The authors have the following interests: All authors are employees of Bristol-Myers Squibb, Co., the funder of this study. In addition, Bristol-Myers Squibb does have an interest in developing an inhibitor of 11Beta-Hydroxysteroid Dehydrogenase Type 1, the experimental gene described in the manuscript entitled; “11beta-hydroxysteroid dehydrogenase type 1 gene knockout attenuates atherosclerosis and in vivo foam cell formation in hyperlipidemic apoE−/− mice”. Patents protecting the intellectual property around the inhibitors are in process. However, no inhibitors, experimental or otherwise, were used in the studies described in this manuscript. There are no further patents, products in development or marketed products to declare. This does not alter the authors' adherence to all the PLOS ONE policies on sharing data and materials.
Conceived and designed the experiments: RG DG JO JC PG WY TWH JR. Performed the experiments: DS JL BG AT AH MY CR LW RL PS NC JT RZ GP SH RY. Analyzed the data: RG JO PG DS JL BG AT AH MY CR LW RL PS NC JT RZ GP SH RY. Contributed reagents/materials/analysis tools: DS JL BG AT AH MY CR LW RL PS NC JT RZ GP SH. Wrote the paper: RG DG.
Chronic glucocorticoid excess has been linked to increased atherosclerosis and general cardiovascular risk in humans. The enzyme 11β-hydroxysteroid dehydrogenase type 1 (11βHSD1) increases active glucocorticoid levels within tissues by catalyzing the conversion of cortisone to cortisol. Pharmacological inhibition of 11βHSD1 has been shown to reduce atherosclerosis in murine models. However, the cellular and molecular details for this effect have not been elucidated.
To examine the role of 11βHSD1 in atherogenesis, 11βHSD1 knockout mice were created on the pro-atherogenic apoE−/− background. Following 14 weeks of Western diet, aortic cholesterol levels were reduced 50% in 11βHSD1−/−/apoE−/− mice vs. 11βHSD1+/+/apoE−/− mice without changes in plasma cholesterol. Aortic 7-ketocholesterol content was reduced 40% in 11βHSD1−/−/apoE−/− mice vs. control. In the aortic root, plaque size, necrotic core area and macrophage content were reduced ∼30% in 11βHSD1−/−/apoE−/− mice. Bone marrow transplantation from 11βHSD1−/−/apoE−/− mice into apoE−/− recipients reduced plaque area 39–46% in the thoracic aorta. In vivo foam cell formation was evaluated in thioglycollate-elicited peritoneal macrophages from 11βHSD1+/+/apoE−/− and 11βHSD1−/−/apoE−/− mice fed a Western diet for ∼5 weeks. Foam cell cholesterol levels were reduced 48% in 11βHSD1−/−/apoE−/− mice vs. control. Microarray profiling of peritoneal macrophages revealed differential expression of genes involved in inflammation, stress response and energy metabolism. Several toll-like receptors (TLRs) were downregulated in 11βHSD1−/−/apoE−/− mice including TLR 1, 3 and 4. Cytokine release from 11βHSD1−/−/apoE−/−-derived peritoneal foam cells was attenuated following challenge with oxidized LDL.
These findings suggest that 11βHSD1 inhibition may have the potential to limit plaque development at the vessel wall and regulate foam cell formation independent of changes in plasma lipids. The diminished cytokine response to oxidized LDL stimulation is consistent with the reduction in TLR expression and suggests involvement of 11βHSD1 in modulating binding of pro-atherogenic TLR ligands.
Glucocorticoids are ubiquitous mammalian hormones involved in the regulation of several fundamental biological processes including energy metabolism, inflammation, arousal, cognition and the response to physiological stress. In humans, the primary active glucocorticoid hormone, cortisol, binds to intracellular glucocorticoid and mineralocorticoid receptors found in target tissues. Dysfunctional regulation of glucocorticoid metabolism resulting in excess cortisol in tissues such as adipose, liver and the vasculature has been implicated as a key mediator in the pathogenesis of obesity, type 2 diabetes and cardiovascular disease
Endogenous cortisol levels (corticosterone in mice) are regulated by two distinct pathways. The hypothalamic-pituitary-adrenal axis is the pathway classically associated with regulation of plasma cortisol. In addition, cortisol tone is also regulated intracellularly by the enzymatic activities of two isoforms of 11β-hydroxysteroid dehydrogenase, type 1 (“11βHSD1”) and type 2 (“11βHSD2”). The latter, 11βHSD2, is mainly expressed in aldosterone-target tissues such as kidney, colon and salivary glands
Glucocorticoid amplification by increased 11βHSD1 activity in highly metabolic tissues such as adipose and liver is proposed to potentiate a phenotype resembling the metabolic syndrome
The beneficial effects of 11βHSD1 inhibition on atherosclerosis have been demonstrated in the mouse with small-molecule 11βHSD1 inhibitors
To clarify these issues, 11βHSD1-deficient mice were created on the apoE knockout background to investigate the propensity of atherosclerosis development. The impact of 11βHSD1 deficiency on plasma lipid profiles, vessel wall atherosclerosis and macrophage function was evaluated in Western diet-fed mice. Intrinsic differences in gene expression profiles and biological pathways affected by 11βHSD1 gene deficiency were evaluated by microarray-based gene profiling of thioglycollate-elicited macrophages from hyperlipidemic mice. The results of these analyses and their implications are described herein.
Animal studies were performed according to guidelines established by the American Association for Accreditation of Laboratory Animal Care and protocols were approved by the Bristol-Myers Squibb-Hopewell Animal Care and Use Committee. 11βHSD1 knockout mice were created at Lexicon Pharmaceuticals (The Woodlands, TX) as described in
Male apoE−/− mice (8–10 weeks of age; B6.129P2-Apoetm1Unc/J) purchased from The Jackson Laboratory were subjected to whole body irradiation to induce bone marrow aplasia using a Mark I Model 30 irradiator (JL Shepherd & Associates, San Francisco, CA). Irradiation was carried out with a single exposure of 1000 rads using a cesium radiation source. Donor bone marrow cells were harvested from age-matched male 11β−/−/apoE−/− and 11β+/+/apoE−/− littermate control mice by flushing isolated femurs and tibias with RPMI supplemented with 20 mM HEPES, 10 U/ml heparin and 100 U/ml penicillin/streptomycin. Approximately four hours after irradiation, mice were anesthetized with isoflurane (2–4%) and immediately reconstituted with 5–7 million bone marrow cells given intravenously (∼200 µl) via the retro-orbital sinus. Following bone marrow transplantation, mice were housed in autoclaved cages supplied with HEPA-filtered air and fed regular chow for 4 weeks to enable bone marrow reconstitution. A small group of irradiated control mice that were injected with cell-free vehicle did not survive beyond two weeks post irradiation. Bone marrow transplant-recipient mice were switched to Western diet (Research Diets, D12079B) for 12 weeks to stimulate atherogenesis.
Confirmation of bone marrow reconstitution was carreid out by RT-PCR analysis of whole blood leukocytes, as described (
Blood pressures (systolic, diastolic and mean arterial pressure) were measured in conscious hyperlipidemic 11β−/−/apoE−/− and 11β+/+/apoE−/− littermate control mice using a non-invasive computerized tail cuff system (CODA Non-Invasive Blood Pressure Monitor, Kent Scientific Corporation, Torrington, CT). Mice were conditioned to tail cuff instrumentation over several days to control for stress. As a normolipidemic reference, blood pressures were also measured in chow-fed male C57BL/6 mice. Blood pressure analyses consisted of fifteen tail cuff pressure acquisitions per run. Data for individual animals represent the average of at least five high-quality acquisitions.
EDTA-anti-coagulated blood samples were taken by retro-orbital bleeding following a 4 hr fast and plasma was isolated by centrifugation. Plasma total cholesterol, triglycerides, HDL-cholesterol and non-HDL-cholesterol were analyzed enzymatically using a Siemens Advia 1800 automated chemistry analyzer (Siemens Healthcare Diagnostics, Flanders NJ).
Whole blood was diluted in 1X (final) Nucleic Acid Purification Lysis Buffer (#4305895, Applied Biosystems Inc., Foster City, CA). Total RNA was isolated using an Applied Biosystems Prism 6100 Nucleic Acid Prep Station according to the manufacturer's instructions.
The degree of atherosclerosis development was assessed by aortic lipid extraction (diet-induced atherosclerosis studies) or en face analysis using oil red O stain (bone marrow transplantation studies). Following euthanasia, hearts and aortas were perfused with heparinzed saline solution (0.9% NaCl) followed by 10% formalin. Hearts were stored in formalin. Thoracic aortae were harvested from mice, adventitial connective tissue removed and aortae were delipidated with ethyl acetate:acetone (2∶1 vol/vol) supplemented with 0.01% 2,6,-di-tert-butyl-4-methylphenol. Samples were gently agitated overnight at 37°C and blown dry under nitrogen. Aortae were dried and delipidated dry weights were measured. The dried lipid film from each aorta was resuspended in 0.3 ml of 10% Triton X-100 with gentle agitation at 37°C for 90 minutes. Samples were analyzed by enzymatic assay for total cholesterol using a Roche Cobas Mira Chemistry Analyzer. Cholesterol content was reported with respect to aorta dry weight.
En face staining of the isolated thoracic aorta was carried out using oil red O. The aortic arch and descending aorta were opened longitudinally and pinned to a black wax plate with Austrian fine stainless steel pins (Fine Science Tools, #26002-15). Each aorta was rinsed with 70% ethanol, stained with oil red O and de-stained in 70% ethanol. Following a brief rinse with de-ionized water, pinned aortae were immersed in phosphate-buffered saline and photographed using a Nikon Digital Camera (Nikon Digital Sight DS-Fi1) mounted on Nikon Dissecting Microscope (SMZ 1000, Micron-Optics, Cedar Knolls, NJ). Image analysis was performed using NIS-Elements: Basic Research Version 3.0 software (Micron-Optics, Cedar Knolls, NJ). Lesion area was expressed as a percentage of the total measured aortic area.
To characterize plaque composition, histological analysis of lesions in the aortic root was performed via the Paigen method
Samples were reconstituted in 150 µL of methanol and briefly vortexed. A volume of 130 µL was transferred to 96 well plates for LCMS analysis. A Waters Acquity UPLC System (Milford, MA) in line with a Thermo Fisher LTQ Orbitrap mass spectrometer (Waltham, MA) was used to separate and detect components with mass accuracy <5 ppm. The mass spectrometer was operated in full scan, positive electrospray (+ESI) mode. Reverse-phase gradient LC conditions were employed using a Water Acquity BEH C18 2.1×100 mm 1.7 mm column and acetonitrile-water mobile phases. Component peak integration was performed using Thermo-Fisher Xcalibur Quan Browser software. An accurate mass window of 10 ppm was used to process the following components: cholesterol = 369.3504 (ms source water loss), 7β-hydroxycholesterol = 385.3494 (ms source water loss) 7-ketocholesterol = 401.3405; D7-7β-hydroxycholesterol (internal standard) = 392.3898 (ms source water loss).
In vivo foam cell studies were performed with mice starting at 8 weeks of age and fed a Western diet for 4 weeks (Western diet, 12079B, 20% fat 0.2% cholesterol, Research Diets). Following the dietary lead-in phase, mice were injected with 1.5 ml of a 4% thioglycollate solution to induce peritonitis. Four days post-injection, mice were euthanized and the abdominal cavity was lavaged with 10 ml of ice-cold phosphate-buffered saline to collect peritoneal macrophages. Approximately 100 µl of each cell suspension was analyzed for total cell count via automated cell counting and cell type using modified Wright-Giemsa staining and light microscope evaluation. For analysis of lipid droplet accumulation, cells were stained using oil red O/hematoxylin
Thioglycollate-elicited peritoneal macrophage/foam cells were prepared as described above. Macrophages were plated in DMEM (1 million cells/ml) supplemented with 20% serum and 200 nM 11-dehydrocorticosterone at 37°C for 5 hours. Adherent cells were washed twice with phosphate-buffered saline and incubated with serum-free DMEM containing 200 nM 11-dehydrocorticosterone. Macrophages were incubated overnight in the presence or absence of ∼30 µg/ml copper-oxidized LDL (#BT-910, Biomedical Technologies, Stoughton, MA). Cell media samples were analyzed for cytokine levels via multiplexing immunoassay (#MPXMCYTO-70 K, Millipore, Bedford, MA) using a Luminex 200 System (Austin, TX). Cells were lysed with CelLytic M reagent (#C2978, Sigma, Creamridge, NJ) and total protein content per well was determined by Bradford assay. Cytokine concentrations were normalized to the cellular protein content per well.
Peritoneal macrophages were collected by lavage as described in the preceding section. Total RNA was isolated from the resulting cell pellet using 1 ml of Tri-Reagent (#AM9738, Ambion, Inc., Austin, TX) according to the manufacturer's instructions (Manual 9738M RevC). The integrity of RNA samples was assessed under non-denaturing conditions on 2% E-gels (#G5018-02, Invitrogen Inc., Carlsbad, CA).
For whole blood expression analysis, cDNA was synthesized using qScript cDNA SuperMix (#95048, Quanta Biosciences Inc., Gaithersburg, MD). PCR reactions were performed in an ABI7900HT PCR system using Applied Biosystems Taqman Gene Expression Assay Mm00476182_m1 (for 11βHSD1) and Mm01611464_g1 (for ribosomal protein L30, the normalization control) in Taqman Universal PCR Mix (#4304337, Applied Biosystems Inc.). For tissue and peritoneal macrophage expression analyses, cDNA was synthesized with High Capacity cDNA Reverse Transcription Kit (#4368813, Applied Biosystems Inc.) PCR reactions were performed via TaqMan assay using ABI7900HT PCR system. PPIA was used as a control for normalization. For the SYBR® Green assay, total RNA was reverse transcribed according to the manufacturer's instructions (#95048, Quanta Biosciences Inc.). PCR reactions were carried out using Power SYBR® Green PCR Master Mix according to the manufacturer's instructions (#4367659, Applied Biosystems Inc.). Ribosomal protein L30 was used as a control. For both protocols, relative mRNA expression was calculated by fold change using the comparative Ct method (2-ΔΔCt)
All target labeling reagents and GeneChip® HT One-Cycle Target Labeling kits were purchased from Affymetrix (Santa Clara, CA). Mouse Genome HT_MG-430 arrays were purchased from Affymetrix. Double-stranded complementary DNAs (cDNAs) were synthesized from 1.2 µg total RNA from each tissue sample through reverse transcription with an oligo-dT primer containing the T7 RNA polymerase promoter using the cDNA Synthesis System from Affymetrix. Biotin-labeled cRNAs were generated from the cDNAs and were processed on a Caliper GeneChip Array Station from Affymetrix. Labeled cRNAs were hybridized on Affymetrix Mouse Genome HT_MG-430 arrays. Array hybridization, washing and scanning were performed according to Affymetrix protocol recommendations. Scanned images were subjected to visual inspection and chip quality reports were generated by Expression console (Affymetrix). The image data was processed using the Robust Multichip Average (RMA) method to determine the specific hybridizing signal for each probe set.
Ingenuity Systems Pathway Analysis (IPA) software version 9.0 (Ingenuity Systems Incorporated; Redwood City, CA;
Data are reported as mean ± standard error of the mean. Statistical analyses were performed using a Student's unpaired t-test, one-way or two-way ANOVA and Tukey's or Dunnett's post-hoc test, as appropriate. Results were considered statistically significant at p≤0.05.
Mice deficient in 11βHSD1 were created by targeted disruption of the 11βHSD1 gene locus (
Gene expression levels in
To study the effects of 11βHSD1 gene deficiency on atherosclerosis development, mice were fed a Western-type diet starting at eight weeks of age and continued for an additional 14 weeks. All mice gained weight normally (
Cholesterol accumulation in the thoracic aortae of mice was evaluated as a measure of atherosclerosis development. In males, aortic total cholesterol levels were reduced by 50% (p<0.01) in 11β−/−/apoE−/− mice vs. control (
Total aortic cholesterol mass from each vessel was normalized to delipidated aorta dry weight.
The role of leukocyte-derived 11βHSD1 on plaque development was evaluated using the bone marrow transplantation model. Bone marrow-derived stem cells from 11β−/−/apoE−/− and 11β+/+/apoE−/− littermate control mice were harvested and injected into irradiated apoE-deficient recipient male mice. Following a 4 week engraftment phase, mice were placed onto Western diet for 12 weeks. Confirmation of efficient transplantation was carried out by quantitative PCR analysis of 11βHSD1 mRNA in whole blood from recipient mice. 11βHSD1 mRNA was essentially undetectable in recipients transplanted with 11β−/−/apoE−/−-derived stem cells vs. control (
Given the reduction in macrophage content in aortic root lesions, the results of the bone marrow transplantation studies and the central role of the macrophage in the pathophysiology of atherosclerosis, two important aspects of macrophage function that are known to contribute to plaque formation were further evaluated in vivo: inflammatory cell migration and cholesterol loading capacity. Inflammatory cell migration was evaluated in vivo using Western diet-fed 11β+/+/apoE−/− and 11β−/−/apoE−/− mice subjected to intraperitoneal thioglycollate challenge. The ∼5 week protocol shown in
Thioglycollate-elicited peritoneal macrophages were also evaluated for the extent of cholesterol loading following thioglycollate challenge. Prior studies using pro-atherogenic hyperlipidemic mice have shown that thioglycollate-induced peritoneal macrophages can accumulate lipid sterols in vivo and possess a phenotype suggestive of foam cells
Affymetrix gene expression profiling was carried out with thioglycollate-elicited peritoneal macrophages from 11β+/+/apoE−/− and 11β−/−/apoE−/− mice. Gene expression profiles were compared by Two-Way ANOVA using Partek® Discovery Suite software. Only the Affymetrix probe sets that displayed a maximum anti-log RMA signal intensity of greater than 16 were considered for post hoc analyses. Differential changes in gene expression levels ranged from ∼+2.3 fold for up-regulated genes (428 genes) and ∼−3.8 fold for down-regulated genes (434 genes). Interestingly, given the observation of decreased in vivo cholesterol loading with 11βHSD1 deficiency, there were no significant changes in transcript levels of genes associated with oxidized LDL scavenging/uptake in macrophages, for example, scavenger receptor A1(SR-A1), A2(SR-A2), B1(SR-B1) and thrombospondin receptor CD36. Similarly, there were no transcript level changes in genes classically associated with macrophage cholesterol homeostasis: acetyl-CoA acetyltransferase 1 (ACAT1), cholesteryl ester hydrolase (CEH), nuclear hormone receptors LXRα and LXRβ and ABC transporter proteins ABCA1 and ABCG1. However, a significant 1.8 fold decrease (p = 0.001) in toll-like receptor 4 (TLR 4) gene expression was detected in 11β−/−/apoE−/− mice vs. control. Further inspection of the TLR gene family revealed significant decreases in TLR 1, 3, 4, 8 and 13 (
Gene Symbol | Gene Name | 11β−/−/vs. control | |
fold change | p value | ||
TLR1 | Toll-like receptor 1 | −1.2 | 1E-02 |
TLR3 | Toll-like receptor 3 | −1.3 | 1E-02 |
TLR4 | Toll-like receptor 4 | −1.8 | 2E-03 |
TLR8 | Toll-like receptor 8 | −1.2 | 1E-03 |
TLR13 | Toll-like receptor 13 | −1.2 | 3E-03 |
Edem3 | ER degradation enhancer, mannosidase alpha-like 3 | −1.6 | 1E-03 |
Jak2 | Janus kinase 2 | −1.3 | 3E-02 |
Map3k2 | Mitogen-activated protein kinase kinase kinase 2 | −1.5 | 3E-03 |
Traf3ip3 | TRAF3 interacting protein 3 | −1.3 | 1E-03 |
Traf5 | TNF receptor-associated factor 5 | −1.3 | 7E-03 |
Toll-like receptors and associated signal transduction pathway genes.
Further analysis of peritoneal macrophage microarray data revealed down regulation of genes associated with TLR signal transduction. As shown in
An evaluation of significantly affected biological pathways with 11βHSD1 gene deficiency was carried by Ingenuity Pathway Analysis (IPA). IPA revealed reductions in genes associated with inflammatory/immune function, suggesting that the overall inflammatory state of 11βHSD1-deficient macrophages was attenuated (
Ingenuity Canonical Pathway | 11β−/−/vs. control | ||
p value | ratio | ||
Oxidative Phosphorylation | 5.01E-14 | 30% | |
Ubiquinone Biosynthesis | 1.00E-11 | 26% | |
Mitochondrial Dysfunction | 1.00E-10 | 23% | |
EIF2 Signaling | 2.45E-09 | 23% | |
Protein Ubiquitination Pathway | 2.88E-06 | 19% | |
Estrogen Receptor Signaling | 4.37E-06 | 23% | |
Purine Metabolism | 1.05E-04 | 12% | |
NRF2-mediated Oxidative Stress Response | 1.91E-04 | 18% | |
Pyrimidine Metabolism | 4.47E-04 | 13% | |
Chemokine Signaling | 1.58E-03 | 22% | |
Glucocorticoid Receptor Signaling | 2.88E-03 | 14% | |
JAK/Stat Signaling | 3.31E-03 | 22% | |
NF-κB Signaling | 4.27E-03 | 17% | |
Apoptosis Signaling | 9.55E-03 | 18% | |
PI3K/AKT Signaling | 1.07E-02 | 15% | |
Activation of IRF by Cytosolic Pattern | 1.15E-02 | 18% | |
Recognition Receptors: Aldosterone Signaling in Epithelial Cells | 7.24E-02 | 13% |
Cytokine release from cultured thioglycollate-elicited peritoneal macrophages from Western diet-fed 11β+/+/apoE−/− and 11β−/−/apoE−/− mice was examined by antibody array analysis following stimulation with oxidized LDL. Incubation conditions and challenge with oxidized LDL occurred in the presence of the 11βHSD1 substrate 11-dehydrocorticosterone. As shown in
Cytokine protein levels detected in cell culture media by protein multiplex analysis following overnight exposure of peritoneal foam cells to ∼30 µg/ml of oxidized LDL (Ox-LDL) vs. untreated controls. Peritoneal foam cells were harvested from Western diet-fed 11βHSD1+/+/apoE−/− (+/+) and 11βHSD1−/−/apoE−/− (−/−) mice. G-CSF, KC, MCP1 and TNF-α levels were normalized to total cellular protein content. Cell culture media contained 200 nM 11-dehydrocorticosterone throughout the course of the experiment. Significance vs. control: *p≤0.05.
Treatment of hyperlipidemic mice with pharmacological inhibitors of 11βHSD1 has been shown to reduce plaque in the presence of reduced plasma lipids. For example, a study in the Western diet-fed apoE knockout mouse showed that eight weeks of treatment with a selective 11βHSD1 inhibitor decreased aortic cholesterol burden by 84% vs. vehicle control
In order to elucidate the physiological and molecular mechanisms underlying the anti-atherosclerotic effect of 11βHSD1 inhibition, the studies described herein were performed. The results indicate that inactivation of 11βHSD1 can positively impact aortic plaque by decreasing i) aortic cholesterol and 7-oxysterol accumulation, ii) aortic root plaque area iii) necrotic core intimal area and iv) CD68-positive macrophage inflammation. These data support a rapidly evolving concept that inhibition of 11βHSD1 can ameliorate atherosclerotic disease via direct effects at the arterial wall, without impacting plasma lipids or other metabolic parameters
Analyses of cholesterol levels in the thoracic aortae of mice revealed significant decreases in cholesterol (
Histological analyses of structure and composition of the atherosclerotic plaques present in the aortic root revealed a 30% reduction in necrotic core area with 11βHSD1 gene deficiency (
The first possibility was addressed by analyzing blood samples from 11β+/+/apoE−/− and 11β−/−/apoE−/− mice (naïve and thioglycollate injected): no differences in monocyte or total leukocyte counts were detected between groups (
Given this conclusion, the third possibility was tested. Mice of both genotypes were placed on a Western diet for 4 weeks. At the end of this period, they were subjected to a peritoneal thioglycollate challenge to elicit recruitment of macrophages into the peritoneum and stimulate foam cell formation with hyperlipidemia. Staining and microscopic evaluation of macrophages showed a distinct foamy appearance and the presence of intracellular lipid droplets due to the accumulation of cholesteryl ester (
Mouse bone marrow transplantation studies have been used to study the role of leukocyte-associated genes on the pathophysiology of atherosclerosis. The results of the analysis described herein clearly indicate the involvement of leukocyte associated-11βHSD1 in atherosclerosis development. In these studies, plaque burden in the thoracic aorta was reduced 39% with 11βHSD1 deficiency vs. control (
The critical observations that leukocyte-specific 11βHSD1 deficiency reduces plaque burden and in vivo cholesterol accumulation in peritoneal macrophages is reduced in 11β−/−/apoE−/− mice suggest that inhibition of 11βHSD1 can modulate an important function of the macrophage central to atherosclerosis pathophysiology. Prior studies by other investigators have shown that hyperlipidemic peritoneal macrophages possess phenotypic and functional attributes of lesional macrophages and appear to be relevant surrogates of macrophages present in atheroma
TLR4 is expressed by foam cells, is present in human coronary artery plaque, is up regulated by oxidized LDL and, most relevant to the results presented herein, is involved in macrophage-mediated lipid accumulation
To discern additional mechanistic hypotheses for the anti-atherosclerotic effect of the 11βHSD1 gene knockout, the microarray data were further analyzed via an Ingenuity Pathway Analysis (IPA). The results of this analysis (
The pattern for chemokine, NF-κB and JAK/STAT signaling, combined with the downregulation of the pattern recognition receptors is consistent with an anti-inflammatory response to 11βHSD1 gene ablation and the relative anti-atherosclerotic phenotype observed in 11β−/−/apoE−/− mice. On the other hand, the broad downregulation in the glucocorticoid signaling pathway is not, on the surface, consistent with an anti-atherosclerotic effect given the known anti-inflammatory effects of pharmacological glucocorticoid therapy. One possibility to explain this discrepancy is that the decrease in signaling via the glucocorticoid receptor (GR) is balanced or dominated by a similar decrease in signaling via the aldosterone-mineralocorticoid receptor pathway. It is well known that aldosterone is a pro-inflammatory agent in macrophages,
The IPA analysis also revealed significant differential changes in expression of genes related to mitochondrial oxidative phosphorylation (
Besides reducing the cholesterol content within the vessel wall, reduction in macrophage foam cell formation can also reduce the progression from simple foam-cell laden fatty streaks to lesions with necrotic cores. It is generally accepted that accumulation of cholesterol and oxidized lipids drives foam cell formation, the subsequent sequelae of pro-inflammatory changes and eventually, apoptotic and/or necrotic cell death
We therefore propose a model whereby the absence of 11βHSD1 attenuates macrophage foam cell formation via downregulation of the toll-like receptors. This in turn reduces uptake of cytotoxic levels of sterols and oxidized lipids, lessening the insult to the cell and diminishing the propensity of macrophages to proceed to necrosis. As fewer macrophages enter necrosis, there will be reduced substrate for necrotic core formation. Besides directly reducing necrotic core formation, reducing macrophage necrosis will also reduce the pro-inflammatory signal that stimulates further macrophage infiltration. This would result in prolongation of the rate at which the plaque matures to the point of being pathological.
In addition, given recent advances in the understanding of the natural history of macrophages in atherogenesis,
The findings reported herein demonstrate that 11βHSD1 inhibition can attenuate development of atherosclerotic disease directly at the vessel wall without necessarily affecting plasma lipid profiles. Decreasing cholesterol loading in macrophages via downregulation of toll-like receptors offers one plausible mechanistic explanation for atheroprotection observed in this model. It is clear that additional investigation at the molecular level and via pharmacological intervention is required to confirm the hypotheses generated by these
The utility of 11βHSD1 inhibition as a treatment for atherosclerosis is becoming increasingly evident. At present, however, efforts to develop 11βHSD1 inhibitors in the clinic have primarily focused on management of type 2 diabetes
(TIF)
(TIF)
(TIF)
(TIF)
(TIF)
(TIF)
(TIF)
(TIF)
(TIF)
(TIF)
We thank Veterinary Sciences at BMS-Hopewell for maintaining exceptional standards for genetically-modified mice. We thank Debra Wescott, Janet Dipiero and Roseann Riley in the Clinical Pathology Department at BMS-Lawrenceville for excellence in processing plasma lipids. We are grateful to Dr. Matthew Fronheiser for enabling our rapid analyses of histological data. We also thank Dr. Todd Kirchgessner, Dr. Cort Madsen, Dr. Rex Parker and all members of BMS-Atherosclerosis for many insightful suggestions given throughout the course of this project. In addition, we offer a special thanks to Dr. Nitin Aggarwal for his help with blood pressure measurements.