The mechanisms by which environmental toxicants alter developmental processes predisposing individuals to adult onset chronic disease are not well-understood. Transplacental arsenic exposure promotes atherogenesis in apolipoprotein E-knockout (ApoE−/−) mice. Because the liver plays a central role in atherosclerosis, diabetes and metabolic syndrome, we hypothesized that accelerated atherosclerosis may be linked to altered hepatic development. This hypothesis was tested in ApoE−/− mice exposed to 49 ppm arsenic in utero from gestational day (GD) 8 to term. GD18 hepatic arsenic was 1.2 µg/g in dams and 350 ng/g in fetuses. The hepatic transcriptome was evaluated by microarray analysis to assess mRNA and microRNA abundance in control and exposed pups at postnatal day (PND) 1 and PND70. Arsenic exposure altered postnatal developmental trajectory of mRNA and microRNA profiles. We identified an arsenic exposure related 51-gene signature at PND1 and PND70 with several hubs of interaction (Hspa8, IgM and Hnf4a). Gene ontology (GO) annotation analyses indicated that pathways for gluconeogenesis and glycolysis were suppressed in exposed pups at PND1, and pathways for protein export, ribosome, antigen processing and presentation, and complement and coagulation cascades were induced by PND70. Promoter analysis of differentially-expressed transcripts identified enriched transcription factor binding sites and clustering to common regulatory sites. SREBP1 binding sites were identified in about 16% of PND70 differentially-expressed genes. Western blot analysis confirmed changes in the liver at PND70 that included increases of heat shock protein 70 (Hspa8) and active SREBP1. Plasma AST and ALT levels were increased at PND70. These results suggest that transplacental arsenic exposure alters developmental programming in fetal liver, leading to an enduring stress and proinflammatory response postnatally that may contribute to early onset of atherosclerosis. Genes containing SREBP1 binding sites also suggest pathways for diabetes mellitus and rheumatoid arthritis, both diseases that contribute to increased cardiovascular disease in humans.
Environmental exposures filtered through the genetic make-up of each individual alter the transcriptional repertoire in organs central to metabolic homeostasis, thereby affecting arterial lipid accumulation, inflammation, and the development of coronary artery disease (CAD). The primary aim of the Stockholm Atherosclerosis Gene Expression (STAGE) study was to determine whether there are functionally associated genes (rather than individual genes) important for CAD development. To this end, two-way clustering was used on 278 transcriptional profiles of liver, skeletal muscle, and visceral fat (n = 66/tissue) and atherosclerotic and unaffected arterial wall (n = 40/tissue) isolated from CAD patients during coronary artery bypass surgery. The first step, across all mRNA signals (n = 15,042/12,621 RefSeqs/genes) in each tissue, resulted in a total of 60 tissue clusters (n = 3958 genes). In the second step (performed within tissue clusters), one atherosclerotic lesion (n = 49/48) and one visceral fat (n = 59) cluster segregated the patients into two groups that differed in the extent of coronary stenosis (P = 0.008 and P = 0.00015). The associations of these clusters with coronary atherosclerosis were validated by analyzing carotid atherosclerosis expression profiles. Remarkably, in one cluster (n = 55/54) relating to carotid stenosis (P = 0.04), 27 genes in the two clusters relating to coronary stenosis were confirmed (n = 16/17, P<10−27and−30). Genes in the transendothelial migration of leukocytes (TEML) pathway were overrepresented in all three clusters, referred to as the atherosclerosis module (A-module). In a second validation step, using three independent cohorts, the A-module was found to be genetically enriched with CAD risk by 1.8-fold (P<0.004). The transcription co-factor LIM domain binding 2 (LDB2) was identified as a potential high-hierarchy regulator of the A-module, a notion supported by subnetwork analysis, by cellular and lesion expression of LDB2, and by the expression of 13 TEML genes in Ldb2–deficient arterial wall. Thus, the A-module appears to be important for atherosclerosis development and, together with LDB2, merits further attention in CAD research.
The WHO predicts that coronary artery disease (CAD) will become the leading cause of death worldwide in 2010. Currently, major research efforts are focused on understanding the genetics of CAD through multi-center, genome-wide association studies of tens of thousands of patients and controls. Such studies can identify common variants of general importance throughout the entire population, which are likely relatively few. The number of rare genetic variants and variants that act in the context of environmental risk factors for CAD is probably much higher. We performed whole-genome expression analyses in several organs to identify functionally associated genes important for CAD development. We found an atherosclerosis module (A-module) consisting of 128 genes, enriched with genetic risk for CAD, involving transendothelial migration of leukocytes (TEML) and LIM domain binding 2 (LDB2) as its high-hierarchy regulator. Our study design represents a novel way of understanding the molecular underpinnings of CAD, focusing on genome-wide expression sensing both environmental and genetic influences. Investigating the relative enrichment of genetic CAD risk in functional groups (modules and networks) is an alternative approach to extract additional relevant information from genome-wide association studies. The A-module and LDB2 are attractive targets for treatments to modulate TEML and atherosclerosis development.
Accumulating evidence implicates a fundamental link between the immune system and atherosclerosis. Toll-like receptors are principal sensors of the innate immune system. Here we report an assessment of the role of the TLR2 pathway in atherosclerosis associated with a high-fat diet and/or bacteria in ApoE+/− mice.
Methods and Results
To explore the role of TLR2 in inflammation- and infection-associated atherosclerosis, 10 week-old ApoE+/−-TLR2+/+, ApoE+/−-TLR2+/− and ApoE+/−-TLR2−/− mice were fed either a high fat diet or a regular chow diet. All mice were inoculated intravenously, once per week for 24 consecutive weeks, with 50 µl live Porphyromonas gingivalis (P.g) (107 CFU) or vehicle (normal saline). Animals were euthanized 24 weeks after the first inoculation. ApoE+/−-TLR2+/+ mice showed a significant increase in atheromatous lesions in proximal aorta and aortic tree compared to ApoE+/−-TLR2+/− and ApoE+/−-TLR2−/− mice for all diet conditions. They also displayed profound changes in plaque composition, as evidenced by increased macrophage infiltration and apoptosis, increased lipid content, and decreased smooth muscle cell mass, all reflecting an unstable plaque phenotype. SAA levels from ApoE+/−-TLR2+/+ mice were significantly higher than from ApoE+/−-TLR2+/− and ApoE+/−-TLR2−/− mice. Serum cytokine analysis revealed increased levels of pro-inflammatory cytokines in ApoE+/−-TLR2+/+ mice compared to ApoE+/−-TLR2+/− and TLR2−/− mice, irrespective of diet or bacterial challenge. ApoE+/−-TLR2+/+ mice injected weekly for 24 weeks with FSL-1 (a TLR2 agonist) also demonstrated significant increases in atherosclerotic lesions, SAA and serum cytokine levels compared to ApoE+/−-TLR2−/− mice under same treatment condition. Finally, mass-spectrometry (MALDI-TOF-MS) of aortic samples analyzed by 2-dimentional gel electrophoresis differential display, identified 6 proteins upregulated greater than 2-fold in ApoE+/−-TLR2+/+ mice fed the high fat diet and inoculated with P.g compared to any other group.
Genetic deficiency of TLR2 reduces diet- and/or pathogen-associated atherosclerosis in ApoE+/− mice, along with differences in plaque composition suggesting greater structural stability while TLR-2 ligand-specific activation triggers atherosclerosis. The present data offers new insights into the pathophysiological pathways involved in atherosclerosis and paves the way for new pharmacological interventions aimed at reducing atherosclerosis.
Low B vitamin status is linked with human vascular disease. We employed a proteomic and biochemical approach to determine whether nutritional folate deficiency and/or hyperhomocysteinemia altered metabolic processes linked with atherosclerosis in ApoE null mice. Animals were fed either a control fat (C; 4 % w/w lard) or a high-fat [HF; 21 % w/w lard and cholesterol (0/15 % w/w)] diet with different B vitamin compositions for 16 weeks. Aorta tissue was prepared and global protein expression, B vitamin, homocysteine and lipoprotein status measured. Changes in the expression of aorta proteins were detected in response to multiple B vitamin deficiency combined with a high-fat diet (P < 0.05) and were strongly linked with lipoprotein concentrations measured directly in the aorta adventitia (P < 0.001). Pathway analysis revealed treatment effects in the aorta-related primarily to cytoskeletal organisation, smooth muscle cell adhesion and invasiveness (e.g., fibrinogen, moesin, transgelin, vimentin). Combined B vitamin deficiency induced striking quantitative changes in the expression of aorta proteins in atherosclerotic ApoE null mice. Deregulated expression of these proteins is associated with human atherosclerosis. Cellular pathways altered by B vitamin status included cytoskeletal organisation, cell differentiation and migration, oxidative stress and chronic inflammation. These findings provide new insight into the molecular mechanisms through which B vitamin deficiency may accelerate atherosclerosis.
Electronic supplementary material
The online version of this article (doi:10.1007/s12263-014-0446-y) contains supplementary material, which is available to authorized users.
Aorta proteome; ApoE null mice; Atherosclerosis; B vitamins; Hyperhomocysteinemia
Peroxisome-proliferator–activated-receptor-γ (PPARγ) acts as a transcriptional regulator of multiple genes involved in glucose and lipid metabolism. In vitro studies showed that activated PPARγ suppresses AT1R-gene expression and vice versa. However, it has not yet been determined in vivo, whether AT1R-PPARγ-interactions play a relevant role in the pathogenesis of diabetic complications and specifically in accelerated atherosclerosis.
Methods and results
ApoE−/− and ApoE−/−/AT1R−/−-mice were rendered diabetic by intraperitoneal injections of streptozotocin. Diabetic and non-diabetic ApoE−/−-mice were further randomized to receive the AT1R antagonist telmisartan, the selective PPARγ antagonist GW9662, telmisartan and GW9662 or vehicle for 18 weeks. Diabetic and non-diabetic ApoE−/−/AT1R−/−-mice were randomized to receive either GW9662 or vehicle. GW9662 treatment in diabetic ApoE−/− and diabetic ApoE−/−/AT1−/−-mice resulted in the highest elevation of fasting blood glucose levels, whereas telmisartan treatment and AT1 deficiency in ApoE−/−-mice showed the lowest fasting blood glucose levels. Diabetic ApoE−/−-mice displayed severe impairment of endothelial function, enhanced oxidative stress and increased atherosclerotic lesion formation. ApoE−/−/AT1R−/− and telmisartan-treated ApoE−/−-mice showed a significantly better endothelial function, decreased oxidative stress and reduced atherosclerotic lesion formation. Treatment of diabetic ApoE−/− and ApoE−/−/AT1R−/−-mice with the selective PPARγ antagonist GW9662 omitted the atheroprotective effects of AT1R deficiency or AT1 antagonism.
Genetic disruption or pharmacological inhibition of the AT1R attenuates atherosclerosis and improves endothelial function in diabetic ApoE−/−-mice via the PPARγ pathway.
Diabetes mellitus; Atherosclerosis; Angiotensin; Receptors
Our understanding of the molecular pathways that underlie melanoma remains incomplete. Although several published microarray studies of clinical melanomas have provided valuable information, we found only limited concordance between these studies. Therefore, we took an in vitro functional genomics approach to understand melanoma molecular pathways.
Affymetrix microarray data were generated from A375 melanoma cells treated in vitro with siRNAs against 45 transcription factors and signaling molecules. Analysis of this data using unsupervised hierarchical clustering and Bayesian gene networks identified proliferation-association RNA clusters, which were co-ordinately expressed across the A375 cells and also across melanomas from patients. The abundance in metastatic melanomas of these cellular proliferation clusters and their putative upstream regulators was significantly associated with patient prognosis. An 8-gene classifier derived from gene network hub genes correctly classified the prognosis of 23/26 metastatic melanoma patients in a cross-validation study. Unlike the RNA clusters associated with cellular proliferation described above, co-ordinately expressed RNA clusters associated with immune response were clearly identified across melanoma tumours from patients but not across the siRNA-treated A375 cells, in which immune responses are not active. Three uncharacterised genes, which the gene networks predicted to be upstream of apoptosis- or cellular proliferation-associated RNAs, were found to significantly alter apoptosis and cell number when over-expressed in vitro.
This analysis identified co-expression of RNAs that encode functionally-related proteins, in particular, proliferation-associated RNA clusters that are linked to melanoma patient prognosis. Our analysis suggests that A375 cells in vitro may be valid models in which to study the gene expression modules that underlie some melanoma biological processes (e.g., proliferation) but not others (e.g., immune response). The gene expression modules identified here, and the RNAs predicted by Bayesian network inference to be upstream of these modules, are potential prognostic biomarkers and drug targets.
Ecto-Nucleotide Pyrophosphatase/Phosphodiesterase 1 (NPP1) generates inorganic pyrophosphate (PPi), a physiologic inhibitor of hydroxyapatite deposition. In a previous study, we found NPP1 expression to be inversely correlated with the degree of atherosclerotic plaque calcification. Moreover, function-impairing mutations of ENPP1, the gene encoding for NPP1, are associated with severe, artery tunica media calcification and myointimal hyperplasia with infantile onset in humans. NPP1 and PPi have the potential to modulate atherogenesis by regulating arterial smooth muscle cell (SMC) differentiation and function, including increase of pro-atherogenic osteopontin (OPN) expression. Hence, this study tested the hypothesis that NPP1 deficiency modulates both atherogenesis and atherosclerotic intimal plaque calcification.
Methods and Results
Npp1/ApoE double deficient mice were generated by crossing mice bearing the ttw allele of Enpp1 (that encodes a truncation mutation) with ApoE null mice and fed with high fat/high cholesterol atherogenic diet. Atherosclerotic lesion area and calcification were examined at 13, 18, 23 and 28 weeks of age. The aortic SMCs isolated from both ttw/ttw ApoE−/− and ttw/+ ApoE−/− mice demonstrated decreased Opn expression. The 28 weeks old ttw/ttw ApoE−/− and ttw/+ ApoE−/− had significantly smaller atherosclerotic lesions compared with wild type congenic ApoE−/− mice. Only ttw/ttw but not ttw/+ mice developed artery media calcification. Furthermore in ttw/+ mice, there was a tendency towards increased plaque calcification compared to ApoE−/− mice without Npp1 deficiency.
We conclude that Npp1 promotes atherosclerosis, potentially mediated by Opn expression in ApoE knockout mice.
atherosclerosis; plaque calcification; osteopontin; inorganic pyrophosphate
Investigating long-term cardiac effects of low doses of ionizing radiation is highly relevant in the context of interventional cardiology and radiotherapy. Epidemiological data report that low doses of irradiation to the heart can result in significant increase in the cardiovascular mortality by yet unknown mechanisms. In addition co-morbidity factor such as hypertension or/and atherosclerosis can enhance cardiac complications. Therefore, we explored the mechanisms that lead to long-term cardiac remodelling and investigated the interaction of radiation-induced damage to heart and cardiovascular systems with atherosclerosis, using wild-type and ApoE-deficient mice.
Methods and Results
ApoE−/− and wild-type mice were locally irradiated to the heart at 0, 0.2 and 2 Gy (RX). Twenty, 40 and 60 weeks post-irradiation, echocardiography were performed and hearts were collected for cardiomyocyte isolation, histopathological analysis, study of inflammatory infiltration and fibrosis deposition. Common and strain-specific pathogenic pathways were found. Significant alteration of left ventricular function (eccentric hypertrophy) occurred in both strains of mice. Low dose irradiation (0.2 Gy) induced premature death in ApoE−/− mice (47% died at 20 weeks). Acute inflammatory infiltrate was observed in scarring areas with accumulation of M1-macrophages and secretion of IL-6. Increased expression of the fibrogenic factors (TGF-β1 and PAI-1) was measured earlier in cardiomyocytes isolated from ApoE−/− than in wt animals.
The present study shows that cardiac exposure to low dose of ionizing radiation induce significant physiological, histopathological, cellular and molecular alterations in irradiated heart with mild functional impairment. Atherosclerotic predisposition precipitated cardiac damage induced by low doses with an early pro-inflammatory polarization of macrophages.
To evaluate global gene expression patterns in the common iliac arteries of monkeys with varied extent of atherosclerosis.
The left common iliac artery was removed from ovariectomized cynomolgus monkeys (n=12) after 6.5 years of consuming a diet containing fat and cholesterol at levels comparable to that consumed in western populations. Arterial gene expression was analyzed by DNA microarray and real time RT-PCR.
Significant differential expression of 986 genes was observed in iliac arteries containing moderate to large atherosclerotic plaques compared to normal/minimally affected reference group arteries. Atherosclerosis-associated genes included cytokines, chemokines, components of signal transduction pathways, and transcriptional activators and repressors, as well as other functional categories. Real time RT-PCR confirmed differential expression of genes chosen from a variety of functional categories. Specifically, expression of genes for estrogen receptor 1 (ESR1), claudin 11, and BH protocadherin 7 (PCDH7) were reduced, whereas expression of genes for apolipoprotein E (ApoE), growth differentiation factor 15 (GDF15), superoxide dismutase 2 (SOD2), SET domain, bifurcated 2 (SETDB2), phospholipase A2 group IIA (PLA2IIA), phospholipase A2 group VII (PLA2VII), and ring finger protein 149 (RNF149) were increased in atherosclerotic arteries.
The gene expression environment in arteries containing atherosclerotic plaques is profoundly different from that of relatively unaffected arteries and reflects the cellular and molecular complexity of atherosclerosis and associated arterial remodeling processes.
atherogenesis; gene expression; diet; cholesterol; intervention potential; atherosclerosis
To study whether 18F-FDG can be used for in vivo imaging of atherogenesis by examining the correlation between 18F-FDG uptake and gene expression of key molecular markers of atherosclerosis in apoE−/− mice.
Nine groups of apoE−/− mice were given normal chow or high-fat diet. At different time-points, 18F-FDG PET/contrast-enhanced CT scans were performed on dedicated animal scanners. After scans, animals were euthanized, aortas removed, gamma counted, RNA extracted from the tissue, and gene expression of chemo (C-X-C motif) ligand 1 (CXCL-1), monocyte chemoattractant protein (MCP)-1, vascular cell adhesion molecule (VCAM)-1, cluster of differentiation molecule (CD)-68, osteopontin (OPN), lectin-like oxidized LDL-receptor (LOX)-1, hypoxia-inducible factor (HIF)-1α, HIF-2α, vascular endothelial growth factor A (VEGF), and tissue factor (TF) was measured by means of qPCR.
The uptake of 18F-FDG increased over time in the groups of mice receiving high-fat diet measured by PET and ex vivo gamma counting. The gene expression of all examined markers of atherosclerosis correlated significantly with 18F-FDG uptake. The strongest correlation was seen with TF and CD68 (p<0.001). A multivariate analysis showed CD68, OPN, TF, and VCAM-1 to be the most important contributors to the uptake of 18F-FDG. Together they could explain 60% of the 18F-FDG uptake.
We have demonstrated that 18F-FDG can be used to follow the progression of atherosclerosis in apoE−/− mice. The gene expression of ten molecular markers representing different molecular processes important for atherosclerosis was shown to correlate with the uptake of 18F-FDG. Especially, the gene expressions of CD68, OPN, TF, and VCAM-1 were strong predictors for the uptake.
The multi-ligand Receptor for AGE (RAGE) contributes to atherosclerosis in apolipoprotein (ApoE) null mice.
To delineate the specific mechanisms by which RAGE accelerated atherosclerosis, we performed Affymetrix gene expression arrays on aortas of non-diabetic and diabetic ApoE null mice expressing RAGE or devoid of RAGE at nine weeks of age, as this reflected a time point at which frank atherosclerotic lesions were not yet present, but that we would be able to identify the genes likely involved in diabetes- and RAGE-dependent atherogenesis.
Methods and Results
We report that there is very little overlap of the genes which are differentially expressed both in the onset of diabetes in ApoE null mice, and in the effect of RAGE deletion in diabetic ApoE null mice. Pathway-Express analysis revealed that the Transforming Growth Factor-β pathway (Tgf-β) and focal adhesion pathways might be expected to play a significant role in both the mechanism by which diabetes facilitates the formation of atherosclerotic plaques in ApoE null mice, and the mechanism by which deletion of RAGE ameliorates this effect. Quantitative polymerase chain reaction studies, Western blotting and confocal microscopy in aortic tissue and in primary cultures of murine aortic smooth muscle cells supported these findings.
Taken together, our work suggests that RAGE-dependent acceleration of atherosclerosis in ApoE null mice is dependent, at least in part, on the action of the ROCK1 branch of the Tgf-β pathway.
AGE; Atherosclerosis; RAGE
Generalized arterial alterations, such as endothelial dysfunction, medial matrix accumulations, and calcifications are associated with type 2 diabetes (T2D). These changes may render the vessel wall more susceptible to injury; however, the molecular characteristics of such diffuse pre-atherosclerotic changes in diabetes are only superficially known.
To identify the molecular alterations of the generalized arterial disease in T2D, DNA microarrays were applied to examine gene expression changes in normal-appearing, non-atherosclerotic arterial tissue from 10 diabetic and 11 age-matched non-diabetic men scheduled for a coronary by-pass operation. Gene expression changes were integrated with GO-Elite, GSEA, and Cytoscape to identify significant biological pathways and networks.
Global pathway analysis revealed differential expression of gene-sets representing matrix metabolism, triglyceride synthesis, inflammation, insulin signaling, and apoptosis. The network analysis showed a significant cluster of dysregulated genes coding for both intra- and extra-cellular proteins associated with vascular cell functions together with genes related to insulin signaling and matrix remodeling.
Our results identify pathways and networks involved in the diffuse vasculopathy present in non-atherosclerotic arterial tissue in patients with T2D and confirmed previously observed mRNA-alterations. These abnormalities may play a role for the arterial response to injury and putatively for the accelerated atherogenesis among patients with diabetes.
Systems biology; Microarray; Diabetes mellitus; Gene expression; Coronary artery disease
Platelet aggregation plays a critical role in myocardial infarction and stroke; however the role of platelet secretion in atherosclerotic vascular disease is poorly understood. Therefore, we examined the hypothesis that platelet dense granule secretion modulates thrombosis, inflammation, and atherosclerotic vascular remodeling after injury.
Methods and Results
Functional deletion of the Hermansky Pudlak Syndrome 3 gene (HPS3−/−) markedly reduces platelet dense granule secretion. HPS3−/− mice have normal platelet counts, platelet morphology, alpha granule number and maximal secretion of the alpha granule marker P-selectin; however, their capacity to form platelet-leukocyte aggregates was significantly reduced (p<0.05). To examine the role of platelet dense granule secretion in these processes, atherosclerosis-prone mice with combined genetic deficiency of ApoE and HPS3 (ApoE−/−,HPS3−/−) were compared to congenic, atherosclerosis-prone mice with normal platelet secretion (ApoE−/−,HPS3+/+). After 16–18 weeks on a high fat diet, both groups of mice had similar fasting cholesterols and body weight. Carotid arteries of ApoE−/−,HPS3+/+ mice rapidly thrombosed after FeCl3-injury but ApoE−/−, HPS3−/− mice were completely resistant to thrombotic arterial occlusion (p<0. 01). Three weeks after injury, neointimal hyperplasia (from alpha smooth muscle actin+ cells) was significantly less (p <0.001) in the arteries from ApoE−/−,HPS3−/− mice. In ApoE−/−, HPS3−/− mice, there were also pronounced reductions in arterial inflammation, as indicated by a 74% decrease in CD45+ leukocytes (p< 0.01) and a 73% decrease in Mac-3+ macrophages (p<0.05).
In atherosclerotic mice, reduced platelet dense granule secretion is associated with marked protection from the development of arterial thrombosis, inflammation and neointimal hyperplasia after vascular injury.
platelets; arteriosclerosis; atherosclerosis; thrombosis; platelet-derived factors; carotid arteries
Cholesterol loaded macrophages in the arterial intima are the earliest histological evidence of atherosclerosis. Studies of mouse models of atherosclerosis have shown that the strain background can have a significant effect on lesion development. We have previously shown that DBA/2 ApoE−/− mice have aortic root lesions 10-fold larger than AKR ApoE−/−mice. The current study analyzes the response to cholesterol loading of macrophages from these two strains. Macrophages from the atherosclerosis susceptible DBA/2 strain had significantly higher levels of total and esterified cholesterol compared to atherosclerosis resistant AKR macrophages, while free cholesterol levels were higher in AKR cells. Gene expression profiles were obtained and data were analyzed for strain, cholesterol loading, and strain-cholesterol loading interaction effects by a fitted linear model. Pathway and transcriptional motif enrichment were identified by gene set enrichment analysis. In addition to observed strain differences in basal gene expression, we identified many transcripts whose expression was significantly altered in response to cholesterol loading, including P2ry13 and P2ry14, Trib3, Hyal1, Vegfa, Ccr5, Ly6a, and Ifit3. Eight pathways were significantly enriched in transcripts regulated by cholesterol loading, among which the lysosome and cytokine-cytokine receptor interaction pathways had the highest number of significantly regulated transcripts. Of the differentially regulated transcripts with a strain-cholesterol loading interaction effect, we identified three genes known to participate in the endoplasmic reticulum (ER) stress response, Ddit3, Trib3 and Atf4. These three transcripts were highly up-regulated by cholesterol in AKR and either down-regulated or unchanged in loaded DBA/2 macrophages, thus associating a robust ER stress response with atherosclerosis resistance. We identified significant transcripts with strain, loading, or strain-loading interaction effect that reside within previously described quantitative trait loci as atherosclerosis modifier candidate genes. In conclusion, we characterized several strain and cholesterol induced differences that may lead to new insights into cellular cholesterol metabolism and atherosclerosis.
Endothelial activation, which is characterized by upregulation of cellular adhesion molecules and pro-inflammatory chemokines and cytokines, and consequent monocyte recruitment to the arterial intima are etiologic factors in atherosclerosis. Redox-active transition metal ions, such as copper and iron, may play an important role in endothelial activation by stimulating redox-sensitive cell signaling pathways. We have shown previously that copper chelation by tetrathiomolybdate (TTM) inhibits LPS-induced acute inflammatory responses in vivo. Here, we investigated whether TTM can inhibit atherosclerotic lesion development in apolipoprotein E-deficient (apoE−/−) mice. We found that 10-week treatment of apoE−/− mice with TTM (33–66 ppm in the diet) reduced serum levels of the copper-containing protein, ceruloplasmin, by 47%, and serum iron by 26%. Tissue levels of “bioavailable” copper, assessed by the copper-to-molybdenum ratio, decreased by 80% in aorta and heart, whereas iron levels of these tissues were not affected by TTM treatment. Furthermore, TTM significantly attenuated atherosclerotic lesion development in whole aorta by 25% and descending aorta by 45% compared to non-TTM treated apoE−/− mice. This anti-atherogenic effect of TTM was accompanied by several anti-inflammatory effects, i.e., significantly decreased serum levels of soluble vascular cell and intercellular adhesion molecules (VCAM-1 and ICAM-1); reduced aortic gene expression of VCAM-1, ICAM-1, monocyte chemotactic protein-1, and pro-inflammatory cytokines; and significantly less aortic accumulation of M1 type macrophages. In contrast, serum levels of oxidized LDL were not reduced by TTM. These data indicate that TTM inhibits atherosclerosis in apoE−/− mice by reducing bioavailable copper and vascular inflammation, not by altering iron homeostasis or reducing oxidative stress.
tetrathiomolybdate; endothelial activation; copper chelation; atherosclerosis; vascular inflammation
Oxidative stress and inflammation are two critical factors that drive the formation of plaques in atherosclerosis. Nrf2 is a redox-sensitive transcription factor that upregulates a battery of antioxidative genes and cytoprotective enzymes that constitute the cellular response to oxidative stress. Our previous studies have shown that disruption of Nrf2 in mice (Nrf2−/−) causes increased susceptibility to pulmonary emphysema, asthma and sepsis due to increased oxidative stress and inflammation. Here we have tested the hypothesis that disruption of Nrf2 in mice causes increased atherosclerosis.
To investigate the role of Nrf2 in the development of atherosclerosis, we crossed Nrf2−/− mice with apoliporotein E-deficient (ApoE−/−) mice. ApoE−/− and ApoE−/− Nrf2−/− mice were fed an atherogenic diet for 20 weeks, and plaque area was assessed in the aortas. Surprisingly, ApoE−/− Nrf2−/− mice exhibited significantly smaller plaque area than ApoE−/− controls (11.5% vs 29.5%). This decrease in plaque area observed in ApoE−/− Nrf2−/− mice was associated with a significant decrease in uptake of modified low density lipoproteins (AcLDL) by isolated macrophages from ApoE−/− Nrf2−/− mice. Furthermore, atherosclerotic plaques and isolated macrophages from ApoE−/− Nrf2−/− mice exhibited decreased expression of the scavenger receptor CD36.
Nrf2 is pro-atherogenic in mice, despite its antioxidative function. The net pro-atherogenic effect of Nrf2 may be mediated via positive regulation of CD36. Our data demonstrates that the potential effects of Nrf2-targeted therapies on cardiovascular disease need to be investigated.
Apoptosis signal-regulating kinase 1 (ASK1)–interacting protein-1 (AIP1) is a signaling adaptor molecule implicated in stress and apoptotic signaling induced by proinflammatory mediators. However, its function in atherosclerosis has not been established. In the present study, we use AIP1-null (AIP1−/−) mice to examine its effect on atherosclerotic lesions in an ApoE-null (ApoE−/−) mouse model of atherosclerosis.
ApoE−/− control mice developed atherosclerosis in the aortic roots and descending aortas upon Western-type diet for 10 weeks, while the atherosclerotic lesions are significantly augmented in ApoE−/−AIP1−/− double knockout (DKO) mice. DKO mice show increases in plasma inflammatory cytokines with no significant alterations in body weight, total cholesterol levels or lipoprotein profiles. Aortas in DKO mice show increased inflammation and endothelial cell (EC) dysfunction with NF-κB activity, correlating with increased accumulation of macrophages in the lesion area. Importantly, macrophages from DKO donors are not sufficient to augment inflammatory responses and atherogenesis when transferred to ApoE-KO recipients. Mechanistic studies suggest that AIP1 is highly expressed in aortic EC but not in macrophages, and AIP1 deletion in EC significantly enhance oxidized LDL-induced NF-κB signaling, gene expression of inflammatory molecules and monocyte adhesion, suggesting that vascular EC are responsible for the increased inflammatory responses observed in DKO mice.
Our data demonstrate that loss of AIP1 in aortic EC primarily contributes to the exacerbated lesion expansion in the ApoE−/−AIP1−/− mice, revealing an important role of AIP1 in limiting inflammation, EC dysfunction and atherosclerosis.
Atherosclerosis; inflammation; endothelial cell; lipids and lipoproteins; macrophage; AIP1
Diabetic subjects are at high risk for developing atherosclerosis through a variety of mechanisms. As the metabolism of glucose results in production of activators of protein kinase C (PKC)β, it was logical to investigate the role of PKCβ in modulation of atherosclerosis in diabetes.
Approach and Results
ApoE−/− and PKCβ −/−/ApoE−/− mice were rendered diabetic with streptozotocin. Quantification of atherosclerosis, gene expression profiling or analysis of signaling molecules was performed on aortic sinus or aortas from diabetic mice. Diabetes-accelerated atherosclerosis increased the level of phosphorylated ERK1/2 and JNK mitogen activated protein (MAP) kinases and augmented vascular expression of inflammatory mediators, as well as increased monocyte/macrophage infiltration and CD11c+ cells accumulation in diabetic ApoE−/− mice; processes which were diminished in diabetic PKCβ −/−/ApoE−/− mice. In addition, pharmacological inhibition of PKCβ reduced atherosclerotic lesion size in diabetic ApoE−/− mice. In vitro, the inhibitors of PKCβ and ERK1/2, as well as small interfering RNA (siRNA) to Egr-1 significantly decreased high glucose-induced expression of CD11c (Itgax), chemokine (C-C motif) ligand 2 (CCL2) and interleukin (IL)-1β in U937 macrophages.
These data link enhanced activation of PKCβ to accelerated diabetic atherosclerosis via a mechanism that includes modulation of gene transcription and signal transduction in the vascular wall; processes that contribute to acceleration of vascular inflammation and atherosclerosis in diabetes. Our results uncover a novel role for PKCβ in modulating CD11c expression and inflammatory response of macrophages in the development of diabetic atherosclerosis. These findings support PKCβ activation as a potential therapeutic target for prevention and treatment of diabetic atherosclerosis.
atherosclerosis; diabetes; PKCβ; CD11c; inflammation
Treponema denticola is a predominantly subgingival oral spirochete closely associated with periodontal disease and has been detected in atherosclerosis. This study was designed to evaluate causative links between periodontal disease induced by chronic oral T. denticola infection and atherosclerosis in hyperlipidemic ApoE−/− mice. ApoE−/− mice (n = 24) were orally infected with T. denticola ATCC 35404 and were euthanized after 12 and 24 weeks. T. denticola genomic DNA was detected in oral plaque samples, indicating colonization of the oral cavity. Infection elicited significantly (P = 0.0172) higher IgG antibody levels and enhanced intrabony defects than sham infection. T. denticola-infected mice had higher levels of horizontal alveolar bone resorption than sham-infected mice and an associated significant increase in aortic plaque area (P ≤ 0.05). Increased atherosclerotic plaque correlated with reduced serum nitric oxide (NO) levels and increased serum-oxidized low-density lipoprotein (LDL) levels compared to those of sham-infected mice. T. denticola infection altered the expression of genes known to be involved in atherosclerotic development, including the leukocyte/endothelial cell adhesion gene (Thbs4), the connective tissue growth factor gene (Ctgf), and the selectin-E gene (Sele). Fluorescent in situ hybridization (FISH) revealed T. denticola clusters in both gingival and aortic tissue of infected mice. This is the first study examining the potential causative role of chronic T. denticola periodontal infection and vascular atherosclerosis in vivo in hyperlipidemic ApoE−/− mice. T. denticola is closely associated with periodontal disease and the rapid progression of atheroma in ApoE−/− mice. These studies confirm a causal link for active oral T. denticola infection with both atheroma and periodontal disease.
Vascular inflammation plays an important role in the development and progression of atherosclerosis. Recently, salusins (salusin-α and salusin-β) have been reported to be associated wtih atherosclerosis. However, its underlying mechanism remains incompletely known. In this study, we observed the effects of salusins on vascular inflammation in apoE-deficient (apoE-/-) mice.
Methods and Results
Six-week old male apoE-/- mice were infused with salusin-α, salusin-β or vehicle for 8 weeks via osmotic mini-pumps. Our results showed that apoE-/- mice receiving vehicle alone developed severe atherosclerotic lesions and dyslipidemia, with significantly up-regulated levels of IL-6, TNF-α, VCAM-1 and MCP-1. For apoE-/- mice receiving 8 weeks of salusin-β infusion, the atherosclerotic lesions were markedly aggravated, and the levels of IL-6, TNF-α, VCAM-1 and MCP-1 were substantially increased, despite a similar plasma lipid concentration with that of apoE-/- mice. However, after 8 week-infusion of salusin-α, apoE-/- mice presented significant amelioration in atherosclerotic lesions, along with remarkably up-regulated level of high-density lipoprotein-cholesterol (HDL-C) and down-regulated levels of IL-6 and TNF-α, but without any effect on the expressions of VCAM-1 and MCP-1. Furthermore, the activation of nuclear factor-κB (NF-κB), an important transcription factor essential for inflammatory molecules, and the degradation of I-κBα, an inhibitor of NF-κB, were markedly increased in apoE-/- mice receiving vehicle alone. Treatment with salusin-β not salusin-α could remarkably accelerate the process of NF-κB nuclear translocation and I-κBα degradation.
Salusin-β, but not salusin-α, promotes vascular inflammation in apoE-deficient mice via the I-κBα/NF-κB pathway. These findings provide further insight into the mechanism of salusins in atherosclerosis and potential targets for the prevention and treatment of atherosclerosis.
Plasma cholesterol lowering (PCL) slows and sometimes prevents progression of atherosclerosis and may even lead to regression. Little is known about how molecular processes in the atherosclerotic arterial wall respond to PCL and modify responses to atherosclerosis regression. We studied atherosclerosis regression and global gene expression responses to PCL (≥80%) and to atherosclerosis regression itself in early, mature, and advanced lesions. In atherosclerotic aortic wall from Ldlr−/−Apob100/100Mttpflox/floxMx1-Cre mice, atherosclerosis regressed after PCL regardless of lesion stage. However, near-complete regression was observed only in mice with early lesions; mice with mature and advanced lesions were left with regression-resistant, relatively unstable plaque remnants. Atherosclerosis genes responding to PCL before regression, unlike those responding to the regression itself, were enriched in inherited risk for coronary artery disease and myocardial infarction, indicating causality. Inference of transcription factor (TF) regulatory networks of these PCL-responsive gene sets revealed largely different networks in early, mature, and advanced lesions. In early lesions, PPARG was identified as a specific master regulator of the PCL-responsive atherosclerosis TF-regulatory network, whereas in mature and advanced lesions, the specific master regulators were MLL5 and SRSF10/XRN2, respectively. In a THP-1 foam cell model of atherosclerosis regression, siRNA targeting of these master regulators activated the time-point-specific TF-regulatory networks and altered the accumulation of cholesterol esters. We conclude that PCL leads to complete atherosclerosis regression only in mice with early lesions. Identified master regulators and related PCL-responsive TF-regulatory networks will be interesting targets to enhance PCL-mediated regression of mature and advanced atherosclerotic lesions.
The main underlying cause of heart attacks and strokes is atherosclerosis. One strategy to prevent these often deadly clinical events is therefore either to slow atherosclerosis progression or better, induce regression of atherosclerotic plaques making them more stable. Plasma cholesterol lowering (PCL) is the most efficient way to induce atherosclerosis regression but sometimes fails to do so. In our study, we used a mouse model with elevated LDL cholesterol levels, similar to humans who develop early atherosclerosis, and a genetic switch to lower plasma cholesterol at any time during atherosclerosis progression. In this model, we examined atherosclerosis gene expression and regression in response to PCL at three different stages of atherosclerosis progression. PCL led to complete regression in mice with early lesions but was incomplete in mice with mature and advanced lesions, indicating that early prevention with PCL in individuals with increased risk for heart attack or stroke would be particularly useful. In addition, by inferring PCL-responsive gene networks in early, mature and advanced atherosclerotic lesions, we identified key drivers specific for regression of early (PPARG), mature (MLL5) and advanced (SRSF10/XRN2) atherosclerosis. These key drivers should be interesting therapeutic targets to enhance PCL-mediated regression of atherosclerosis.
In vitro data indicate that human LDL modified by Group V secretory phospholipase A2 (GV sPLA2) is pro-atherogenic. Consistent with this, gain and loss of function studies demonstrated that GV sPLA2 promotes atherosclerosis in LDLR-/- mice. The current study investigates whether GV sPLA2 promotes atherosclerotic processes in apoE-/- mice.
Methods and results
LDL (d = 1.019-1.063) from apoE-/- and LDLR-/- mice fed chow or Western diet were hydrolyzed by GV sPLA2. Phosphatidylcholine on LDL from LDLR-/- mice fed either a chow or Western diet was hydrolyzed to a greater extent (61.1±0.4% and 45.3±4.6%) than the corresponding fractions from apoE-/- mice (41.7±3.6% and 39.4±1.2%). ApoE-/- LDL induced macrophage foam cell formation in vitro without modification by GV sPLA2, whereas hydrolysis of LDLR-/- LDL was a pre-requisite for foam cell formation. In contrast to findings in LDLR-/- mice, GV sPLA2 deficiency did not significantly reduce atherosclerosis in apoE-/- mice, although collagen content was significantly reduced in lesions of apoE-/- mice lacking GV sPLA2.
The ability of GV sPLA2 to promote atherosclerotic lipid deposition in apoE-/- and LDLR-/- mice may be related to its ability to increase the atherogenic potential of LDL from these mice as assessed in vitro.
sphingomyelin; foam cells; cholesterol ester; atherosclerosis
To test if blood monocytosis in mice with atherosclerosis affects infarct healing.
Monocytes are cellular protagonists of tissue repair and their specific subtypes regulate the healing program after MI. Inflammatory Ly-6Chi monocytes dominate on day 1-4 and digest damaged tissue; reparative Ly-6Clo monocytes dominate on day 5-10 and promote angiogenesis and scar formation. However, the monocyte repertoire is disturbed in atherosclerotic mice: Ly-6Chi monocytes expand selectively, which may disrupt the resolution of inflammation.
Methods and Results
Ex vivo analysis of 5 day-old infarcts showed >10-times more Ly-6Chi monocytes in atherosclerotic (apoE-/-) mice compared to wild type mice. The injured tissue in apoE-/- mice also showed a more pronounced inflammatory gene expression profile (e.g. increased TNF-α and MPO and decreased TGF-β) and a higher abundance of proteases, which are associated with the activity of Ly-6Chi monocytes. To relate inflammatory activity to left ventricular remodeling, we used a combination of noninvasive molecular and physiologic imaging. FMT-CT on day 5 post MI showed higher proteolysis and phagocytosis in infarcts of atherosclerotic mice. Serial MRI showed accelerated deterioration of EF between day 1 and 21 after MI in apoE-/-. Finally, we could recapitulate these features in wild-type mice with artificially–induced Ly-6Chi monocytosis.
Ly-6Chi monocytosis disturbs resolution of inflammation in murine infarcts and consequently enhances left ventricular remodeling. These findings position monocyte subsets as potential therapeutic targets to augment tissue repair after infarction and to prevent post-MI heart failure.
Ly-6Chi monocytes are key contributors to atherosclerosis in mice. However, how Ly-6Chi monocytes selectively accumulate in atherosclerotic lesions is largely unknown. Monocyte homing to sites of atherosclerosis is primarily initiated by rolling on P- and E-selectin expressed on endothelium. We hypothesize that P-selectin glycoprotein ligand-1 (PSGL-1), the common ligand of P- and E-selectin on leukocytes, contributes to the preferential homing of Ly-6Chi monocytes to atherosclerotic lesions.
Methods and Results
To test this hypothesis, we examined the expression and function of PSGL-1 on Ly-6Chi and Ly-6Clo monocytes from wild-type mice, ApoE-/- mice, and mice lacking both ApoE and PSGL-1 genes (ApoE-/-/PSGL-1-/-). We found that Ly-6Chi monocytes expressed a higher level of PSGL-1, and had enhanced binding to fluid-phase P- and E-selectin, compared to Ly-6Clo monocytes. Under in vitro flow conditions, more Ly-6Chi monocytes rolled on P-, E-, and L-selectin at slower velocities than Ly-6Clo cells. In an ex vivo perfused carotid artery model, Ly-6Chi monocytes interacted preferentially with atherosclerotic endothelium compared with Ly-6Clo monocytes in a PSGL-1-dependent manner. In vivo, ApoE-/- mice lacking PSGL-1 had impaired Ly-6Chi monocyte recruitment to atherosclerotic lesions. Moreover, ApoE-/-/PSGL-1-/- mice exhibited significantly reduced monocyte infiltration in wire injury-induced neointima and in atherosclerotic lesions. ApoE-/-/PSGL-1-/- mice also developed smaller neointima and atherosclerotic plaques.
These data indicate that PSGL-1 is a new marker for Ly-6Chi monocytes and a major determinant for Ly-6Chi cell recruitment to sites of atherosclerosis in mice.
atherosclerosis; leukocytes; endothelium; cell adhesion molecules
Angiotensin II (AII) is a major determinant of atherosclerosis. Although macrophages are the most abundant cells in atherosclerotic plaques and express AII type 1 receptor (AT1), the pathophysiologic role of macrophage AT1 in atherogenesis remains uncertain. We examined the contribution of macrophage AT1 to accelerated atherosclerosis in an AII-responsive setting induced by uninephrectomy (UNx).
Methods and Results
AT1−/− or AT1+/+ marrow from apolipoprotein E deficient (apoE−/−) mice was transplanted into recipient apoE−/− mice with subsequent UNx or sham operation: apoE−/−/AT1+/+→apoE−/− + Sham; apoE−/−/AT1+/+→apoE−/− + UNx; apoE−/−/AT1−/−→apoE−/− + Sham; apoE−/−/AT1−/−→apoE−/− + UNx. No differences in body weight, blood pressure, lipid profile, and serum creatinine were observed between the two UNx groups. ApoE−/−/AT1+/+→apoE−/− + UNx had significantly more atherosclerosis (16907 ± 21473 vs 116071 ± 8180 μm2, p<0.05). By contrast, loss of macrophage AT1 which reduced local AT1 expression, prevented any effect of UNx on atherosclerosis (77174 ± 9947 vs 75714 ± 11333 μm2, p=NS). Although UNx did not affect total macrophage content in the atheroma, lesions in apoE−/−/AT1−/−→apoE−/− + UNx had fewer classically activated macrophage phenotype (M1) and more alternatively activated phenotype (M2). Further, UNx did not affect plaque necrosis or apoptosis in apoE−/−/AT1−/−→apoE−/− whereas it significantly increased both (by 2- and 6-fold, respectively) in apoE−/−/AT1+/+→apoE−/− mice. Instead, apoE−/−/AT1−/−→apoE−/− had 5-fold-increase in macrophage-associated apoptotic bodies, indicating enhanced efferocytosis. In vitro studies confirmed blunted susceptibility to apoptosis, especially in M2 macrophages, and a more efficient phagocytic function of AT1−/− macrophages vs AT1+/+.
AT1 receptor of bone marrow-derived macrophages worsens the extent and complexity of renal injury–induced atherosclerosis by shifting the macrophage phenotype to more M1 and less M2 through mechanisms that include increased apoptosis and impaired efferocytosis.
kidney; atherosclerosis; macrophage; angiotensin II type 1 receptor; efferocytosis