Andrew C. Li

The peroxisome proliferator-activated receptor-γ is a negative regulator of macrophage activation

The peroxisome proliferator-activated receptor-γ (PPAR-γ) is a member of the nuclear receptor superfamily of ligand-dependent transcription factors that is predominantly expressed in adipose tissue, adrenal gland and spleen. PPAR-γ has been demonstrated to regulate adipocyte differentiation and glucose homeostasis in response to several structurally distinct compounds, including thiazolidinediones and fibrates. Naturally occurring compounds such as fatty acids and the prostaglandin D2 metabolite 15-deoxy-Δ12,14prostaglandin J2 (15d-PGJ2) bind to PPAR-γ and stimulate transcription of target genes. Prostaglandin D2metabolites have not yet been identified in adipose tissue, butaremajor products of arachidonic-acid metabolism in macrophages, raising the possibility that they might serve as endogenous PPAR-γ ligands in this cell type. Here we show that PPAR-γ is markedly upregulated in activated macrophages and inhibits the expression of the inducible nitric oxide synthase, gelatinase B and scavenger receptor A genes in response to 15d-PGJ2 and synthetic PPAR-γ ligands. PPAR-γ inhibits gene expression in part by antagonizing the activities of the transcription factors AP-1, STAT and NF-κB. These observations suggest that PPAR-γ and locally produced prostaglandin D2 metabolites are involved in the regulation of inflammatory responses, and raise the possibility that synthetic PPAR-γ ligands may be of therapeutic value in human diseases such as atherosclerosis and rheumatoid arthritis in which activated macrophages exert pathogenic effects.

A SUMOylation-dependent pathway mediates transrepression of inflammatory response genes by PPAR-gamma.

Peroxisome proliferator-activated receptor-γ (PPAR-γ) has essential roles in adipogenesis and glucose homeostasis, and is a molecular target of insulin-sensitizing drugs. Although the ability of PPAR-γ agonists to antagonize inflammatory responses by transrepression of nuclear factor kappa B (NF-κB) target genes is linked to antidiabetic and antiatherogenic actions, the mechanisms remain poorly understood. Here we report the identification of a molecular pathway by which PPAR-γ represses the transcriptional activation of inflammatory response genes in mouse macrophages. The initial step of this pathway involves ligand-dependent SUMOylation of the PPAR-γ ligand-binding domain, which targets PPAR-γ to nuclear receptor corepressor (NCoR)–histone deacetylase-3 (HDAC3) complexes on inflammatory gene promoters. This in turn prevents recruitment of the ubiquitylation/19S proteosome machinery that normally mediates the signal-dependent removal of corepressor complexes required for gene activation. As a result, NCoR complexes are not cleared from the promoter and target genes are maintained in a repressed state. This mechanism provides an explanation for how an agonist-bound nuclear receptor can be converted from an activator of transcription to a promoter-specific repressor of NF-κB target genes that regulate immunity and homeostasis.

Peroxisome proliferator–activated receptor γ ligands inhibit development of atherosclerosis in LDL receptor–deficient mice

The peroxisome proliferator‐activated receptor γ (PPARγ) is a nuclear receptor that regulates fat-cell development and glucose homeostasis and is the molecular target of a class of insulin-sensitizing agents used for the management of type 2 diabetes mellitus. PPARγ is highly expressed in macrophage foam cells of atherosclerotic lesions and has been demonstrated in cultured macrophages to both positively and negatively regulate genes implicated in the development of atherosclerosis. We report here that the PPARγ-specific agonists rosiglitazone and GW7845 strongly inhibited the development of atherosclerosis in LDL receptor‐deficient male mice, despite increased expression of the CD36 scavenger receptor in the arterial wall. The antiatherogenic effect in male mice was correlated with improved insulin sensitivity and decreased tissue expression of TNF-α and gelatinase B, indicating both systemic and local actions of PPARγ. These findings suggest that PPARγ agonists may exert antiatherogenic effects in diabetic patients and provide impetus for efforts to develop PPARγ ligands that separate proatherogenic activities from antidiabetic and antiatherogenic activities. J. Clin. Invest. 106:523‐531 (2000).

The macrophage foam cell as a target for therapeutic intervention

Specialized functions of macrophages have evolved to protect the body from infection. However, the same mechanisms that enable phagocytosis of pathogens and activation of leukocytes also permit the uptake of lipoproteins and release of reactive oxygen species and immune mediators that collectively contribute to atherosclerosis. New approaches to inhibit lipid accumulation in macrophage foam cells and reduce inflammatory responses may be of therapeutic value in preventing coronary artery disease.

Differential inhibition of macrophage foam-cell formation and atherosclerosis in mice by PPARα, β/δ, and γ

PPARα, β/δ, and γ regulate genes involved in the control of lipid metabolism and inflammation and are expressed in all major cell types of atherosclerotic lesions. In vitro studies have suggested that PPARs exert antiatherogenic effects by inhibiting the expression of proinflammatory genes and enhancing cholesterol efflux via activation of the liver X receptor–ABCA1 (LXR-ABCA1) pathway. To investigate the potential importance of these activities in vivo, we performed a systematic analysis of the effects of PPARα, β, and γ agonists on foam-cell formation and atherosclerosis in male LDL receptor–deficient (LDLR–/–) mice. Like the PPARγ agonist, a PPARα-specific agonist strongly inhibited atherosclerosis, whereas a PPARβ-specific agonist failed to inhibit lesion formation. In concert with their effects on atherosclerosis, PPARα and PPARγ agonists, but not the PPARβ agonist, inhibited the formation of macrophage foam cells in the peritoneal cavity. Unexpectedly, PPARα and PPARγ agonists inhibited foam-cell formation in vivo through distinct ABCA1-independent pathways. While inhibition of foam-cell formation by PPARα required LXRs, activation of PPARγ reduced cholesterol esterification, induced expression of ABCG1, and stimulated HDL-dependent cholesterol efflux in an LXR-independent manner. In concert, these findings reveal receptor-specific mechanisms by which PPARs influence macrophage cholesterol homeostasis. In the future, these mechanisms may be exploited pharmacologically to inhibit the development of atherosclerosis.

Regulated Accumulation of Desmosterol Integrates Macrophage Lipid Metabolism and Inflammatory Responses

Inflammation and macrophage foam cells are characteristic features of atherosclerotic lesions, but the mechanisms linking cholesterol accumulation to inflammation and LXR-dependent response pathways are poorly understood. To investigate this relationship, we utilized lipidomic and transcriptomic methods to evaluate the effect of diet and LDL receptor genotype on macrophage foam cell formation within the peritoneal cavities of mice. Foam cell formation was associated with significant changes in hundreds of lipid species and unexpected suppression, rather than activation, of inflammatory gene expression. We provide evidence that regulated accumulation of desmosterol underlies many of the homeostatic responses, including activation of LXR target genes, inhibition of SREBP target genes, selective reprogramming of fatty acid metabolism, and suppression of inflammatory-response genes, observed in macrophage foam cells. These observations suggest that macrophage activation in atherosclerotic lesions results from extrinsic, proinflammatory signals generated within the artery wall that suppress homeostatic and anti-inflammatory functions of desmosterol.

An ADIOL-ERβ-CtBP Transrepression Pathway Negatively Regulates Microglia-Mediated Inflammation

Microglia and astrocytes play essential roles in the maintenance of homeostasis within the central nervous system, but mechanisms that control the magnitude and duration of responses to infection and injury remain poorly understood. Here, we provide evidence that 5-androsten-3β,17β-diol (ADIOL) functions as a selective modulator of estrogen receptor (ER)β to suppress inflammatory responses of microglia and astrocytes. ADIOL and a subset of synthetic ERβ-specific ligands, but not 17β-estradiol, mediate recruitment of CtBP corepressor complexes to AP-1-dependent promoters, thereby repressing genes that amplify inflammatory responses and activate Th17 T cells. Reduction of ADIOL or ERβ expression results in exaggerated inflammatory responses to TLR4 agonists. Conversely, the administration of ADIOL or synthetic ERβ-specific ligands that promote CtBP recruitment prevents experimental autoimmune encephalomyelitis in an ERβ-dependent manner. These findings provide evidence for an ADIOL/ERβ/CtBP-transrepression pathway that regulates inflammatory responses in microglia and can be targeted by selective ERβ modulators.

Lipoprotein Accumulation in Macrophages via Toll-Like Receptor-4–Dependent Fluid Phase Uptake

Toll-like receptor (TLR)4 recognizes microbial pathogens, such as lipopolysaccharide, and mediates lipopolysaccharide-induced proinflammatory cytokine secretion, as well as microbial uptake by macrophages. In addition to exogenous pathogens, TLR4 recognizes modified self, such as minimally oxidized low-density lipoprotein (mmLDL). Here we report that mmLDL and its active components, cholesteryl ester hydroperoxides, induce TLR4-dependent fluid phase uptake typical of macropinocytosis. We show that mmLDL induced recruitment of spleen tyrosine kinase (Syk) to a TLR4 signaling complex, TLR4 phosphorylation, activation of a Vav1-Ras-Raf-MEK-ERK1/2 signaling cascade, phosphorylation of paxillin, and activation of Rac, Cdc42, and Rho. These mmLDL-induced and TLR4- and Syk-dependent signaling events and cytoskeletal rearrangements lead to enhanced uptake of small molecules, dextran, and, most importantly, both native and oxidized LDL, resulting in intracellular lipid accumulation. An intravenous injection of fluorescently labeled mmLDL in wild-type mice resulted in its rapid accumulation in circulating monocytes, which was significantly attenuated in TLR4-deficient mice. These data describe a novel mechanism leading to enhanced lipoprotein uptake in macrophages that would contribute to foam cell formation and atherosclerosis. These data also suggest that cholesteryl ester hydroperoxides are an endogenous ligand for TLR4. Because TLR4 is highly expressed on the surface of circulating monocytes in patients with chronic inflammatory conditions, and cholesteryl ester hydroperoxides are present in plasma, lipid uptake by monocytes in circulation may contribute to the pathological roles of monocytes in chronic inflammatory diseases.

Impaired Development of Atherosclerosis in Hyperlipidemic Ldlr−/− and ApoE−/− Mice Transplanted With Abcg1−/− Bone Marrow

Objective—The lungs of Abcg1−/− mice accumulate macrophage foam cells that contain high levels of unesterified and esterified cholesterol, consistent with a role for ABCG1 in facilitating the efflux of cholesterol from macrophages to high-density lipoprotein (HDL) and other exogenous sterol acceptors. Based on these observations, we investigated whether loss of ABCG1 affects foam cell deposition in the artery wall and the development of atherosclerosis. Methods and Results—Bone marrow from wild-type or Abcg1−/− mice was transplanted into Ldlr−/− or ApoE−/− mice. After administration of a high-fat/high-cholesterol diet, plasma and tissue lipid levels and atherosclerotic lesion size were quantified and compared. Surprisingly, transplantation of Abcg1−/− bone marrow cells resulted in a significant reduction in lesion size in both mouse models, despite the fact that lipid levels increased in the lung, spleen, and kidney. Lesions of Ldlr−/− mice transplanted with Abcg1−/− cells contained increased numbers of apoptotic cells. Consistent with this observation, in vitro studies demonstrated that Abcg1−/− macrophages were more susceptible to oxidized low-density lipoprotein (ox-LDL)-dependent apoptosis than Abcg1+/+ cells. Conclusions—Diet-induced atherosclerosis is impaired when atherosclerotic-susceptible mice are transplanted with Abcg1−/− bone marrow. The demonstration that Abcg1−/− macrophages undergo accelerated apoptosis provides a mechanism to explain the decrease in the atherosclerotic lesions.

Control of angiogenesis by AIBP-mediated cholesterol efflux

Cholesterol is a structural component of the cell and is indispensable for normal cellular function, although its excess often leads to abnormal proliferation, migration, inflammatory responses and/or cell death. To prevent cholesterol overload, ATP-binding cassette (ABC) transporters mediate cholesterol efflux from the cells to apolipoprotein A-I (apoA-I) and the apoA-I-containing high-density lipoprotein (HDL). Maintaining efficient cholesterol efflux is essential for normal cellular function. However, the role of cholesterol efflux in angiogenesis and the identity of its local regulators are poorly understood. Here we show that apoA-I binding protein (AIBP) accelerates cholesterol efflux from endothelial cells to HDL and thereby regulates angiogenesis. AIBP- and HDL-mediated cholesterol depletion reduces lipid rafts, interferes with VEGFR2 (also known as KDR) dimerization and signalling and inhibits vascular endothelial growth factor-induced angiogenesis in vitro and mouse aortic neovascularization ex vivo. Notably, Aibp, a zebrafish homologue of human AIBP, regulates the membrane lipid order in embryonic zebrafish vasculature and functions as a non-cell-autonomous regulator of angiogenesis. aibp knockdown results in dysregulated sprouting/branching angiogenesis, whereas forced Aibp expression inhibits angiogenesis. Dysregulated angiogenesis is phenocopied in Abca1 (also known as Abca1a) Abcg1-deficient embryos, and cholesterol levels are increased in Aibp-deficient and Abca1 Abcg1-deficient embryos. Our findings demonstrate that secreted AIBP positively regulates cholesterol efflux from endothelial cells and that effective cholesterol efflux is critical for proper angiogenesis.