster City, CA; PN4322171), following the manufacturer's protocol. cDNA was subsequently purified from the reaction mix using the QIAquick PCR Purification kit (Qiagen PN28104) according to the instructions provided with the kit.
All quantitative RT-PCR (qPCR, TaqMan®) reactions described below were run on an Applied Biosystems 7500 Real Time PCR System using the following cycling parameters: Step 1: 50°C, 2 minutes; Step 2: 95°C, 10 minutes; Step 3: 95°C, 15 seconds; Step 4: 60°C, 1 minute; repeat Steps 3 and 4, 39 more times. Amplification of transcripts for the genes of interest in each sample was compared to the same assay run on a "standard curve" consisting of a dilution series of cDNA prepared from RNA from an appropriate tissue source, unless otherwise noted. Standard curve cDNA concentrations were determined empirically so that the CT values for the input experimental samples fell within the experimental range of the respective standard curve for each transcript of interest. Input cDNA amounts were determined by titration experiments for each transcript. Amounts were chosen that best allowed for changes in CT due to experimental conditions while remaining on the standard curve. Data analysis was performed according to the Relative Standard Curve Method .
Quantitative RT-PCR on mouse RNA samples utilized the following assays from Applied Biosystems: ABCA1, Mm00442646_m1; ABCG1, Mm00437390_m1. The mouse GAPDH transcript was measured for each sample to normalize the amount of input RNA for each reaction, using the Applied Biosystems Rodent GAPDH Control Reagent Kit (# 4308313). Amplification of the genes in each sample was compared to the same assay run on a "standard curve" consisting of a dilution series of cDNA prepared from RNA from a mixture of mouse tissues (Mouse Universal RNA, Clontech # 636657).
Quantitative RT-PCR on rat RNA samples utilized the following oligonucleotide probe/primer sets: ABCA1, probe FAM-AGGATGTGGTGGAGCAGGCG and primers, forward 5'-GGGTGGCTTCGCCTACTTG-3' and reverse-5'-GACGCCCGTTTTCTTCTCAG-3'; ABCG1, probe FAM-TCACACATCGGGATCGGTCTC and primers, forward 5'-GTACTGACACACCTGCGAATCAC-3' and reverse-5'-TCGTTCCCAATCCCAAGGTA-3'. The rat GAPDH transcript was measured for each sample to normalize the amount of input RNA for each reaction, using the Applied Biosystems Rodent GAPDH Control Reagent Kit (# 4308313).
For measuring monkey transcripts, primate-specific primer and probe sets for ABCA1 and ABCG1 were designed with Primer Express Software (Applied Biosystems, Foster City, CA). The ABCG1 probe, FAM-CTGGTGACGAGAGGCTTCCTCAGTCC and primers, forward 5'-GGCAGAATTTAAAACTGCAACACA-3' and reverse-5'-GGTGCCTGGTACTAAGGAGCAA-3', were designed using Rhesus macaque nucleotide sequence (Genbank Accession # BV209042). Human ABCA1 TaqMan® reagents, reported previously  were used for ABCA1 quantitation following their validation using total RNA from cynomolgus monkey liver (Biochain, Hayward, CA) and results were normalized to human 18S rRNA (Applied Biosystems Eukaryotic 18S rRNA Control Assay Hs99999901_s1) following validation of this 18S rRNA assay on monkey RNA.
For measuring human transcripts, the following quantitative RT-PCR assays were obtained from Applied Biosystems: ABCA1, Hs00194045_m1; ABCG1, Hs00245154_m1; PLTP, Hs00272126_m1. The human GAPDH transcript was measured for each sample to normalize the amount of input RNA for each reaction, using the Human GAPDH Control Reagent Kit (# 402869). Amplification of the genes in each sample was compared to the same assay run on a "standard curve" consisting of a dilution series of cDNA prepared from RNA from a mixture of human tissues (Human Universal RNA, Clontech # 636538).
Measurement of ABCA1 and ABCG1 transcripts in blood samples from the human clinical study of LXR-623 in healthy human subjects was performed using the same Applied Biosystems human TaqMan assays as described above (ABCA1, Hs00194045_m1; ABCG1, Hs00245154_m1; GAPDH, Endogenous Control Kit # 402869). However, an ''external standard'' approach was utilized, in which TaqMan data from each assay is compared to a standard curve generated with known quantities of pre-prepared transcript for each target. ABCA1, ABCG1 and GAPDH cDNAs in pXL5 cloning vectors were obtained from Origene (Rockville, MD). Pure synthetic standards for each transcript were prepared by in vitro transcription and purified. Transcripts were quantitated, diluted to 109 copies/mL, aliquoted and stored at -80°C until use. Data generated from samples were compared to standard curves utilizing these synthetic standards, quantitated and normalized in terms of number of target transcripts per 106 GAPDH molecules.
For human TaqMan assays, two-step RT-PCR reactions were performed using the TaqMan Gold RT-PCR Kit from Applied Biosystems (cat # N808-0233) according to the manufacturer's instructions. The kit includes TaqMan PCR Core Reagents (catalog # N808-0228), TaqMan Reverse Transcription Reagents (catalog # N808-0234) and TaqMan GAPDH Control Reagents (catalog # 402869). qPCR reactions were run on an Applied Biosystems 7500 Real Time PCR System using the following cycling parameters: Step 1: 50°C, 2 minutes; Step 2: 95°C, 10 minutes; Step 3: 95°C, 15 seconds; Step 4: 60°C, 1 minute; repeat Steps 3 and 4, 39 more times. Data analysis was performed according to the Relative Standard Curve Method .
Microarray analysis of global gene expression
PBMC were purified from normal human donors (n = 4), and separately treated ex vivo as described above with either 2 uM LXR-623 or vehicle (0.1% DMSO) for 18 hours. RNA was purified as described above, and amplified and labeled using the Ovation Biotin Labeling and RNA Amplification System (NuGEN, San Carlos, CA). The labeled RNA was then used for microarray analysis using the GeneChip® HG U133 2.0 Plus array (Affymetrix, Santa Clara, CA). Expression profiling was performed on the GeneChips® as described previously . Hybridization signal intensities of probe sets representing each gene were measured for individual samples in each cohort group (LXR-623 treated vs. vehicle), and an average signal intensity for that gene was then calculated and compared to the average signal values from the other cohort. Genes were judged to be changed significantly by treatment if the change in the mean hybridization signal intensity for the probe set(s) representing that gene were > 2 fold higher or lower in the treatment group than in the control group, with a p-value < 0.05 as determined by Student's t test.
Analysis of protein expression by immunoblotting
PBMC was isolated from human blood collected in 8 ml CPT-citrate tubes (within an hour of collection), and plated onto 100 mm tissue culture dishes in RPMI containing 10% FBS, 2 uM L-glutamine and 50 IU/ml penicillin and 50 ug/ml streptomycin at a density of 10 million cells/plate. After allowing cells to settle for 90 minutes, the cells were treated with or without LXR agonists (2 uM) for 24 hours or 48 hours. Cells were lysed at the end of the incubation in 1 × Cell lysis buffer (Cell Signaling Technologies) containing Pefabloc SC (protease inhibitor) on ice for 10 minutes (500 ul/plate). Both adherent and non-adherent cells were collected. Equal volumes (16.25 ul) of cell lysate were loaded into each well of NuPAGE 4–12% Bis-Tris gels (Invitrogen), and Full-Range molecular weight markers (RPN800, GE Healthcare) were used to assess molecular weights. Separated proteins were electroblotted onto a nitrocellulose membrane (Invitrogen). The membranes were blocked in 5% Blot-QuickBlocker (Gbiosciences, St. Louis, MO) for one hour followed by washing in washing buffer (PBS, 0.1% Tween20). To determine the equivalence of protein loading between samples, actin protein in each sample was detected by Western blotting using an anti-actin antibody (Actin (1–19)-HRP, Santa Cruz, 1:2000). In addition, protein loading was assessed by staining the membrane with Ponceau S (Sigma). Duplicate membranes were blotted separately with anti-ABCA1 (Novus Biologicals, NB400-10555, 1:500), anti-ABCG1 (Abcam AB36969, 1:2500), or anti-LXRα (Novus NB300-612, 1:400). Unbound antibodies were removed by washing the membrane three times for 15 minutes each in washing buffer and were then incubated with secondary antibodies (anti-goat-HRP, Chemicon or anti-rabbit-HRP, NEF812001 Perkin Elmer 1:2000) for one hour followed by another three washes in the washing buffers as above. Proteins of interest were detected by chemiluminescence using ECL Western blotting detection reagents (Amersham). Correct bands were identified by molecular weight, and specificity was confirmed by comparing with a duplicate blot incubated with a different antibody.
LXRs are expressed in peripheral blood mononuclear cells
LXR agonists induce gene expression in rodent peripheral blood cells in vivo
Although gene induction in peripheral blood was correlated with plasma drug levels, the critical physiological effects of LXR activation are thought to reside within tissues such as the intestine, liver, or macrophages within the atherosclerotic lesion. Gene expression or drug concentration within these tissues cannot be easily monitored. To determine whether activation of gene expression in peripheral blood cells could provide insight into gene regulation within tissues, the induction of ABCA1 and ABCG1 within the spleen, an organ highly enriched in immune system cells, was compared to induction in peripheral blood cells. For GW3965, there was a strong correlation between the induction of ABCA1 or ABCG1 in the blood and spleen (Figure 2D). However, for LXR-623 the spleen appeared to have increased sensitivity relative to the peripheral blood at low plasma concentrations (Figure 2E). Whether this difference between ligands reflects differing levels of LXRα and LXRβ expression in blood cells versus spleen, or is due to some other factor such as differing coactivator abundance, remains to be determined. These initial results indicate that induction of LXR target gene regulation in the peripheral blood may serve as an indicator of target gene induction in relevant tissues.
ABCA1 and ABCG1 transcription in peripheral blood cells of non-human primates is regulated in a dose-dependent manner by oral dosing of LXR-623
Human peripheral blood mononuclear cells respond to ex vivo LXR-623 exposure by increasing expression of LXR target genes
Up-Regulated Human Peripheral Blood Biomarkers of LXR-623 Activity
ATP-binding cassette, sub-family G (WHITE), member 1
stearoyl-CoA desaturase (delta-9-desaturase)
ATP-binding cassette, sub-family A (ABC1), member 1
sterol regulatory element binding transcription factor 1
phospholipid transfer protein
low density lipoprotein receptor (familial hypercholesterolemia)
nuclear receptor subfamily 1, group H, member 3
fatty acid desaturase 1
Multiple cell types in human PBMC express functional LXRα and LXRβ
ABCA1 and ABCG1 expression is increased in peripheral blood of human subjects following oral administration of LXR-623
Assessment of temporal profiles in each of the biomarker transcripts in peripheral blood collected from a representative subject receiving LXR-623 (Figure 7, panels C and D) revealed that peak transcriptional levels of ABCA1 and ABCG1 were detected 4 hours post-dosing, after which the levels of ABCA1 and ABCG1 decreased back to baseline levels by twenty-four to forty-eight hours. Strong dose-response and exposure-response relationships were observed for ABCA1 and ABCG1 transcriptional biomarkers in subjects receiving ascending doses of LXR-623, and these will be reported in a publication describing all of the results of the single ascending dose study in detail (A. Katz et al, submitted).
The intent of this work was to identify easily accessible, rapid, and robust indicators of LXR agonist exposure and activity to aid in the clinical development of synthetic LXR modulator compounds. An ideal surrogate tissue for such analyses is peripheral blood, but it was unclear whether LXR agonist activity could be monitored in peripheral blood. It was well known from the literature that activated macrophages (usually tissue-associated and not freely circulating in blood) respond to LXR agonists by increasing the expression of certain LXR target genes such as the ABC-cassette genes. Landis et al.  had previously reported that treatment of purified human primary monocytes in culture with a combination of oxidized LDL and 9-cis-retinoic caused the induction of TNFα expression and secretion, suggesting that LXRs may be expressed and functional in peripheral blood cells. But subsequent experiments to show that the monocytes' response to LXR agonist treatment was mediated by LXR binding to an LXR response element (LXRE) in the promoter of the TNFα gene were performed in cells transfected with an expression vector containing LXRα , so proof that circulating monocytes expressed functional LXRs was not conclusively established. There have been some reports of LXR expression and response to agonists in T-cells [43, 44]. More recently, Siest et al  showed weak and variable expression of LXRα and LXRβ mRNA in PBMC from normal human donors using custom microarrays. However, this technique is relatively insensitive compared to qPCR, and no data were provided on the functionality of LXRs in PBMC. Therefore, we sought to determine whether transcriptional biomarkers of LXR activity could be monitored in peripheral blood.
Data presented here show that human peripheral blood mononuclear cells express LXRα and LXRβ. Surprisingly, functional LXR expression was found in T- and B-cells as well as in monocytes ex vivo. Evaluation of the transcriptional response of peripheral blood to synthetic LXR agonists in vivo was first performed in rats and mice, where expression of LXR target genes ABCA1 and ABCG1 was found to be significantly increased by different LXR agonist compounds, and as early as one hour following a single oral dose of LXR-623. These observations were then confirmed with experiments in higher species, in which monkeys given daily doses of LXR agonist compound showed robust and persistent expression changes in ABCA1 and ABCG1 in peripheral blood RNA after 7 days of dosing. These results were then extended to humans using blood cells from healthy subjects treated ex vivo with LXR-623. In both rats and humans given a single dose of LXR-623, the induction of ABCA1 and ABCG1 expression in peripheral blood cells tracked closely with plasma drug levels. Intriguingly, the elevation of ABCA1 and ABCG1 mRNA was not sustained beyond the peak of plasma LXR-623 concentration, suggesting a short in vivo t1/2 for these two mRNAs and the dependence of mRNA levels primarily upon transcription rate. This attribute is advantageous for pharmacodynamic biomarkers.
We applied global transcriptional profiling to human PBMC's treated with LXR-623 in culture to evaluate the repertoire of gene expression in peripheral blood and to determine whether the spectrum of transcriptional changes appeared to have biological relevance. It was found that many LXR target genes known to be regulated in macrophage, liver, or duodenum were also regulated in peripheral blood cells, and these genes were known to be involved in reverse cholesterol transport and lipid metabolism. This observation, combined with an apparent correlation between blood and spleen response to LXR agonists in the rat, suggests that the LXR response that can be monitored in peripheral blood may have clinical significance and might ultimately provide surrogate transcriptional markers of biological efficacy.
ABCA1 and ABCG1 were evaluated as pharmacodynamic markers of LXR-623 exposure in a single ascending dose study of LXR-623 in healthy human volunteers. In human whole blood RNA, ABCA1 and ABCG1 responded with similar temporal profiles following LXR-623 exposure in a representative human subject, indicating that the compound was appropriately engaging its target in vivo and eliciting the expected biological response. Future studies will attempt to correlate peripheral blood response to LXR agonist compound with ultimate biological efficacy endpoints.
Peripheral blood cells show promise as a surrogate tissue for monitoring the activity of LXR modulator compounds in target organs. Several candidate biomarkers of LXR agonist exposure and activity have been identified in peripheral blood, and two of these (ABCA1 and ABCG1) have been demonstrated to change substantially (up to 200 fold change) and rapidly (≤ 4 hours) upon compound treatment. These transcriptional markers have been shown to be upregulated in peripheral blood cells from rodents, primates, and humans, and the magnitude of transcriptional induction of these biomarkers in peripheral blood cells closely corresponds to LXR agonist compound concentration in serum. These LXR biomarkers have already proven to be useful for the evaluation of a novel synthetic LXR agonist in a human clinical study.
The authors wish to thank Drs. Arie Katz and Xu Meng for providing samples for measurement of ABCA1 and ABCG1 transcript levels from a representative subject participating in the LXR-623 SAD study, and Ms. Aamani Parchuri for technical assistance. This work was supported by Wyeth Pharmaceuticals.
- Jaye M: LXR agonists for the treatment of atherosclerosis. Curr Opin Investig Drugs. 2003, 4: 1053-1058.PubMedGoogle Scholar
- Joseph SB, Tontonoz P: LXRs: new therapeutic targets in atherosclerosis?. Curr Opin Pharmacol. 2003, 3: 192-197. 10.1016/S1471-4892(03)00009-2.View ArticlePubMedGoogle Scholar
- Tontonoz P, Mangelsdorf DJ: Liver × receptor signaling pathways in cardiovascular disease. Mol Endocrinol. 2003, 17: 985-993. 10.1210/me.2003-0061.View ArticlePubMedGoogle Scholar
- Janowski BA, Willy PJ, Devi TR, Falck JR, Mangelsdorf DJ: An oxysterol signalling pathway mediated by the nuclear receptor LXR alpha. Nature. 1996, 383: 728-731. 10.1038/383728a0.View ArticlePubMedGoogle Scholar
- 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. 10.1101/gad.9.9.1033.View ArticlePubMedGoogle Scholar
- Willy PJ, Mangelsdorf DJ: Unique requirements for retinoid-dependent transcriptional activation by the orphan receptor LXR. Genes Dev. 1997, 11: 289-298. 10.1101/gad.11.3.289.View ArticlePubMedGoogle Scholar
- Steffensen KR, Gustafsson JA: Putative metabolic effects of the liver × receptor (LXR). Diabetes. 2004, 53 (Suppl 1): S36-42. 10.2337/diabetes.53.2007.S36.View ArticlePubMedGoogle Scholar
- Repa JJ, Turley SD, Lobaccaro JA, Medina J, Li L, Lustig K, Shan B, Heyman RA, Dietschy JM, Mangelsdorf DJ: Regulation of absorption and ABC1-mediated efflux of cholesterol by RXR heterodimers. Science. 2000, 289: 1524-1529. 10.1126/science.289.5484.1524.View ArticlePubMedGoogle Scholar
- Gupta S, Pandak WM, Hylemon PB: LXR alpha is the dominant regulator of CYP7A1 transcription. Biochem Biophys Res Commun. 2002, 293: 338-343. 10.1016/S0006-291X(02)00229-2.View ArticlePubMedGoogle Scholar
- Repa JJ, Liang G, Ou J, Bashmakov Y, Lobaccaro JM, Shimomura I, Shan B, Brown MS, Goldstein JL, Mangelsdorf DJ: Regulation of mouse sterol regulatory element-binding protein-1c gene (SREBP-1c) by oxysterol receptors, LXRalpha and LXRbeta. Genes Dev. 2000, 14: 2819-2830. 10.1101/gad.844900.PubMed CentralView ArticlePubMedGoogle Scholar
- Quinet EM, Savio DA, Halpern AR, Chen L, Schuster GU, Gustafsson JA, Basso MD, Nambi P: Liver × receptor (LXR)-beta regulation in LXRalpha-deficient mice: implications for therapeutic targeting. Mol Pharmacol. 2006, 70: 1340-1349. 10.1124/mol.106.022608.View ArticlePubMedGoogle Scholar
- Lund EG, Peterson LB, Adams AD, Lam MH, Burton CA, Chin J, Guo Q, Huang S, Latham M, Lopez JC, Menke JG, Milot DP, Mitnaul LJ, Rex-Rabe SE, Rosa RL, Tian JY, Wright SD, Sparrow CP: Different roles of liver × receptor alpha and beta in lipid metabolism: effects of an alpha-selective and a dual agonist in mice deficient in each subtype. Biochem Pharmacol. 2006, 71: 453-463. 10.1016/j.bcp.2005.11.004.View ArticlePubMedGoogle Scholar
- Millatt LJ, Bocher V, Fruchart JC, Staels B: Liver × receptors and the control of cholesterol homeostasis: potential therapeutic targets for the treatment of atherosclerosis. Biochim Biophys Acta. 2003, 1631: 107-118.View ArticlePubMedGoogle Scholar
- Schwartz K, Lawn RM, Wade DP: ABC1 gene expression and ApoA-I-mediated cholesterol efflux are regulated by LXR. Biochem Biophys Res Commun. 2000, 274: 794-802. 10.1006/bbrc.2000.3243.View ArticlePubMedGoogle Scholar
- Venkateswaran A, Repa JJ, Lobaccaro JM, Bronson A, Mangelsdorf DJ, Edwards PA: Human white/murine ABC8 mRNA levels are highly induced in lipid-loaded macrophages. A transcriptional role for specific oxysterols. J Biol Chem. 2000, 275: 14700-14707. 10.1074/jbc.275.19.14700.View ArticlePubMedGoogle Scholar
- Naik SU, Wang X, Da Silva JS, Jaye M, Macphee CH, Reilly MP, Billheimer JT, Rothblat GH, Rader DJ: Pharmacological activation of liver × receptors promotes reverse cholesterol transport in vivo. Circulation. 2006, 113: 90-97. 10.1161/CIRCULATIONAHA.105.560177.View ArticlePubMedGoogle Scholar
- Klucken J, Buchler C, Orso E, Kaminski WE, Porsch-Ozcurumez M, Liebisch G, Kapinsky M, Diederich W, Drobnik W, Dean M, Allikmets R, Schmitz G: ABCG1 (ABC8), the human homolog of the Drosophila white gene, is a regulator of macrophage cholesterol and phospholipid transport. Proc Natl Acad Sci USA. 2000, 97: 817-822. 10.1073/pnas.97.2.817.PubMed CentralView ArticlePubMedGoogle Scholar
- Levy E, Spahis S, Sinnett D, Peretti N, Maupas-Schwalm F, Delvin E, Lambert M, Lavoie MA: Intestinal cholesterol transport proteins: an update and beyond. Curr Opin Lipidol. 2007, 18: 310-318. 10.1097/MOL.0b013e32813fa2e2.View ArticlePubMedGoogle Scholar
- Hu B, Collini M, Unwalla R, Miller C, Singhaus R, Quinet E, Savio D, Halpern A, Basso M, Keith J, Clerin V, Chen L, Resmini C, Liu QY, Feingold I, Huselton C, Azam F, Farnegardh M, Enroth C, Bonn T, Goos-Nilsson A, Wilhelmsson A, Nambi P, Wrobel J: Discovery of phenyl acetic acid substituted quinolines as novel liver × receptor agonists for the treatment of atherosclerosis. J Med Chem. 2006, 49: 6151-6154. 10.1021/jm0609566.View ArticlePubMedGoogle Scholar
- Hu B, Jetter J, Kaufman D, Singhaus R, Bernotas R, Unwalla R, Quinet E, Savio D, Halpern A, Basso M, Keith J, Clerin V, Chen L, Liu QY, Feingold I, Huselton C, Azam F, Goos-Nilsson A, Wilhelmsson A, Nambi P, Wrobel J: Further modification on phenyl acetic acid based quinolines as liver × receptor modulators. Bioorg Med Chem. 2007, 15: 3321-3333. 10.1016/j.bmc.2007.03.013.View ArticlePubMedGoogle Scholar
- Joseph SB, McKilligin E, Pei L, Watson MA, Collins AR, Laffitte BA, Chen M, Noh G, Goodman J, Hagger GN, Tran J, Tippin TK, Wang X, Lusis AJ, Hsueh WA, Law RE, Collins JL, Willson TM, Tontonoz P: Synthetic LXR ligand inhibits the development of atherosclerosis in mice. Proc Natl Acad Sci USA. 2002, 99: 7604-7609. 10.1073/pnas.112059299.PubMed CentralView ArticlePubMedGoogle Scholar
- Schultz JR, Tu H, Luk A, Repa JJ, Medina JC, Li L, Schwendner S, Wang S, Thoolen M, Mangelsdorf DJ, Lustig KD, Shan B: Role of LXRs in control of lipogenesis. Genes Dev. 2000, 14: 2831-2838. 10.1101/gad.850400.PubMed CentralView ArticlePubMedGoogle Scholar
- Terasaka N, Hiroshima A, Koieyama T, Ubukata N, Morikawa Y, Nakai D, Inaba T: T-0901317 a synthetic liver × receptor ligand, inhibits development of atherosclerosis in LDL receptor-deficient mice. FEBS Lett. 2003, 536: 6-11. 10.1016/S0014-5793(02)03578-0.View ArticlePubMedGoogle Scholar
- Mertens A, Verhamme P, Bielicki JK, Phillips MC, Quarck R, Verreth W, Stengel D, Ninio E, Navab M, Mackness B, Mackness M, Holvoet P: Increased low-density lipoprotein oxidation and impaired high-density lipoprotein antioxidant defense are associated with increased macrophage homing and atherosclerosis in dyslipidemic obese mice: LCAT gene transfer decreases atherosclerosis. Circulation. 2003, 107: 1640-1646. 10.1161/01.CIR.0000056523.08033.9F.View ArticlePubMedGoogle Scholar
- Westhuyzen J: The oxidation hypothesis of atherosclerosis: an update. Ann Clin Lab Sci. 1997, 27: 1-10.PubMedGoogle Scholar
- Iuliano L: The oxidant stress hypothesis of atherogenesis. Lipids. 2001, 36 (Suppl): S41-44. 10.1007/s11745-001-0680-1.View ArticlePubMedGoogle Scholar
- van Leuven SI, Kastelein JJ, D'Cruz DP, Hughes GR, Stroes ES: Atherogenesis in rheumatology. Lupus. 2006, 15: 117-121. 10.1191/0961203306lu2273rr.View ArticlePubMedGoogle Scholar
- Robbesyn F, Salvayre R, Negre-Salvayre A: Dual role of oxidized LDL on the NF-kappaB signaling pathway. Free Radic Res. 2004, 38: 541-551. 10.1080/10715760410001665244.View ArticlePubMedGoogle Scholar
- Collins JL, Fivush AM, Watson MA, Galardi CM, Lewis MC, Moore LB, Parks DJ, Wilson JG, Tippin TK, Binz JG, Plunket KD, Morgan DG, Beaudet EJ, Whitney KD, Kliewer SA, Willson TM: Identification of a nonsteroidal liver × receptor agonist through parallel array synthesis of tertiary amines. J Med Chem. 2002, 45: 1963-1966. 10.1021/jm0255116.View ArticlePubMedGoogle Scholar
- Schroeder A, Mueller O, Stocker S, Salowsky R, Leiber M, Gassmann M, Lightfoot S, Menzel W, Granzow M, Ragg T: The RIN: an RNA integrity number for assigning integrity values to RNA measurements. BMC Molecular Biology. 2006, 7: 3-10.1186/1471-2199-7-3.PubMed CentralView ArticlePubMedGoogle Scholar
- ABI PRISM 7700 Sequence Detection System User Bulletin #2: Relative Quantitation of Gene Expression. [http://www3.appliedbiosystems.com/cms/groups/mcb_support/documents/generaldocuments/cms_040980.pdf]
- Quinet EM, Savio DA, Halpern AR, Chen L, Miller CP, Nambi P: Gene-selective modulation by a synthetic oxysterol ligand of the liver × receptor. J Lipid Res. 2004, 45: 1929-1942. 10.1194/jlr.M400257-JLR200.View ArticlePubMedGoogle Scholar
- LaVallie ER, Chockalingam PS, Collins-Racie LA, Freeman BA, Keohan CC, Leitges M, Dorner AJ, Morris EA, Majumdar MK, Arai M: Protein kinase Czeta is up-regulated in osteoarthritic cartilage and is required for activation of NF-kappaB by tumor necrosis factor and interleukin-1 in articular chondrocytes. J Biol Chem. 2006, 281: 24124-24137. 10.1074/jbc.M601905200.View ArticlePubMedGoogle Scholar
- Chawla A, Boisvert WA, Lee CH, Laffitte BA, Barak Y, Joseph SB, Liao D, Nagy L, Edwards PA, Curtiss LK, Evans RM, Tontonoz P: A PPAR gamma-LXR-ABCA1 pathway in macrophages is involved in cholesterol efflux and atherogenesis. Mol Cell. 2001, 7: 161-171. 10.1016/S1097-2765(01)00164-2.View ArticlePubMedGoogle Scholar
- Costet P, Luo Y, Wang N, Tall AR: Sterol-dependent transactivation of the ABC1 promoter by the liver × receptor/retinoid × receptor. J Biol Chem. 2000, 275: 28240-28245.PubMedGoogle Scholar
- 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 USA. 2001, 98: 507-512. 10.1073/pnas.021488798.PubMed CentralView ArticlePubMedGoogle Scholar
- Sun Y, Hao M, Luo Y, Liang CP, Silver DL, Cheng C, Maxfield FR, Tall AR: Stearoyl-CoA desaturase inhibits ATP-binding cassette transporter A1-mediated cholesterol efflux and modulates membrane domain structure. J Biol Chem. 2003, 278: 5813-5820. 10.1074/jbc.M208687200.View ArticlePubMedGoogle Scholar
- Mak PA, Laffitte BA, Desrumaux C, Joseph SB, Curtiss LK, Mangelsdorf DJ, Tontonoz P, Edwards PA: Regulated expression of the apolipoprotein E/C-I/C-IV/C-II gene cluster in murine and human macrophages. A critical role for nuclear liver × receptors alpha and beta. J Biol Chem. 2002, 277: 31900-31908. 10.1074/jbc.M202993200.View ArticlePubMedGoogle Scholar
- Cao G, Beyer TP, Yang XP, Schmidt RJ, Zhang Y, Bensch WR, Kauffman RF, Gao H, Ryan TP, Liang Y, Eacho PI, Jiang XC: Phospholipid transfer protein is regulated by liver × receptors in vivo. J Biol Chem. 2002, 277: 39561-39565. 10.1074/jbc.M207187200.View ArticlePubMedGoogle Scholar
- Ishimoto K, Tachibana K, Sumitomo M, Omote S, Hanano I, Yamasaki D, Watanabe Y, Tanaka T, Hamakubo T, Sakai J, Kodama T, Doi T: Identification of human low-density lipoprotein receptor as a novel target gene regulated by liver × receptor alpha. FEBS Lett. 2006, 580: 4929-4933. 10.1016/j.febslet.2006.08.010.View ArticlePubMedGoogle Scholar
- Whitney KD, Watson MA, Goodwin B, Galardi CM, Maglich JM, Wilson JG, Willson TM, Collins JL, Kliewer SA: Liver × receptor (LXR) regulation of the LXRalpha gene in human macrophages. J Biol Chem. 2001, 276: 43509-43515. 10.1074/jbc.M106155200.View ArticlePubMedGoogle Scholar
- Landis MS, Patel HV, Capone JP: Oxysterol activators of liver × receptor and 9-cis-retinoic acid promote sequential steps in the synthesis and secretion of tumor necrosis factor-alpha from human monocytes. J Biol Chem. 2002, 277: 4713-4721. 10.1074/jbc.M108807200.View ArticlePubMedGoogle Scholar
- Walcher D, Kummel A, Kehrle B, Bach H, Grub M, Durst R, Hombach V, Marx N: LXR Activation Reduces Proinflammatory Cytokine Expression in Human CD4-Positive Lymphocytes. Arterioscler Thromb Vasc Biol. 2006, 26: 1022-1028. 10.1161/01.ATV.0000210278.67076.8f.View ArticlePubMedGoogle Scholar
- Hindinger C, Hinton DR, Kirwin SJ, Atkinson RD, Burnett ME, Bergmann CC, Stohlman SA: Liver × receptor activation decreases the severity of experimental autoimmune encephalomyelitis. Journal of Neuroscience Research. 2006, 84: 1225-1234. 10.1002/jnr.21038.View ArticlePubMedGoogle Scholar
- Siest G, Jeannesson E, Marteau J-B, Samara A, Marie B, Pfister M, Visvikis-Siest S: Transcription Factor and Drug-Metabolizing Enzyme Gene Expression in Lymphocytes from Healthy Human Subjects. Drug Metab Dispos. 2008, 36: 182-189. 10.1124/dmd.107.017228.View ArticlePubMedGoogle Scholar
This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.