Ldlr Mice Research Articles

Macrophage-specific overexpression of interleukin-5 attenuates atherosclerosis in LDL receptor-deficient mice.

Interleukin-5 (IL-5) increases the secretion of natural T15/EO6 IgM antibodies that inhibit the uptake of oxidized low-density lipoprotein (LDL) by macrophages. This study aimed to determine whether macrophage-specific expression of IL-5 in LDL receptor-deficient mice (Ldlr(-/-)) could improve cholesterol metabolism and reduce atherosclerosis. To induce macrophage-specific IL-5 expression, the pLVCD68-IL5 lentivirus was delivered into Ldlr(-/-) mice via bone marrow transplantation. The recipient mice were fed a Western-type diet for 12 weeks to induce lesion formation. We found that IL-5 was efficiently and specifically overexpressed in macrophages in recipients of pLVCD68-IL5-transduced bone marrow cells (BMC). Plasma titers of T15/EO6 IgM antibodies were significantly elevated by 58% compared with control mice transplanted with pLVCD68 lacking the IL-5 coding sequence. Plaque areas of aortas in IL-5-overexpressing mice were reduced by 43% and associated with a 2.4-fold decrease in lesion size at the aortic roots when compared with mice receiving pLVCD68-transduced BMCs. The study showed that macrophage-specific overexpression of IL-5 inhibited the progression of atherosclerotic lesions. These findings suggest that modulation of IL-5 cytokine expression represents a potential strategy for intervention of familial hypercholesterolemia and other cardiovascular diseases.

Source and role of intestinally derived lysophosphatidic acid in dyslipidemia and atherosclerosis

We previously reported that i) a Western diet increased levels of unsaturated lysophosphatidic acid (LPA) in small intestine and plasma of LDL receptor null (LDLR(-/-)) mice, and ii) supplementing standard mouse chow with unsaturated (but not saturated) LPA produced dyslipidemia and inflammation. Here we report that supplementing chow with unsaturated (but not saturated) LPA resulted in aortic atherosclerosis, which was ameliorated by adding transgenic 6F tomatoes. Supplementing chow with lysophosphatidylcholine (LysoPC) 18:1 (but not LysoPC 18:0) resulted in dyslipidemia similar to that seen on adding LPA 18:1 to chow. PF8380 (a specific inhibitor of autotaxin) significantly ameliorated the LysoPC 18:1-induced dyslipidemia. Supplementing chow with LysoPC 18:1 dramatically increased the levels of unsaturated LPA species in small intestine, liver, and plasma, and the increase was significantly ameliorated by PF8380 indicating that the conversion of LysoPC 18:1 to LPA 18:1 was autotaxin dependent. Adding LysoPC 18:0 to chow increased levels of LPA 18:0 in small intestine, liver, and plasma but was not altered by PF8380 indicating that conversion of LysoPC 18:0 to LPA 18:0 was autotaxin independent. We conclude that i) intestinally derived unsaturated (but not saturated) LPA can cause atherosclerosis in LDLR(-/-) mice, and ii) autotaxin mediates the conversion of unsaturated (but not saturated) LysoPC to LPA.

Myeloid A Disintegrin and Metalloproteinase Domain 10 Deficiency Modulates Atherosclerotic Plaque Composition by Shifting the Balance from Inflammation toward Fibrosis

A disintegrin and metalloproteinase domain 10 (ADAM10) is a metalloprotease involved in cleavage of various cell surface molecules, such as adhesion molecules, chemokines, and growth factor receptors. Although we have previously shown an association of ADAM10 expression with atherosclerotic plaque progression, a causal role of ADAM10 in atherosclerosis has not been investigated. Bone marrow from conditional knockout mice lacking Adam10 in the myeloid lineage or from littermate controls was transplanted into lethally irradiated low density lipoprotein receptor Ldlr(-/-) mice on an atherogenic diet. Myeloid Adam10 deficiency did not affect plaque size, but it increased plaque collagen content. Matrix metalloproteinase 9 and 13 expression and matrix metalloproteinase 2 gelatinase activity were significantly impaired in Adam10-deficient macrophages, whereas their capacity to stimulate collagen production was unchanged. Furthermore, relative macrophage content in advanced atherosclerotic lesions was decreased. In vitro, Adam10-deficient macrophages showed reduced migration toward monocyte chemoattractant protein-1 and transmigration through collagen. In addition, Adam10-deficient macrophages displayed increased anti-inflammatory phenotype with elevated IL-10, and reduced production of proinflammatory tumor necrosis factor, IL-12, and nitric oxide in response to lipopolysaccharide. These data suggest a critical role of Adam10 for leukocyte recruitment, inflammatory mediator production, and extracellular matrix degradation. Thereby, myeloid ADAM10 may play a causal role in modulating atherosclerotic plaque stability.

Role of Hic-5 in the formation of microvilli-like structures and the monocyte-endothelial interaction that accelerates atherosclerosis.

The adhesion of circulating monocytes to endothelial cells (ECs) is an early and critical event in the formation of atherosclerotic plaques. Hydrogen peroxide-inducible clone 5 (Hic-5) serves as an adaptor molecule in cell adhesion complexes. However, the role of endothelial Hic-5 in monocyte-EC interaction and atherogenesis remains unclear. We examined the roles of endothelial Hic-5 in monocyte-EC interaction and atherogenesis using mouse models of atherosclerosis and cultured human umbilical vein endothelial cells (HUVECs). Hic-5 was expressed in ECs, but not in monocytes/macrophages. An ex vivo monocyte adhesion assay revealed that adhesion of THP-1 monocytes to aortas isolated from Apoe(-/-) and LDLR(-/-) mice stimulated by TNF-α or oxidized LDL was suppressed by Hic-5 deficiency. Scanning electron microscopic observations of aortas harvested from Apoe(-/-) mice revealed that TNF-α- or oxidized LDL-induced microvilli-like structures were markedly suppressed by Hic-5 deficiency. Relative Hic-5 deficiency suppressed 60% of the atherosclerotic lesions in aortas from Apoe(-/-) and LDLR(-/-) mice. In contrast, overexpression of Hic-5 in HUVECs promoted induction of microvilli-like structures and adherence of THP-1 cells in an adhesion receptor such as intercellular adhesion molecule-1- and vascular cell adhesion molecule-1-dependent manner. Hic-5 in ECs plays an important role in the formation of microvilli-like structures and in the interaction between ECs and monocytes, leading to monocyte recruitment and subsequent development of atherosclerosis.

Hematopoietic α7 nicotinic acetylcholine receptor deficiency increases inflammation and platelet activation status, but does not aggravate atherosclerosis

The autonomic nervous system attenuates inflammation through activation of the α7 nicotinic acetylcholine receptor (α7nAChR), a pathway termed the cholinergic anti-inflammatory reflex. Interestingly, α7nAChR is expressed on immune cells and platelets, both of which play a crucial role in the development of atherosclerosis. To investigate the role of hematopoietic α7nAChR in inflammation and platelet function in atherosclerotic ldlr(-/-) mice and to identify its consequences for atherosclerotic lesion development. Bone marrow from α7nAChR(-/-) mice or wild-type littermates was transplanted into irradiated ldlr(-/-) mice. After a recovery period of 8 weeks, the mice were fed an atherogenic Western-type diet for 7 weeks. Hematopoietic α7nAChR deficiency clearly increased the number of leukocytes in the peritoneum (2.6-fold, P < 0.001), blood (2.9-fold; P < 0.01), mesenteric lymph nodes (2.0-fold; P < 0.001) and spleen (2.2-fold; P < 0.01), indicative of an increased inflammatory status. Additionally, expression of inflammatory mediators was increased in peritoneal leukocytes (TNFα, 1.6-fold, P < 0.01; CRP, 1.8-fold, P < 0.01) as well as in the spleen (TNFα, 1.6-fold, P < 0.01). The lack of α7nAChR on platelets from these mice increased the expression of active integrin αIIb β3 upon stimulation by ADP (1.9-fold, P < 0.01), indicating increased activation status, while incubation of human platelets with an α7nAChR agonist decreased aggregation (-35%, P < 0.05). Despite the large effects of hematopoietic α7nAChR deficiency on inflammatory status and platelet function, it did not affect atherosclerosis development or composition of lesions. Hematopoietic α7nAChR is important for attenuation of inflammatory responses and maintaining normal platelet reactivity, but loss of hematopoietic α7nAChR does not aggravate development of atherosclerosis.

Hematopoietic Akt2 deficiency attenuates the progression of atherosclerosis.

Atherosclerosis is the major cause of death and disability in diabetic and obese subjects with insulin resistance. Akt2, a phosphoinositide-dependent serine-threonine protein kinase, is highly express in insulin-responsive tissues; however, its role during the progression of atherosclerosis remains unknown. Thus, we aimed to investigate the contribution of Akt2 during the progression of atherosclerosis. We found that germ-line Akt2-deficient mice develop similar atherosclerotic plaques as wild-type mice despite higher plasma lipids and glucose levels. It is noteworthy that transplantation of bone marrow cells isolated from Akt2(-/-) mice to Ldlr(-/-) mice results in marked reduction of the progression of atherosclerosis compared with Ldlr(-/-) mice transplanted with wild-type bone marrow cells. In vitro studies indicate that Akt2 is required for macrophage migration in response to proatherogenic cytokines (monocyte chemotactic protein-1 and macrophage colony-stimulating factor). Moreover, Akt2(-/-) macrophages accumulate less cholesterol and have an alternative activated or M2-type phenotype when stimulated with proinflammatory cytokines. Together, these results provide evidence that macrophage Akt2 regulates migration, the inflammatory response and cholesterol metabolism and suggest that targeting Akt2 in macrophages might be beneficial for treating atherosclerosis.

Reducing Macrophage Proteoglycan Sulfation Increases Atherosclerosis and Obesity through Enhanced Type I Interferon Signaling

Heparan sulfate proteoglycans (HSPGs) are an important constituent of the macrophage glycocalyx and extracellular microenvironment. To examine their role in atherogenesis, we inactivated the biosynthetic gene N-acetylglucosamine N-deacetylase-N-sulfotransferase 1 (Ndst1) in macrophages and crossbred the strain to Ldlr(-/-) mice. When placed on an atherogenic diet, Ldlr(-/-)Ndst1(f/f)LysMCre(+) mice had increased atherosclerotic plaque area and volume compared to Ldlr(-/-) mice. Diminished sulfation of heparan sulfate resulted inenhanced chemokine expression; increased macrophages in plaques; increased expression of ACAT2, a key enzyme in cholesterol ester storage; and increased foam cell conversion. Motif analysis of promoters of upregulated genes suggested increased type I interferon signaling, which was confirmed by elevation of STAT1 phosphorylation induced by IFN-β. The proinflammatory macrophages derived from Ndst1(f/f)LysMCre(+) mice also sensitized the animals to diet-induced obesity. We propose that macrophage HSPGs control basal activation of macrophages by maintaining type I interferon reception in a quiescent state through sequestration of IFN-β.

Reduced Levels of microRNAs miR-124a and miR-150 Are Associated with Increased Proinflammatory Mediator Expression in Krüppel-like Factor 2 (KLF2)-deficient Macrophages

Previous studies have shown that the myeloid-specific deficiency of the transcription factor Krüppel-like factor 2 (KLF2) accelerates atherosclerosis in hypercholesterolemic Ldlr(-/-) mice due to the enhanced adhesion of myeloid cells to activated endothelial cells in the vessel wall. This study revealed elevated basal inflammation with elevated plasma levels of Ccl2, Ccl4, Ccl5, and Ccl11 in the myeloid-specific KLF2 knock-out (myeKlf2(-/-)) mice. Peritoneal macrophages isolated from myeKlf2(-/-) mice showed increased mRNA levels of several inflammatory mediators, including Ccl2, Ccl5, Ccl7, Cox-2, Cxcl1, and IL-6. In contrast, the levels of two microRNAs, miR-124a and miR-150, were lower in Klf2(-/-) macrophages compared with Klf2(+/+) macrophages. Additional studies showed a direct inverse relationship between miR-124a levels with Ccl2 expression, with anti-miR-124a increasing Ccl2 mRNA levels in Klf2(+/+) macrophages, whereas the restoration of miR-124a levels in Klf2(-/-) macrophages significantly reduced Ccl2 mRNA expression. Likewise, the inverse relationship was observed between miR-150 levels and Cxcl1 expression in Klf2(+/+) and Klf2(-/-) mice. Moreover, miR150 likely regulates the miR124a expression and thus augments expression of inflammatory mediators in myeKlf2(-/-) macrophages. This study documented that the transcription factor KLF2 modulates inflammatory chemokine production via regulation of microRNA expression levels in immune cells.

The role of the FPR2/ALX receptor in atherosclerosis development and plaque stability.

AimsThe formyl peptide receptor (FPR) subtype FPR2/ALX transduces pro-inflammatory responses and participates in the resolution of inflammation depending on activation. The aim of the present study was to unravel the role of FPR2/ALX signalling in atherosclerosis.Methods and resultsExpression of FPR2/ALX was analysed in 127 human carotid atherosclerotic lesions and revealed that this receptor was expressed on macrophages, smooth muscle cells (SMCs), and endothelial cells. Furthermore, FPR2/ALX mRNA levels were significantly up-regulated in atherosclerotic lesions compared with healthy vessels. In multiple regression, age, creatinine, and clinical signs of increased cerebral ischaemia were independent predictors of FPR2/ALX expression. To provide mechanistic insights into these observations, we generated Ldlr−/−xFpr2−/− mice, which exhibited delayed atherosclerosis development and less macrophage infiltration compared with Ldlr−/−xFpr2+/+ mice. These findings were reproduced by transplantation of Fpr2−/− bone marrow into Ldlr−/− mice and further extended by in vitro experiments, demonstrating a lower inflammatory state in Fpr2−/− macrophages. FPR2/ALX expression correlated with chemo- and cytokines in human atherosclerotic lesions and leucocytes. Finally, atherosclerotic lesions in Ldlr−/−xFpr2−/− mice exhibited decreased collagen content, and Fpr2−/− SMCs exhibited a profile of increased collagenase and decreased collagen production pathways.ConclusionFPR2/ALX is proatherogenic due to effects on bone marrow-derived cells, but promoted a more stable plaque phenotype through effects on SMCs. Taken together, these results suggest a dual role of FPR2/ALX signalling in atherosclerosis by way of promoting disease progression and but increasing plaque stability.

α7 Nicotinic acetylcholine receptor is expressed in human atherosclerosis and inhibits disease in mice--brief report.

Cholinergic pathways of the autonomic nervous system are known to modulate inflammation. Because atherosclerosis is a chronic inflammatory condition, we tested whether cholinergic signaling operates in this disease. We have analyzed the expression of the α7 nicotinic acetylcholine receptor (α7nAChR) in human atherosclerotic plaques and studied its effects on the development of atherosclerosis in the hypercholesterolemic Ldlr(-/-) mouse model. α7nAChR protein was detected on T cells and macrophages in surgical specimens of human atherosclerotic plaques. To study the role of α7nAChR signaling in atherosclerosis, male Ldlr(-/-) mice were lethally irradiated and reconstituted with bone marrow from wild-type or α7nAChR-deficient animals. Ablation of hematopoietic cell α7nAChR increased aortic atherosclerosis by 72%. This was accompanied by increased aortic interferon-γ mRNA, implying increased Th1 activity in the absence of α7nAChR signaling. The present study shows that signaling through hematopoietic α7nAChR inhibits atherosclerosis and suggests that it operates by modulating immune inflammation. Given the observation that α7nAChR is expressed by T cells and macrophages in human plaques, our findings support the notion that cholinergic regulation may act to inhibit disease development also in man.

Glycogen Synthase Kinase 3α Deficiency Attenuates Atherosclerosis and Hepatic Steatosis in High Fat Diet–Fed Low Density Lipoprotein Receptor–Deficient Mice

Studies have implicated signaling through glycogen synthase kinase (GSK) 3α/β in the activation of pro-atherogenic pathways and the accelerated development of atherosclerosis. By using a mouse model, we examined the role of GSK3α in the development and progression of accelerated atherosclerosis. We crossed Gsk3a/GSK3α-knockout mice with low-density lipoprotein receptor (Ldlr) knockout mice. Five-week-old Ldlr(-/-);Gsk3a(+/+), Ldlr(-/-);Gsk3a(+/-), and Ldlr(-/-);Gsk3a(-/-) mice were fed a chow diet or a high-fat diet for 10 weeks and then sacrificed. GSK3α deficiency had no detectible effect on any measured parameters in chow-fed mice. High-fat-diet fed Ldlr(-/-) mice that were deficient for GSK3α had significantly less hepatic lipid accumulation and smaller atherosclerotic lesions (60% smaller in Ldlr(-/-);Gsk3a(+/-) mice, 80% smaller in Ldlr(-/-);Gsk3a(-/-) mice; P<0.05), compared with Ldlr(-/-);Gsk3a(+/+) controls. GSK3α deficiency was associated with a significant increase in plasma IL-10 concentration and IL-10 expression in isolated macrophages. A twofold to threefold enhancement in endoplasmic reticulum stress-induced IL-10 expression was observed in Thp-1-derived macrophages that were pretreated with the GSK3α/β inhibitor CT99021. Together, these results suggest that GSK3α plays a pro-atherogenic role, possibly by mediating the effects of endoplasmic reticulum stress in the activation of pro-atherogenic pathways.

Osteocalcin protects against nonalcoholic steatohepatitis in a mouse model of metabolic syndrome.

Nonalcoholic fatty liver disease, particularly its more aggressive form, nonalcoholic steatohepatitis (NASH), is associated with hepatic insulin resistance. Osteocalcin, a protein secreted by osteoblast cells in bone, has recently emerged as an important metabolic regulator with insulin-sensitizing properties. In humans, osteocalcin levels are inversely associated with liver disease. We thus hypothesized that osteocalcin may attenuate NASH and examined the effects of osteocalcin treatment in middle-aged (12-mo-old) male Ldlr(-/-) mice, which were fed a Western-style high-fat, high-cholesterol diet for 12 weeks to induce metabolic syndrome and NASH. Mice were treated with osteocalcin (4.5 ng/h) or vehicle for the diet duration. Osteocalcin treatment not only protected against Western-style high-fat, high-cholesterol diet-induced insulin resistance but substantially reduced multiple NASH components, including steatosis, ballooning degeneration, and fibrosis, with an overall reduction in nonalcoholic fatty liver disease activity scores. Further, osteocalcin robustly reduced expression of proinflammatory and profibrotic genes (Cd68, Mcp1, Spp1, and Col1a2) in liver and suppressed inflammatory gene expression in white adipose tissue. In conclusion, these results suggest osteocalcin inhibits NASH development by targeting inflammatory and fibrotic processes.

Hematopoietic overexpression of Cyp27a1 reduces hepatic inflammation independently of 27-hydroxycholesterol levels in Ldlr−/− mice

Non-alcoholic steatohepatitis (NASH) is characterized by hepatic lipid accumulation and inflammation. Currently, the underlying mechanisms, leading to hepatic inflammation, are still unknown. The breakdown of free cholesterol inside Kupffer cells (KCs) by the mitochondrial enzyme CYP27A1 produces 27-hydroxycholesterol (27HC). We recently demonstrated that administration of 27HC to hyperlipidemic mice reduced hepatic inflammation. In line, hematopoietic deletion of Cyp27a1 resulted in increased hepatic inflammation. In the current manuscript, the effect of hematopoietic overexpression of Cyp27a1 on the development of NASH and cholesterol trafficking was investigated. We hypothesized that Cyp27a1 overexpression in KCs will lead to reduced hepatic inflammation. Irradiated Ldlr(-/-) mice were transplanted (tp) with bone marrow from mice overexpressing Cyp27a1 (Cyp27a1(over)) and wild type (Wt) mice and fed either chow or a high-fat, high-cholesterol (HFC) diet for 3 months. Additionally, gene expression was assessed in bone marrow-derived macrophages (BMDM) from Cyp27a1(over) and Wt mice. In line with our hypothesis, hepatic inflammation in HFC-fed Cyp27a1(over)-tp mice was reduced and KCs were less foamy compared to Wt-tp mice. Remarkably, these changes occurred even though plasma and liver levels of 27HC did not differ between both groups. BMDM from Cyp27a1(over) mice revealed reduced inflammatory gene expression and increased expression of cholesterol transporters compared to Wt BMDM after lipopolysaccharide (LPS) stimulation. Our data suggest that overexpression of Cyp27a1 in KCs reduces hepatic inflammation independently of 27HC levels in plasma and liver, further pointing towards KCs as specific target for improving the therapy of NASH.

Inhibiting DNA Methylation by 5-Aza-2'-deoxycytidine ameliorates atherosclerosis through suppressing macrophage inflammation.

Inflammation marks all stages of atherogenesis. DNA hypermethylation in the whole genome or specific genes is associated with inflammation and cardiovascular diseases. Therefore, we aimed to study whether inhibiting DNA methylation by DNA methyltransferase inhibitor 5-aza-2'-deoxycytidine (5-aza-dC) ameliorates atherosclerosis in low-density lipoprotein receptor knockout (Ldlr(-/-)) mice. Ldlr(-/-) mice were fed an atherogenic diet and adminisered saline or 5-aza-dC (0.25 mg/kg) for up to 30 weeks. 5-aza-dC treatment markedly decreased atherosclerosis development in Ldlr(-/-) mice without changes in body weight, plasma lipid profile, macrophage cholesterol levels and plaque lipid content. Instead, this effect was associated with decreased macrophage inflammation. Macrophages with 5-aza-dC treatment had downregulated expression of genes involved in inflammation (TNF-α, IL-6, IL-1β, and inducible nitric oxidase) and chemotaxis (CD62/L-selectin, chemokine [C-C motif] ligand 2/MCP-1 [CCL2/MCP-1], CCL5, CCL9, and CCL2 receptor CCR2). This resulted in attenuated macrophage migration and adhesion to endothelial cells and reduced macrophage infiltration into atherosclerotic plaques. 5-aza-dC also suppressed macrophage endoplasmic reticulum stress, a key upstream signal that activates macrophage inflammation and apoptotic pathways. Finally, 5-aza-dC demethylated liver X receptor α (LXRα) and peroxisome proliferator-activated receptor γ1 (PPARγ1) promoters, which are both enriched with CpG sites. This led to overexpression of LXRα and PPARγ, which may be responsible for 5-aza-dC's anti-inflammatory and atheroprotective effect. Our findings provide strong evidence that DNA methylation may play a significant role in cardiovascular diseases and serve as a therapeutic target for prevention and treatment of atherosclerosis.

MHC Class II-restricted antigen presentation by plasmacytoid dendritic cells drives proatherogenic T cell immunity.

Plasmacytoid dendritic cells (pDCs) bridge innate and adaptive immune responses and are important regulators of immuno-inflammatory diseases. However, their role in atherosclerosis remains elusive. Here, we used genetic approaches to investigate the role of pDCs in atherosclerosis. Selective pDC deficiency in vivo was achieved using CD11c-Cre × Tcf4(-/flox) bone marrow transplanted into Ldlr(-/-) mice. Compared with control Ldlr(-/-) chimeric mice, CD11c-Cre × Tcf4(-/flox) mice had reduced atherosclerosis levels. To begin to understand the mechanisms by which pDCs regulate atherosclerosis, we studied chimeric Ldlr(-/-) mice with selective MHCII deficiency on pDCs. Significantly, these mice also developed reduced atherosclerosis compared with controls without reductions in pDC numbers or changes in conventional DCs. MHCII-deficient pDCs showed defective stimulation of apolipoprotein B100-specific CD4(+) T cells in response to native low-density lipoprotein, whereas production of interferon-α was not affected. Finally, the atheroprotective effect of selective MHCII deficiency in pDCs was associated with significant reductions of proatherogenic T cell-derived interferon-γ and lesional T cell infiltration, and was abrogated in CD4(+) T cell-depleted animals. This study supports a proatherogenic role for pDCs in murine atherosclerosis and identifies a critical role for MHCII-restricted antigen presentation by pDCs in driving proatherogenic T cell immunity.

Systemic Atherosclerosis Causes Detrusor Overactivity: Functional and Morphological Changes in Hyperlipoproteinemic apoE–/–LDLR–/– Mice

The prevalence of systemic atherosclerosis and overactive bladder/detrusor overactivity increases almost simultaneously with age but an association between these diseases has not yet been proved. We evaluated changes in bladder function and morphology, including vascularization, in apoE(-/-)LDLR(-/-) double knockout mice with systemic atherosclerosis but without central nervous system involvement. Cystometry was performed in awake, freely moving 60-week-old apoE(-/-)LDLR(-/-) mice and C57BL/6N controls. The mice were sacrificed and perfused with Microfil® contrast medium. The bladder was excised, dissected and scanned by nano-computerized tomography, including 3-dimensional reconstruction. Samples then underwent histomorphological analysis. In apoE(-/-)LDLR(-/-) mice cystometry revealed a significant decrease in the peak-peak interval, micturition interval, functional bladder capacity and micturition volume. However, maximum bladder pressure increased. Nano-computerized tomography revealed a significant reduction in bladder wall thickness, segment volume, vascular volume and the vascular volume fraction. Histomorphologically bladder specimens showed a thickened media of intramural vessels, activated endothelial cells and intramural inflammatory cells. To our knowledge this study presents a new in vivo mouse model of nonneurogenic detrusor overactivity caused by systemic atherosclerosis. Decreased bladder wall vascularization seems to be a major factor for detrusor overactivity onset. Capillaries are rarified with reduced lumina due to thickened media. Activated endothelial cells and the infiltration of inflammatory cells in apoE(-/-)LDLR(-/-) mice underlines once more that atherosclerosis is an inflammatory process that may also be relevant to the onset of detrusor overactivity.

Innate immune receptor NOD2 promotes vascular inflammation and formation of lipid-rich necrotic cores in hypercholesterolemic mice.

Atherosclerosis is an inflammatory disease associated with the activation of innate immune TLRs and nucleotide-binding oligomerization domain-containing protein (NOD)-like receptor pathways. However, the function of most innate immune receptors in atherosclerosis remains unclear. Here, we show that NOD2 is a crucial innate immune receptor influencing vascular inflammation and atherosclerosis severity. 10-week stimulation with muramyl dipeptide (MDP), the NOD2 cognate ligand, aggravated atherosclerosis, as indicated by the augmented lesion burden, increased vascular inflammation and enlarged lipid-rich necrotic cores in Ldlr(-/-) mice. Myeloid-specific ablation of NOD2, but not its downstream kinase, receptor-interacting serine/threonine-protein kinase 2, restrained the expansion of the lipid-rich necrotic core in Ldlr(-/-) chimeric mice. In vitro stimulation of macrophages with MDP enhanced the uptake of oxidized low-density lipoprotein and impaired cholesterol efflux in concordance with upregulation of scavenger receptor A1/2 and downregulation of ATP-binding cassette transporter A1. Ex vivo stimulation of human carotid plaques with MDP led to increased activation of inflammatory signaling pathways p38 MAPK and NF-κB-mediated release of proinflammatory cytokines. Altogether, this study suggests that NOD2 contributes to the expansion of the lipid-rich necrotic core and promotes vascular inflammation in atherosclerosis.

The omega-3 fatty acid docosahexaenoic acid favorably modulates the inflammatory pathways and macrophage polarization within aorta of LDLR(-/-) mice.

The omega-3 fatty acid docosahexaenoic acid (DHA) has potent anti-atherogenic properties but its mechanisms of action at the vascular level remain poorly explored. Knowing the broad range of molecular targets of omega-3 fatty acids, microarray analysis was used to open-mindedly evaluate the effects of DHA on aorta gene expression in LDLR(-/-) mice and better understand its local anti-atherogenic action. Mice were fed an atherogenic diet and received daily oral gavages with oils rich in oleic acid or DHA. Bioinformatics analysis of microarray data first identified inflammation and innate immunity as processes the most affected by DHA supplementation within aorta. More precisely, several down-regulated genes were associated with the inflammatory functions of macrophages (e.g., CCL5 and CCR7), cell movement (e.g., ICAM-2, SELP, and PECAM-1), and the major histocompatibility complex (e.g., HLA-DQA1 and HLA-DRB1). Interestingly, several genes were identified as specific biomarkers of macrophage polarization, and their changes suggested a preferential orientation toward a M2 reparative phenotype. This observation was supported by the upstream regulator analysis highlighting the involvement of three main regulators of macrophage polarization, namely PPARγ (z-score=2.367, p=1.50×10(-13)), INFγ (z-score=-2.797, p=2.81×10(-14)), and NFκB (z-score=2.360, p=6.32×10(-9)). Moreover, immunohistological analysis of aortic root revealed an increased abundance of Arg1 (+111%, p=0.01), a specific biomarker of M2 macrophage. The present study showed for the first time that DHA supplementation during atherogenesis is associated with protective modulation of inflammation and innate immunity pathways within aorta putatively through the orientation of plaque macrophages toward a M2 reparative phenotype.

Sesamol and sesame (Sesamum indicum) oil enhance macrophage cholesterol efflux via up-regulation of PPARγ1 and LXRα transcriptional activity in a MAPK-dependent manner.

Cholesterol clearance by macrophages is a vital process to eliminate excess cholesterol from the body. Internalization of modified cholesterol by macrophages triggers overexpression of peroxisome proliferator-activated receptor γ1 (PPARγ1) and liver X receptor α (LXRα), two transcription factors that are critically involved in macrophage cholesterol efflux. Recent studies demonstrate that oral administration of sesamol derivative (INV-403) and sesame oil leads to a significant attenuation of atherosclerosis in Watanabe heritable hyperlipidemic rabbits and LDLR(-/-) mice, respectively. However, the exact molecular mechanisms underlying such anti-atherogenic effects remain largely unrevealed. Luciferase reporter assays were performed to assess the effects of sesamol and sesame oil on PPARγ1 and LXRα gene expression. The potential of sesamol and sesame oil to modulate cholesterol efflux was evaluated using (3)H-cholesterol efflux assays. Sesamol and sesame oil treatments lead to a significant up-regulation of PPARγ1 and LXRα expression and transcriptional activity in a MAPK-dependent manner. Importantly, primary macrophages display a significantly enhanced cholesterol efflux potential upon treatment with sesamol and sesame oil, and this stimulatory effect is mediated by MAPK signaling. Our findings suggest that the previously reported anti-atherogenic effects of sesamol and sesame oil could be attributed, at least in part, to enhanced PPARγ1 and LXRα expression and transcriptional activity leading to improved macrophage cholesterol efflux. Our study is novel in elucidating the molecular and cellular mechanisms underlying the protective effects of sesamol and sesame oil against atherosclerosis.

The low density lipoprotein receptor modulates the effects of hypogonadism on diet-induced obesity and related metabolic perturbations

Here, we investigated how LDL receptor deficiency (Ldlr(-/-)) modulates the effects of testosterone on obesity and related metabolic dysfunctions. Though sham-operated Ldlr(-/-) mice fed Western-type diet for 12 weeks became obese and showed disturbed plasma glucose metabolism and plasma cholesterol and TG profiles, castrated mice were resistant to diet-induced obesity and had improved glucose metabolism and reduced plasma TG levels, despite a further deterioration in their plasma cholesterol profile. The effect of hypogonadism on diet-induced weight gain of Ldlr(-/-) mice was independent of ApoE and Lrp1. Indirect calorimetry analysis indicated that hypogonadism in Ldlr(-/-) mice was associated with increased metabolic rate. Indeed, mitochondrial cytochrome c and uncoupling protein 1 expression were elevated, primarily in white adipose tissue, confirming increased mitochondrial metabolic activity due to thermogenesis. Testosterone replacement in castrated Ldlr(-/-) mice for a period of 8 weeks promoted diet-induced obesity, indicating a direct role of testosterone in the observed phenotype. Treatment of sham-operated Ldlr(-/-) mice with the aromatase inhibitor exemestane for 8 weeks showed that the obesity of castrated Ldlr(-/-) mice is independent of estrogens. Overall, our data reveal a novel role of Ldlr as functional modulator of metabolic alterations associated with hypogonadism.