Andres Laguna-Fernandez

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

Aims The 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 results Expression 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. Conclusion FPR2/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.

Inhibition of indoleamine 2,3-dioxygenase promotes vascular inflammation and increases atherosclerosis in Apoe-/- mice.

AIMS Atherosclerosis is a chronic inflammatory disease that is initiated by the retention and accumulation of low-density lipoprotein in the artery, leading to maladaptive response of cells from the immune system and vessel wall. Strong evidence implicates indoleamine 2,3-dioxygenase (IDO), the first and rate-limiting enzyme of the kynurenine pathway of tryptophan (Trp) degradation, with immune regulation and anti-inflammatory mechanisms in different diseases. However, the role of IDO and the endogenous degradation of Trp have never been directly examined in atherosclerosis development. We used the IDO inhibitor 1-methyl-Trp (1-MT) to determine the role of IDO-mediated Trp metabolism in vascular inflammation and atherosclerosis. METHODS AND RESULTS Apoe(-/-) mice were treated with 1-MT in drinking water for 8 weeks. Systemic IDO inhibition led to a significant increase in atherosclerotic lesions that were ∼58 and 54% larger in the aortic arch and root, respectively. 1-MT treatment enhanced vascular inflammation, up-regulated VCAM-1 and CCL2, and increased CD68 macrophage accumulation into the plaque. Notably, the rise in VCAM-1 expression was not limited to the plaque but also found in smooth muscle cells (SMCs) of the tunica media. Furthermore, we found that IDO-dependent Trp metabolism by SMCs regulates VCAM-1 expression, and that 1-MT-induced acceleration of atherosclerosis and vascular inflammation can be reversed by exogenous administration of the Trp metabolite 3-hydroxyanthranilic acid (3-HAA). CONCLUSION IDO-mediated Trp metabolism regulates vascular inflammation and plaque formation in hypercholesterolaemic Apoe(-/-) mice. Our data establish that this pathway plays a major role in the pathological process of atherogenesis.

Aspirin‐triggered lipoxin A4 inhibits atherosclerosis progression in apolipoprotein E−/− mice

Atherosclerosis is characterized by a chronic non‐resolving inflammation in the arterial wall. Aspirin‐triggered lipoxin A4 (ATL) is a potent anti‐inflammatory mediator, involved in the resolution of inflammation. However, the therapeutic potential of immune targeting by means of ATL in atherosclerosis has not previously been explored. The aim of the present study was to determine the effects of ATL and its receptor Fpr2 on atherosclerosis development and progression in apolipoprotein E deficient (ApoE−/−) mice.

Aspirin-triggered lipoxin inhibits atherosclerosis progression in ApoE(-/-) mice.

Atherosclerosis is characterized by a chronic non‐resolving inflammation in the arterial wall. Aspirin‐triggered lipoxin A4 (ATL) is a potent anti‐inflammatory mediator, involved in the resolution of inflammation. However, the therapeutic potential of immune targeting by means of ATL in atherosclerosis has not previously been explored. The aim of the present study was to determine the effects of ATL and its receptor Fpr2 on atherosclerosis development and progression in apolipoprotein E deficient (ApoE−/−) mice.

ERV1/ChemR23 Signaling Protects from Atherosclerosis by Modifying oxLDL Uptake and Phagocytosis in Macrophages

Background: In addition to enhanced proinflammatory signaling, impaired resolution of vascular inflammation plays a key role in atherosclerosis. Proresolving lipid mediators formed through the 12/15 lipoxygenase pathways exert protective effects against murine atherosclerosis. n-3 Polyunsaturated fatty acids, including eicosapentaenoic acid (EPA), serve as the substrate for the formation of lipid mediators, which transduce potent anti-inflammatory and proresolving actions through their cognate G-protein–coupled receptors. The aim of this study was to identify signaling pathways associated with EPA supplementation and lipid mediator formation that mediate atherosclerotic disease progression. Methods: Lipidomic plasma analysis were performed after EPA supplementation in Apoe−/− mice. Erv1/Chemr23−/−xApoe−/− mice were generated for the evaluation of atherosclerosis, phagocytosis, and oxidized low-density lipoprotein uptake. Histological and mRNA analyses were done on human atherosclerotic lesions. Results: Here, we show that EPA supplementation significantly attenuated atherosclerotic lesion growth induced by Western diet in Apoe−/− mice and was associated with local cardiovascular n-3 enrichment and altered lipoprotein metabolism. Our systematic plasma lipidomic analysis identified the resolvin E1 precursor 18-monohydroxy EPA as a central molecule formed during EPA supplementation. Targeted deletion of the resolvin E1 receptor Erv1/Chemr23 in 2 independent hyperlipidemic murine models was associated with proatherogenic signaling in macrophages, increased oxidized low-density lipoprotein uptake, reduced phagocytosis, and increased atherosclerotic plaque size and necrotic core formation. We also demonstrate that in macrophages the resolvin E1–mediated effects in oxidized low-density lipoprotein uptake and phagocytosis were dependent on Erv1/Chemr23. When analyzing human atherosclerotic specimens, we identified ERV1/ChemR23 expression in a population of macrophages located in the proximity of the necrotic core and demonstrated augmented ERV1/ChemR23 mRNA levels in plaques derived from statin users. Conclusions: This study identifies 18-monohydroxy EPA as a major plasma marker after EPA supplementation and demonstrates that the ERV1/ChemR23 receptor for its downstream mediator resolvin E1 transduces protective effects in atherosclerosis. ERV1/ChemR23 signaling may represent a previously unrecognized therapeutic pathway to reduce atherosclerotic cardiovascular disease.

Association of a variant in the gene encoding for ERV1/ChemR23 with reduced inflammation in visceral adipose tissue from morbidly obese individuals

Obesity comorbidities are closely associated with chronic low-grade adipose tissue inflammation. A number of SNPs associated with inflammation has been identified, underscoring the impact of genetic determinants on this process. Here, we screened SNPs in genes with pro-inflammatory (IL-1β, IL-6, STAT3 and JAK2), anti-inflammatory (IL-10 and SOCS3) and pro-resolving (ERV1/ChemR23) properties in 101 obese and 99 non-obese individuals. Among the SNPs analyzed, we identified that individuals carrying a C allele in the rs1878022 polymorphism of the ERV1/ChemR23 gene, which encodes for the receptor of the pro-resolving mediator RvE1, had increased ERV1/ChemR23 protein expression and reduced levels of the inflammatory cytokine IL-6 in adipose tissue. Moreover, patients carrying the C allele in homozygosity had lower plasma levels of IL-6, IFN-α2, IL-15, IL-1ra, IL-10, GM-CSF, G-CSF and VEGF and enhanced leukocyte responsiveness to RvE1. C-carriers also exhibited decreased TAG to HDL ratio, a surrogate marker of insulin resistance and a predictor of incident fatty liver. Finally, we confirmed in vivo that the ERV1/ChemR23 receptor regulates systemic and tissue inflammation since mice lacking ERV1/ChemR23 expression showed increased IL-6 levels in adipose tissue and peritoneal macrophages. Together, our study identified an ERV1/ChemR23 variant that protects patients with obesity from excessive inflammatory burden.

Lipoxygenases and Cardiovascular Diseases

The lipoxygenase (LO) family of enzymes metabolize fatty acids into bioactive lipid mediators that exert potent actions on inflammatory reactions related to several cardiovascular diseases, such as atherosclerosis. The polyunsaturated omega-6 fatty acid arachidonic acid serves as a substrate for 5-LO, 12- and 15-LO, which catalyzes the formation of several bioactive lipid mediators. For example, 5-LO-derived leukotrienes transduce pro-inflammatory signaling in leukocytes and within the vascular wall. Targeting leukotriene receptors reduces experimental atherosclerosis, and pharmacoepidemiological studies indicate that leukotriene receptor antagonism is associated with a decreased cardiovascular risk. In contrast, sequential lipoxygenation of arachidonic acid yields lipoxins, which are anti-inflammatory and transduce the resolution of inflammation. The FPR2/ALX receptor is activated by both lipoxins and peptide agonists, and in murine models of atherosclerosis, FPR2/ALX deletion decreases atherosclerotic lesion size but increases atherosclerotic plaque instability. Finally, omega-3 essential fatty acids may serve as substrate for the LO enzymes yielding mediators that promote inflammation resolution, and omega-3 supplementation reduces experimental atherosclerosis. In conclusion, it is important to fully consider and explore all possible pathways of LO metabolism and their downstream metabolites when considering the role of the 5-, 12- and 15-LO pathways in cardiovascular disease.