scFv-Anti-LDL(-)-Metal-Complex Multi-Wall Functionalized-Nanocapsules as a Promising Tool for the Prevention of Atherosclerosis Progression.

Atherosclerosis can be originated from the accumulation of modified cholesterol-rich lipoproteins in the arterial wall. The electronegative LDL, LDL(-), plays an important role in the pathogenesis of atherosclerosis once this cholesterol-rich lipoprotein can be internalized by macrophages, contributing to the formation of foam cells, and provoking an immune-inflammatory response. Herein, we engineered a nanoformulation containing highly pure surface-functionalized nanocapsules using a single-chain fragment variable (scFv) reactive to LDL(-) as a ligand and assessed whether it can affect the LDL(-) uptake by primary macrophages and the progression of atherosclerotic lesions in Ldlr -/- mice. The engineered and optimized scFv-anti-LDL(-)-MCMN-Zn nanoformulation is internalized by human and murine macrophages in vitro by different endocytosis mechanisms. Moreover, macrophages exhibited lower LDL(-) uptake and reduced mRNA and protein levels of IL1B and MCP1 induced by LDL(-) when treated with this new nanoformulation. In a mouse model of atherosclerosis employing Ldlr -/- mice, intravenous administration of scFv-anti-LDL(-)-MCMN-Zn nanoformulation inhibited atherosclerosis progression without affecting vascular permeability or inducing leukocytes-endothelium interactions. Together, these findings suggest that a scFv-anti-LDL(-)-MCMN-Zn nanoformulation holds promise to be used in future preventive and therapeutic strategies for atherosclerosis.

AMPK activates LXRα and ABCA1 expression in human macrophages.

ATP-binding cassette transporter A1 (ABCA1) is a key modulator of macrophage cholesterol homeostasis. We studied the impact of AMP-activated protein kinase (AMPK) on ABCA1 expression in primary human and THP-1 macrophages. Pharmacological or genetic activation of AMPK increased mRNA and protein expression of ABCA1 and its transcriptional activator liver X receptor (LXR) α, resulting in increased cholesterol efflux to apolipoprotein AI-containing medium. On the other side, an AMPK knockdown decreased ABCA1 and LXRα mRNA and protein. Silencing LXRα, but not LXRß, attenuated ABCA1 expression after AMPK activation, and luciferase reporter as well as chromatin immunoprecipitation analyses showed the binding of LXRα to the LXR responsive element in the ABCA1 promoter. Inhibition of extracellular-signal regulated kinase and mechanistic target of rapamycin signalling increased ABCA1 expression, at the same time making it unresponsive to AMPK activation. Considering other potential regulators of ABCA1 expression, we excluded histone deacetylase HDAC9 and FOXO3 involvement in mediating AMPK effects on ABCA1. Our data link AMPK activation to an increased cholesterol efflux capacity of macrophages, suggesting an atheroprotective effect of macrophage AMPK.

FABP4 inhibition suppresses PPARγ activity and VLDL-induced foam cell formation in IL-4-polarized human macrophages.

OBJECTIVE: Macrophages, converted to lipid-loaded foam cells, accumulate in atherosclerotic lesions. Macrophage lipid metabolism is transcriptionally regulated by peroxisome proliferator-activated receptor gamma (PPARγ), and its target gene fatty acid binding protein 4 (FABP4) accelerates the progression of atherosclerosis in mouse models. Since expression of PPARγ and FABP4 is increased upon interleukin-4 (IL-4)-induced macrophage polarization, we aimed to investigate the role of FABP4 in human IL-4-polarized macrophages. METHODS AND RESULTS: We investigated the impact of FABP4 on PPARγ-dependent gene expression in primary human monocytes differentiated to macrophages in the presence of IL-4. IL-4 increased PPARγ and its target genes lipoprotein lipase (LPL) and FABP4 compared to non-polarized or LPS/interferon γ-stimulated macrophages. LPL expression correlated with increased very low density lipoprotein (VLDL)-induced triglyceride accumulation in IL-4-polarized macrophages, which was sensitive to inhibition of lipolysis or PPARγ antagonism. Inhibition of FABP4 during differentiation using chemical inhibitors BMS309403 and HTS01037 or FABP4 siRNA decreased the expression of FABP4 and LPL, and reduced lipid accumulation in macrophages treated with VLDL. FABP4 or LPL inhibition also reduced the expression of inflammatory mediators chemokine (C-C motif) ligand 2 (CCL2) and IL-1ß in response to VLDL in IL-4-polarized macrophages. PPARγ luciferase reporter assays confirmed that FABP4 supports fatty acid-induced PPARγ activation. CONCLUSION: Our findings suggest that IL-4 induces a lipid-accumulating macrophage phenotype by activating PPARγ and subsequent LPL expression. Inhibition of FABP4 decreases VLDL-induced foam cell formation, indicating that anti-atherosclerotic effects achieved by FABP4 inhibition in mouse models may be feasible in the human system as well.

AMP-Activated Protein Kinase Interacts with the Peroxisome Proliferator-Activated Receptor Delta to Induce Genes Affecting Fatty Acid Oxidation in Human Macrophages.

AMP-activated protein kinase (AMPK) maintains energy homeostasis by suppressing cellular ATP-consuming processes and activating catabolic, ATP-producing pathways such as fatty acid oxidation (FAO). The transcription factor peroxisome proliferator-activated receptor δ (PPARδ) also affects fatty acid metabolism, stimulating the expression of genes involved in FAO. To question the interplay of AMPK and PPARδ in human macrophages we transduced primary human macrophages with lentiviral particles encoding for the constitutively active AMPKα1 catalytic subunit, followed by microarray expression analysis after treatment with the PPARδ agonist GW501516. Microarray analysis showed that co-activation of AMPK and PPARδ increased expression of FAO genes, which were validated by quantitative PCR. Induction of these FAO-associated genes was also observed upon infecting macrophages with an adenovirus coding for AMPKγ1 regulatory subunit carrying an activating R70Q mutation. The pharmacological AMPK activator A-769662 increased expression of several FAO genes in a PPARδ- and AMPK-dependent manner. Although GW501516 significantly increased FAO and reduced the triglyceride amount in very low density lipoproteins (VLDL)-loaded foam cells, AMPK activation failed to potentiate this effect, suggesting that increased expression of fatty acid catabolic genes alone may be not sufficient to prevent macrophage lipid overload.

AICAR inhibits PPARγ during monocyte differentiation to attenuate inflammatory responses to atherogenic lipids.

AIMS: Transcriptional regulation through peroxisome proliferator-activated receptor γ (PPARγ) is critical for an altered lipid metabolism during monocyte to macrophage differentiation. Here, we investigated how 5-aminoimidazole-4-carboxamide riboside (AICAR), an activator of AMP-dependent protein kinase (AMPK), affects PPARγ during monocyte differentiation. METHODS AND RESULTS: During the differentiation of THP-1 monocytic cells or primary human monocytes to macrophages, we observed that AICAR inhibited the expression of PPARγ target genes, such as fatty acid-binding protein 4 or CD36. This effect was independent of AICAR conversion to AICAR ribotide and AMPK activation. While AICAR increased PPARγ mRNA expression that paralleled differentiation, it inhibited PPARγ protein synthesis without affecting PPARγ protein stability. Monocytes differentiated to macrophages in the presence of AICAR revealed an attenuated uptake of oxidized low-density lipoprotein (oxLDL) and reduced oxLDL-triggered c-Jun N-terminal kinase (JNK) activation. JNK and endoplasmic reticulum stress responses to the saturated fatty acid palmitate were attenuated as well, an effect mimicked by the knockdown of PPARγ. Although PPARγ has been reported to support alternative macrophage activation, AICAR did not inhibit interleukin-4-induced gene expression in differentiating monocytes. CONCLUSION: Inhibition of PPARγ-dependent gene expression during monocyte differentiation may contribute to an AICAR-elicited macrophage phenotype characterized by reduced inflammatory responses to modified lipoproteins and saturated fatty acids.