Atherosclerosis is a chronic progressive inflammatory disease where advanced lesions can eventually completely obstruct blood flow resulting in clinical events, such as a myocardial infarction or stroke. Monocytes and macrophages are the dominant biologically active immune cells involved in atherosclerosis disease and play a pivotal role during initiation, progression, and regression of disease. Altering macrophage inflammation is critical to induce regression of atherosclerosis and microRNAs (miRs) have emerged as key regulators of the macrophage phenotype. MiRs are small noncoding RNAs that regulate gene expression. They are dysregulated during atherosclerosis development and are key regulators of macrophage function and polarization. MiRs are short nucleotide transcripts that are very stable in circulation and thus have potential as therapeutics and/or biomarkers in the context of atherosclerosis. Of relevance to this review is that inhibition of macrophage-specific miR-155 may be a viable therapeutic strategy to decrease inflammation associated with atherosclerosis. However, further studies on these miRs and advancements in miR therapeutic delivery are required for these therapeutics to advance to the clinical setting. Conjugated linoleic acid (CLA), a pro-resolving lipid mediator, is an agonist of the peroxisome proliferator-activated receptor (PPAR)-γ. The biological activities of CLA have been documented to have anti-atherogenic effects in experimental models of atherosclerosis, inducing regression and impacting on monocyte and macrophage cells. Our work and that of others on PPAR-γ agonists and polyunsaturated fatty acids have shown that these mediators regulate candidate miRNAs and promote pro-resolving atherosclerotic plaque microenvironments.

Atherosclerosis, the underlying cause of heart attack and strokes, is a progressive dyslipidaemic and inflammatory disease where monocyte-derived macrophage cells play a pivotal role. Although most of the mechanisms that contribute to the progression of atherosclerosis have been identified, there is limited information on those governing regression. Conjugated linoleic acid (CLA) is a generic term denoting a group of naturally occurring isomers of linoleic acid (18:2, n6) that differ in the position or geometry (i.e. cis or trans) of their double bonds. The most predominant isomers in ruminant fats are cis-9, trans-11 CLA (c9,t11-CLA), which accounts for more than 80% of CLA isomers in dairy products and trans-10, cis-12 CLA (t10,c12-CLA). Dietary administration of a blend of the two most abundant isomers of CLA has been shown to inhibit the progression and induce the regression of preestablished atherosclerosis. Studies investigating the mechanisms involved in CLA-induced atheroprotective effects are continually emerging. The purpose of this review is to discuss comprehensively the effects of CLA on monocyte/macrophage function in atherosclerosis and to identify possible mechanisms through which CLA mediates its atheroprotective effects. AtherosclerosisAtherosclerosis is a complex inflammatory disease that is characterized by the progressive formation of lipid laden fibrous plaques within the arterial wall [1]. This chronic disease arises from a maladaptive inflammatory response, an impaired resolution process and a defective lipid metabolism [2]. Progressive damage to the vessel wall culminates in arterial occlusion resulting in stenosis or lesion rupture triggering thrombosis. Atherosclerosis is the underlying cause of ischaemic events and often the first clinical manifestation of atherosclerosis is myocardial infarction or stroke [3] The development of the atherosclerotic lesion is initiated by endothelial dysfunction at arterial branch points or locations of altered blood flow [2]. These endothelial alterations facilitate the passage and retention of macromolecules such as LDL molecules within the intima layer [17]. Subsequent oxidation of LDL within the subendothelium by reactive oxygen species (ROS) triggers an inflammatory response [18] characterized by the recruitment of inflammatory cells to the site of endothelial damage. The majority of leukocytes within the developing atherosclerotic lesion are monocytes and macrophages [19]. Oxidized LDL (oxLDL), direct arterial injury, cytokines and growth factors stimulate the secretion of chemokines such as monocyte chemoattractant protein-1 (MCP-1) and interleukin (IL)-8 from endothelial cells, smooth muscle cells and leukocytes [20]. Both MCP-1 and oxLDL recruit monocytes to the vessel wall [18,21].

Extracellular vesicles (EVs) are emerging as key players in different stages of atherosclerosis. Here we provide evidence that EVs released by mixed aggregates of monocytes and platelets in response to TNF‐α display pro‐inflammatory actions on endothelial cells and atherosclerotic plaques. Tempering platelet activation with Iloprost, Aspirin or a P2Y12 inhibitor impacted quantity and phenotype of EV produced. Proteomics of EVs from cells activated with TNF‐α alone or in the presence of Iloprost revealed a distinct composition, with interesting hits like annexin‐A1 and gelsolin. When added to human atherosclerotic plaque explants, EVs from TNF‐α stimulated monocytes augmented release of cytokines. In contrast, EVs generated by TNF‐α together with Iloprost produced minimal plaque activation. Notably, patients with coronary artery disease that required percutaneous coronary intervention had elevated plasma numbers of monocyte, platelet as well as double positive EV subsets. In conclusion, EVs released following monocyte/platelet activation may play a potential role in the development and progression of atherosclerosis. Whereas attenuating platelet activation modifies EV composition released from monocyte/platelet aggregates, curbing their pro‐inflammatory actions may offer therapeutic avenues for the treatment of atherosclerosis.

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BackgroundAtherosclerosis is a chronic inflammatory disease driven by macrophage accumulation in medium and large sized arteries. Macrophage polarization and inflammation are governed by microRNAs (miR) that regulate the expression of inflammatory proteins and cholesterol trafficking. Previous transcriptomic analysis led us to hypothesize that miR-155-5p (miR-155) is regulated by conjugated linoleic acid (CLA), a pro-resolving mediator which induces regression of atherosclerosis in vivo. In parallel, as extracellular vesicles (EVs) and their miR content have potential as biomarkers, we investigated alterations in urinary-derived EVs (uEVs) during the progression of human coronary artery disease (CAD).MethodsmiR-155 expression was quantified in aortae from ApoE−/− mice fed a 1% cholesterol diet supplemented with CLA blend (80:20, cis-9,trans-11:trans-10,cis-12 respectively) which had been previously been shown to induce atherosclerosis regression. In parallel, human polarized THP-1 macrophages were used to investigate the effects of CLA blend on miR-155 expression. A miR-155 mimic was used to investigate its inflammatory effects on macrophages and on ex vivo human carotid endarterectomy (CEA) plaque specimens (n = 5). Surface marker expression and miR content were analyzed in urinary extracellular vesicles (uEVs) obtained from patients diagnosed with unstable (n = 12) and stable (n = 12) CAD.ResultsHere, we report that the 1% cholesterol diet increased miR-155 expression while CLA blend supplementation decreased miR-155 expression in the aorta during atherosclerosis regression in vivo. CLA blend also decreased miR-155 expression in vitro in human THP-1 polarized macrophages. Furthermore, in THP-1 macrophages, miR-155 mimic decreased the anti-inflammatory signaling proteins, BCL-6 and phosphorylated-STAT-3. In addition, miR-155 mimic downregulated BCL-6 in CEA plaque specimens. uEVs from patients with unstable CAD had increased expression of miR-155 in comparison to patients with stable CAD. While the overall concentration of uEVs was decreased in patients with unstable CAD, levels of CD45+ uEVs were increased. Additionally, patients with unstable CAD had increased CD11b+ uEVs and decreased CD16+ uEVs.ConclusionmiR-155 suppresses anti-inflammatory signaling in macrophages, is decreased during regression of atherosclerosis in vivo and is increased in uEVs from patients with unstable CAD suggesting miR-155 has potential as a prognostic indicator and a therapeutic target.

Extracellular vesicles (EVs) are nanoparticles found in all biological fluids, capable of transporting biological material around the body. Extensive research into the physiological role of EVs has led to the development of the Minimal Information for Studies of Extracellular Vesicles (MISEV) framework in 2018. This framework guides the standardisation of protocols in the EV field. To date, the focus has been on EVs of human origin. As comparative medicine progresses, there has been a drive to study similarities between diseases in humans and animals. To successfully research EVs in felines, we must validate the application of the MISEV guidelines in this group. EVs were isolated from the plasma of healthy humans and felines. EV characterisation was carried out according to the MISEV guidelines. Human and feline plasma showed a similar concentration of EVs, comparable expression of known EV markers and analogous particle to protein ratios. Mass spectrometry analyses showed that the proteomic signature of EVs from humans and felines were similar. Asymmetrical flow field flow fractionation, showed two distinct subpopulations of EVs isolated from human plasma, whereas only one subpopulation was isolated from feline plasma. Metabolomic profiling showed similar profiles for humans and felines. In conclusion, isolation, and characterisation of EVs from humans and felines show that MISEV2018 guidelines may also be applied to felines. Potential comparative medicine studies of EVs may provide a model for studying naturally occurring diseases in both humans and felines.

CO 2 , the primary gaseous product of respiration, is a major physiologic gas, the biology of which is poorly understood. Elevated CO 2 is a feature of the microenvironment in multiple inflammatory diseases that suppresses immune cell activity. However, little is known about the CO 2 -sensing mechanisms and downstream pathways involved. We found that elevated CO 2 correlates with reduced monocyte and macrophage migration in patients undergoing gastrointestinal surgery and that elevated CO 2 reduces migration in vitro. Mechanistically, CO 2 reduces autocrine inflammatory gene expression, thereby inhibiting macrophage activation in a manner dependent on decreased intracellular pH. Pharmacologic or genetic inhibition of carbonic anhydrases (CAs) uncouples a CO 2 -elicited intracellular pH response and attenuates CO 2 sensitivity in immune cells. Conversely, CRISPRdriven upregulation of the isoenzyme CA2 confers CO 2 sensitivity in nonimmune cells. Of interest, we found that patients with chronic lung diseases associated with elevated systemic CO 2 (hypercapnia) display a greater risk of developing anastomotic leakage following gastrointestinal surgery, indicating impaired wound healing. Furthermore, low intraoperative pH levels in these patients correlate with reduced intestinal macrophage infiltration. In conclusion, CO 2 is an immunomodulatory gas sensed by immune cells through a CA2-coupled change in intracellular pH.

Monocytes/macrophages drive progression and regression of atherosclerosis. Conjugated linoleic acid (CLA), an anti‐inflammatory lipid, mediates atheroprotective effects. We investigated how CLA alters monocyte/macrophage phenotype during attenuated progression and regression of atherosclerosis. Apolipoprotein E knockout (ApoE−/−) mice were fed a high‐fat (60%) high‐cholesterol (1%) diet (HFHCD) for 2 wk, followed by 6‐wk 1% CLA 80: 20 supplementation to investigate disease progression. Simultaneously, ApoE−/− mice were fed a 12‐wk HFHCD with/without CLA for the final 4 wk to investigate regression. Aortic lesions were quantified by en face staining. Proteomic analysis, real‐time quantitative PCR and flow cytometry were used to interrogate monocyte/macrophage phenotypes. CLA supplementation inhibited atherosclerosis progression coincident with decreased proinflammatory and increased anti‐inflammatory macrophages. However, CLA‐induced regression was associated with increased proinflammatory monocytes resulting in increased proresolving M2 bone marrow‐derived macrophages, splenic macrophages, and dendritic cells in lesion‐draining lymph nodes. Proteomic analysis confirmed regulation of a proinflammatory bone marrow response, which was abolished upon macrophage differentiation. Thus, in attenuation and regression of atherosclerosis, regardless of the monocyte signature, during monocyte to macrophage differentiation, proresolving macrophages prevail, mediating vascular repair. This study provides novel mechanistic insight into the monocyte/macrophage phenotypes in halted atherosclerosis progression and regression of atherosclerosis.— Bruen, R., Curley, S., Kajani, S., Lynch, G., O'Reilly, M. E., Dillon, E. T., Fitzsimons, S., Mthunzi, L., McGillicuddy, F. C., Belton, O. Different monocyte phenotypes result in proresolving macrophages in conjugated linoleic acid‐induced attenuated progression and regression of atherosclerosis. FASEB J. 33, 11006–11020 (2019). http://www.fasebj.org