Mediator 1 Is Atherosclerosis Protective by Regulating Macrophage Polarization.
ABSTRACT: MED1 (mediator 1) interacts with transcription factors to regulate transcriptional machinery. The role of MED1 in macrophage biology and the relevant disease state remains to be investigated.To study the molecular mechanism by which MED1 regulates the M1/M2 phenotype switch of macrophage and the effect on atherosclerosis, we generated MED1/apolipoprotein E (ApoE) double-deficient (MED1?Mac/ApoE-/-) mice and found that atherosclerosis was greater in MED1?Mac/ApoE-/- mice than in MED1fl/fl/ApoE-/- littermates. The gene expression of M1 markers was increased and that of M2 markers decreased in both aortic wall and peritoneal macrophages from MED1?Mac/ApoE-/- mice, whereas MED1 overexpression rectified the changes in M1/M2 expression. Moreover, LDLR (low-density lipoprotein receptor)-deficient mice received bone marrow from MED1?Mac mice showed greater atherosclerosis. Mechanistically, MED1 ablation decreased the binding of PPAR? (peroxisome proliferator-activated receptor ?) and enrichment of H3K4me1 and H3K27ac to upstream region of M2 marker genes. Furthermore, interleukin 4 induction of PPAR? and MED1 increased the binding of PPAR? or MED1 to the PPAR response elements of M2 marker genes.Our data suggest that MED1 is required for the PPAR?-mediated M2 phenotype switch, with M2 marker genes induced but M1 marker genes suppressed. MED1 in macrophages has an antiatherosclerotic role via PPAR?-regulated transactivation.
Project description:NADPH oxidase (Nox)-derived reactive oxygen species (ROS) are instrumental in all inflammatory phases of atherosclerosis. Dysregulated histone deacetylase (HDAC)-related epigenetic pathways have been mechanistically linked to alterations in gene expression in experimental models of cardiovascular disorders. Hitherto, the relation between HDAC and Nox in atherosclerosis is not known. We aimed at uncovering whether HDAC plays a role in mediating Nox up-regulation, oxidative stress, inflammation, and atherosclerotic lesion progression. Human non-atherosclerotic and atherosclerotic arterial samples, ApoE-/- mice, and in vitro polarized monocyte-derived M1/M2-macrophages (Mac) were examined. Male ApoE-/- mice, maintained on normal or high-fat, cholesterol-rich diet, were randomized to receive 10 mg/kg suberoylanilide hydroxamic acid (SAHA), a pan-HDAC inhibitor, or its vehicle, for 4 weeks. In the human/animal studies, real-time PCR, Western blot, lipid staining, lucigenin-enhanced chemiluminescence assay, and enzyme-linked immunosorbent assay were employed. The protein levels of class I, class IIa, class IIb, and class IV HDAC isoenzymes were significantly elevated both in human atherosclerotic tissue samples and in atherosclerotic aorta of ApoE-/- mice. Treatment of ApoE-/- mice with SAHA reduced significantly the extent of atherosclerotic lesions, and the aortic expression of Nox subtypes, NADPH-stimulated ROS production, oxidative stress and pro-inflammatory markers. Significantly up-regulated HDAC and Nox subtypes were detected in inflammatory M1-Mac. In these cells, SAHA reduced the Nox1/2/4 transcript levels. Collectively, HDAC inhibition reduced atherosclerotic lesion progression in ApoE-/- mice, possibly by intertwined mechanisms involving negative regulation of Nox expression and inflammation. The data propose that HDAC-oriented pharmacological interventions could represent an effective therapeutic strategy in atherosclerosis.
Project description:Angiotensin II is a major determinant of atherosclerosis. Although macrophages are the most abundant cells in atherosclerotic plaques and express angiotensin II 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 angiotensin II-responsive setting induced by uninephrectomy (UNx).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 2 UNx groups. ApoE(-/-)/AT1(+/+) ?apoE(-/-)+UNx had significantly more atherosclerosis (16907±21473 versus 116071±8180 ?m(2), P<0.05). By contrast, loss of macrophage AT1 which reduced local AT1 expression, prevented any effect of UNx on atherosclerosis (77174±9947 versus 75714±11333 ?m(2), 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 versus 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.
Project description:Lifestyle choices play a significant role in the etiology of atherosclerosis. Male Apoe-/- mice that develop spontaneous atherosclerotic lesions were fed 0%, 0.3%, and 0.4% mangosteen extracts, composed largely of α-mangostin (MG), for 17 weeks. Body weight gains were significantly decreased in both MG-treated groups compared to the control, but the general condition remained good throughout the study. The levels of total cholesterol (decreased very-low-density lipoprotein in lipoprotein profile) and triglycerides decreased significantly in the MG-treated mice in conjunction with decreased hepatic HMG-CoA synthase and Fatty acid transporter. Additionally, increased serum lipoprotein lipase activity and histopathology further showed a significant reduction in atherosclerotic lesions at both levels of MG exposure. Real-time PCR analysis for macrophage indicators showed a significant elevation in the levels of Cd163, an M2 macrophage marker, in the lesions of mice receiving 0.4% MG. However, the levels of Nos2, associated with M1 macrophages, showed no change. In addition, quantitative immunohistochemical analysis of macrophage subtypes showed a tendency for increased M2 populations (CD68⁺/CD163⁺) in the lesions of mice given 0.4% MG. In further analysis of the cytokine-polarizing macrophage subtypes, the levels of Interleukin13 (Il13), associated with M2 polarization, were significantly elevated in lesions exposed to 0.4% MG. Thus, MG could suppress the development of atherosclerosis in Apoe-/- mice, possibly through an M2 macrophage-mediated mechanism.
Project description:Atherosclerosis is a chronic inflammatory process that leads to plaque formation in large and medium sized vessels. T helper 1 (Th1) cells constitute the majority of plaque infiltrating pro-atherogenic T cells and are induced via IFN?-dependent activation of T-box (Tbet) and/or IL-12-dependent activation of signal transducer and activator of transcription 4 (STAT4). We thus aimed to define a role for STAT4 in atherosclerosis. STAT4-deficiency resulted in a ?71% reduction (p < 0.001) in plaque burden in Stat4(-/-)Apoe(-/-) vs Apoe(-/-) mice fed chow diet and significantly attenuated atherosclerosis (?31%, p < 0.01) in western diet fed Stat4(-/-)Apoe(-/-) mice. Surprisingly, reduced atherogenesis in Stat4(-/-)Apoe(-/-) mice was not due to attenuated IFN? production in vivo by Th1 cells, suggesting an at least partially IFN?-independent pro-atherogenic role of STAT4. STAT4 is expressed in T cells, but also detected in macrophages (M?s). Stat4(-/-)Apoe(-/-)in vitro differentiated M1 or M2 M?s had reduced cytokine production compare to Apoe(-/-) M1 and M2 M?s that was accompanied by reduced induction of CD69, I-A(b), and CD86 in response to LPS stimulation. Stat4(-/-)Apoe(-/-) M?s expressed attenuated levels of CCR2 and demonstrated reduced migration toward CCL2 in a transwell assay. Importantly, the percentage of aortic CD11b(+)F4/80(+)Ly6C(hi) M?s was reduced in Stat4(-/-)Apoe(-/-) vs Apoe(-/-) mice. Thus, this study identifies for the first time a pro-atherogenic role of STAT4 that is at least partially independent of Th1 cell-derived IFN?, and primarily involving the modulation of M? responses.
Project description:Atherosclerosis (AS) in diabetic patients is often associated with low stability, which might be largely attributed to unfavorable macrophage polarization and increased inflammatory response induced by hyperglycaemia. Ginsenoside Rg3 is one of the main active principles of Panax Ginseng, which has been reported to be a natural ligand of peroxisome proliferator-activated receptor-gamma (PPAR?), a key nuclear transcriptional factor involved in inflammation and macrophage differentiation. However, it remains unclear if Rg3 could exert protective effects on plaque stability in diabetes. In this study, we investigated the role of ginsenoside 20(S)-Rg3 in macrophage polarization and AS plaque stability using advanced glycation end products-treated macrophages and diabetic AS mice models. In vitro, advanced glycation end products (AGEs) treatment promoted the expression of proinflammatory molecules and M1 surface markers, whereas 20(S)-Rg3 could reverse the M1 polarization to the M2 phenotype. In vivo, the administration of 20(S)-Rg3 promoted AS lesion stability and reduced the plaque burden, accompanied by increased M2 macrophages and reduced M1 macrophages. In addition, PPAR? antagonist GW9662 co-administration mostly blocked these effects, suggesting the important role of PPAR? pathways in mediating 20(S)-Rg3 effects in macrophage polarization and atherosclerosis progression. Together, these results demonstrated an immunomodulatory role of ginsenoside 20(S)-Rg3 in promoting macrophages to a profile of the M2 type through PPAR?-dependent mechanisms, and indicated a potential role of 20(S)-Rg3 in the prevention and treatment of diabetic atherosclerosis.
Project description:We previously reported that hemopexin (Hx), a heme scavenger, is significantly increased and associated with proinflammatory high-density lipoprotein under atherogenic conditions. Although it is established that Hx together with macrophages plays a role in mitigating oxidative damage, the role of Hx in the development of atherosclerosis is unknown.We used Hx and apoE double-knockout mice (HxE(-/-)) to determine the role of Hx in the development of atherosclerosis. HxE(-/-) mice had significantly more free heme, reactive oxygen species, and proinflammatory high-density lipoprotein in their circulation, when compared with control apoE(-/-) mice. Atherosclerotic plaque area (apoE(-/-)=9.72±2.5×10(4) ?m(2) and HxE(-/-)=27.23±3.6×10(4) ?m(2)) and macrophage infiltration (apoE(-/-)=38.8±5.8×10(3) ?m(2) and HxE(-/-)=103.4±17.8×10(3) ?m(2)) in the aortic sinus were significantly higher in the HxE(-/-) mice. Atherosclerotic lesions in the aortas were significantly higher in the HxE(-/-) mice compared with apoE(-/-) mice. Analysis of polarization revealed that macrophages from HxE(-/-) mice were more M1-like. Ex vivo studies demonstrated that HxE(-/-) macrophage cholesterol efflux capacity was significantly reduced when compared with apoE(-/-) mice. Injection of human Hx into HxE(-/-) mice reduced circulating heme levels and human Hx pretreatment of naive bone marrow cells ex vivo resulted in a shift from M1- to M2-like macrophages.We conclude that Hx plays a novel protective role in alleviating heme-induced oxidative stress, improving inflammatory properties of high-density lipoprotein, macrophage phenotype and function, and inhibiting the development of atherosclerosis in apoE(-/-) mice.
Project description:Altered macrophage kinetics is a pivotal mechanism of visceral obesity-induced inflammation and cardiometabolic risk. Because monocytes can differentiate into either proatherogenic M1 macrophages or anti-inflammatory M2 macrophages, approaches that limit M1 while promoting M2 differentiation represent a unique therapeutic strategy. We hypothesized that adiponectin may prime human monocytes toward the M2 phenotype. Adiponectin promoted the alternative activation of human monocytes into anti-inflammatory M2 macrophages as opposed to the classically activated M1 phenotype. Adiponectin-treated cells displayed increased M2 markers, including the mannose receptor (MR) and alternative macrophage activation-associated CC chemokine-1. Incubation of M1 macrophages with adiponectin-treated M2-derived culture supernatant resulted in a pronounced inhibition of tumor necrosis factor-alpha and monocyte chemotactic protein-1 secretion. Activation of human monocytes into M2 macrophages by adiponectin was mediated, in addition to AMP-activated protein kinase and peroxisome proliferator-activated receptor (PPAR)-gamma, via PPAR-alpha. Furthermore, macrophages isolated from adiponectin knockout mice demonstrated diminished levels of M2 markers such as MR, which were restored with adiponectin treatment. We report a novel immunoregulatory mechanism through which adiponectin primes human monocyte differentiation into anti-inflammatory M2 macrophages. Conditions associated with low adiponectin levels, such as visceral obesity and insulin resistance, may promote atherosclerosis, in part through aberrant macrophage kinetics.
Project description:To investigate the role of Krüppel-like factor 4 (KLF4), an essential transcriptional regulator of macrophage polarization (M1/M2), in the pathogenesis of atherosclerosis.Despite the acknowledged importance of macrophages in atherosclerosis, the role of M1 (classically activated or proinflammatory) versus M2 (alternatively activated or anti-inflammatory) macrophages in this process remains incompletely understood. We recently identified KLF4 as a regulator of macrophage subset specification; that is, KLF4 promotes M2 and inhibits M1 phenotype. Here, we provide evidence that KLF4-deficient macrophages exhibit enhanced proinflammatory activation and foam cell formation in response to oxidized lipids. In vivo, myeloid KLF4-deficient mice (ApoE(-/-) background) develop significantly more vascular inflammation and atherosclerotic lesion formation.Our findings identify myeloid KLF4 as an essential regulator of vascular inflammation and experimental atherogenesis.
Project description:Complement is a central effector system within the immune system and is implicated in a range of inflammatory disorders. CD59 is a key regulator of complement membrane attack complex (MAC) assembly. The atherogenic role of terminal complement has long been suspected but is still unclear. Here, we demonstrate that among mice deficient in apolipoprotein (Apo)E, the additional loss of murine CD59 (mCd59ab(-/-)/ApoE(-/-)) accelerated advanced atherosclerosis featuring occlusive coronary atherosclerosis, vulnerable plaque, and premature death and that these effect could be attenuated by overexpression of human CD59 in the endothelium. Complement inhibition using a neutralizing anti-mouse C5 antibody attenuated atherosclerosis in mCd59ab(-/-)/ApoE(-/-) mice. Furthermore, MAC mediated endothelial damage and promoted foam cell formation. These combined results highlight the atherogenic role of MAC and the atheroprotective role of CD59 and suggest that inhibition of MAC formation may provide a therapeutic approach for the treatment of atherosclerosis.
Project description:Abnormal lipid-mediated hepatic inflammatory-immune dysfunction and chronic low grade inflammation play an important role in the pathogenesis of non-alcoholic fatty liver disease (NAFLD). Macrophage polarization is an important mechanism for the regulation of inflammatory response. Since PPAR-? has emerged as a master regulator of macrophage polarization, we aimed to investigate the lipid-induced macrophage/Kupffer cell polarization in vivo and in vitro, and explore the association between PPAR-? activity and macrophages M1/M2 polarization shifting. Here we showed that long-term high-fat diet increased Kupffer cells content with M1-predominant phenotype and increasing production of pro-inflammatory cytokines. Saturated fatty acids polarized Kupffer cells/macrophages to an M1-predominant phenotype while n-3 PUFA polarized Kupffer cells/macrophages to an M2 phenotype, which was associated with activation of NF-?B signal pathway and PPAR-? respectively. Furthermore, up-regulation of PPAR-? shifted lipid-induced macrophages polarization from M1-predominant phenotype to M2 phenotype. Macrophages polarization switch was associated with the interaction between PPAR-? and NF-?Bp65 signal pathway. Rosiglitazone restored high-fat diet-induced imblance of Kupffer cells M1/M2 polarization and alleviated hepatic steatosis as well as local pro-inflammatory response. These findings suggest that manipulation of PPAR-? activity has the potential to balance lipid-induced M1/M2 macrophage/Kupffer cell polarization, and leading to prevent the development of NAFLD.