Carbon Dioxide Sensing by Immune Cells Occurs through Carbonic Anhydrase 2-Dependent Changes in Intracellular pH.

CO2, the primary gaseous product of respiration, is a major physiologic gas, the biology of which is poorly understood. Elevated CO2 is a feature of the microenvironment in multiple inflammatory diseases that suppresses immune cell activity. However, little is known about the CO2-sensing mechanisms and downstream pathways involved. We found that elevated CO2 correlates with reduced monocyte and macrophage migration in patients undergoing gastrointestinal surgery and that elevated CO2 reduces migration in vitro. Mechanistically, CO2 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 CO2-elicited intracellular pH response and attenuates CO2 sensitivity in immune cells. Conversely, CRISPR-driven upregulation of the isoenzyme CA2 confers CO2 sensitivity in nonimmune cells. Of interest, we found that patients with chronic lung diseases associated with elevated systemic CO2 (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, CO2 is an immunomodulatory gas sensed by immune cells through a CA2-coupled change in intracellular pH.

Dose-Dependent Association of Inflammatory Cytokines with Carotid Atherosclerosis in Transient Ischaemic Attack: Implications for Clinical Trials.

INTRODUCTION: The 5-year recurrence risk after ischaemic stroke and transient ischaemic attack (TIA) is 25-30%. Although inflammation may be a target for prevention trials, the contribution of plaque inflammation to acute cerebrovascular events remains unclear. We investigated the association of acute inflammatory cytokines and high-sensitivity C-reactive protein (CRP) with recently symptomatic carotid atherosclerosis in a prospective cohort study. METHODS: Blood and Imaging markers of TIA BIO-TIA) is a multicentre prospective study of imaging and inflammatory markers in patients with TIA. Exclusion criteria were infection and other co-morbid illnesses associated with inflammation. CRP and serum cytokines (interleukin [IL]-6, IL-1ß, IL-8, IL-10, IL-12, interferon-γ [IFN-γ] and tumour necrosis factor-α [TNF-α]) were measured. All patients had carotid imaging. RESULTS: Two hundred and thirty-eight TIA cases and 64 controls (TIA mimics) were included. Forty-nine (20.6%) cases had symptomatic internal carotid artery stenosis. Pro-inflammatory cytokine levels increased in a dose-dependent manner across controls, TIA without carotid stenosis (CS), and TIA with CS (IL-1ß, ptrend = 0.03; IL-6, ptrend < 0.0001; IL-8, ptrend = 0.01; interferon (IFN)-γ, ptrend = 0.005; TNF-α, ptrend = 0.003). Results were unchanged when DWI-positive cases were excluded. On multivariable linear regression, only age (p = 0.01) and CS (p = 0.04) independently predicted log-IL-6. On multivariable Cox regression, CRP was the only independent predictor of 90-day stroke recurrence (adjusted hazard ratio per 1-unit increase 1.03 [95% CI: 1.01-1.05], p = 0.003). CONCLUSION: Symptomatic carotid atherosclerosis was associated with elevated cytokines in TIA patients after controlling for other sources of inflammation. High-sensitivity CRP was associated with recurrent ischaemic stroke at 90 days. These findings implicate acute plaque inflammation in the pathogenesis of cerebral thromboembolism and support a rationale for randomized trials of anti-inflammatory therapy for stroke patients, who were excluded from coronary trials.

Extracellular vesicles from monocyte/platelet aggregates modulate human atherosclerotic plaque reactivity.

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.

microRNA-155 Is Decreased During Atherosclerosis Regression and Is Increased in Urinary Extracellular Vesicles During Atherosclerosis Progression.

Background: Atherosclerosis 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). Methods: miR-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. Results: Here, 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. Conclusion: miR-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.

Different monocyte phenotypes result in proresolving macrophages in conjugated linoleic acid-induced attenuated progression and regression of atherosclerosis.

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.

Liraglutide Attenuates Preestablished Atherosclerosis in Apolipoprotein E-Deficient Mice via Regulation of Immune Cell Phenotypes and Proinflammatory Mediators.

We have shown that the glucagon-like peptide-1 receptor agonist (GLP-1RA) liraglutide (Lir) inhibits development of early atherosclerosis in vivo by modulating immune cell function. We hypothesized that Lir could attenuate pre-established disease by modulating monocyte or macrophage phenotype to induce atheroprotective responses. Human atherosclerotic plaques obtained postendarterectomy and human peripheral blood macrophages were treated ex vivo with Lir. In parallel, apolipoprotein E-deficient (ApoE-/-) mice received a high-fat, high-cholesterol diet to induce atherosclerosis for 8 weeks, after which ApoE-/- mice received 300 µg/kg of Lir daily or vehicle control for a further 4 weeks to investigate the attenuation of atherosclerosis. Lir inhibited proinflammatory monocyte chemoattractant protein-1 secretion from human endarterectomy samples and monocyte chemoattractant protein-1, tumor necrosis factor-α, and interleukin (IL)-1ß secretion from human macrophages after ex vivo treatment. An increase in CD206 mRNA and IL-10 secretion was also detected, which implies resolution of inflammation. Importantly, Lir significantly attenuated pre-established atherosclerosis in ApoE-/- mice in the whole aorta and aortic root. Proteomic analysis of ApoE-/- bone marrow cells showed that Lir upregulated the proinflammatory cathepsin protein family, which was abolished in differentiated macrophages. In addition, flow cytometry analysis of bone marrow cells induced a shift toward reduced proinflammatory and increased anti-inflammatory macrophages. We concluded that Lir attenuates pre-established atherosclerosis in vivo by altering proinflammatory mediators. This is the first study to describe a mechanism through which Lir attenuates atherosclerosis by increasing bone marrow proinflammatory protein expression, which is lost in differentiated bone marrow-derived macrophages. This study contributes to our understanding of the anti-inflammatory and cardioprotective role of GLP-1RAs. SIGNIFICANCE STATEMENT: It is critical to understand the mechanisms through which liraglutide (Lir) mediates a cardioprotective effect as many type 2 diabetic medications increase the risk of myocardial infarction and stroke. We have identified that Lir reduces proinflammatory immune cell populations and mediators from plaque-burdened murine aortas in vivo and augments proresolving bone marrow-derived macrophages in attenuation of atherosclerotic disease, which provides further insight into the atheroprotective effect of Lir.

ApoE elevation is associated with the persistence of psychotic experiences from age 12 to age 18: Evidence from the ALSPAC birth cohort.

Apolipoproteins, which play important roles in lipid metabolism, innate immunity and synaptic signalling, have been implicated in first episode psychosis and schizophrenia. This is the first study to investigate plasma apolipoprotein expression in children with psychotic experiences that persist into adulthood. Here, using semi-targeted proteomic analysis we compared plasma apolipoprotein expression levels in age 12 subjects who reported psychotic experiences at both age 12 and age 18 (n = 37) with age-matched subjects who only experienced psychotic experiences (PEs) at age 12 (n = 38). Participants were recruited from the UK Avon Longitudinal Study of Parents and Children (ALSPAC) cohort who participated in psychiatric assessment interviews at ages 12 and 18. We identified apoE, a protein with significant regulatory activity on cholesterol metabolism in the brain, to be significantly up regulated (p < 0.003) in those with persistent psychotic experiences. We confirmed this finding in these samples using ELISA. Our findings indicate elevated plasma apoE in age 12 children who experience PEs is associated with persistence psychotic experiences.

miR-155 in the Resolution of Atherosclerosis.

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.

Lipoxins Protect Against Inflammation in Diabetes-Associated Atherosclerosis.

Increasing evidence points to the fact that defects in the resolution of inflammatory pathways predisposes individuals to the development of chronic inflammatory diseases, including diabetic complications such as accelerated atherosclerosis. The resolution of inflammation is dynamically regulated by the production of endogenous modulators of inflammation, including lipoxin A4 (LXA4). Here, we explored the therapeutic potential of LXA4 and a synthetic LX analog (Benzo-LXA4) to modulate diabetic complications in the streptozotocin-induced diabetic ApoE-/- mouse and in human carotid plaque tissue ex vivo. The development of diabetes-induced aortic plaques and inflammatory responses of aortic tissue, including the expression of vcam-1, mcp-1, il-6, and il-1ß, was significantly attenuated by both LXA4 and Benzo-LXA4 in diabetic ApoE-/- mice. Importantly, in mice with established atherosclerosis, treatment with LXs for a 6-week period, initiated 10 weeks after diabetes onset, led to a significant reduction in aortic arch plaque development (19.22 ± 2.01% [diabetic]; 12.67 ± 1.68% [diabetic + LXA4]; 13.19 ± 1.97% [diabetic + Benzo-LXA4]). Secretome profiling of human carotid plaque explants treated with LXs indicated changes to proinflammatory cytokine release, including tumor necrosis factor-α and interleukin-1ß. LXs also inhibited platelet-derived growth factor-stimulated vascular smooth muscle cell proliferation and transmigration and endothelial cell inflammation. These data suggest that LXs may have therapeutic potential in the context of diabetes-associated vascular complications.

Liraglutide dictates macrophage phenotype in apolipoprotein E null mice during early atherosclerosis.

BACKGROUND: Macrophages play a pivotal role in atherosclerotic plaque development. Recent evidence has suggested the glucagon-like peptide-1 receptor (GLP-1R) agonist, liraglutide, can attenuate pro-inflammatory responses in macrophages. We hypothesized that liraglutide could limit atherosclerosis progression in vivo via modulation of the inflammatory response. METHODS: Human THP-1 macrophages and bone marrow-derived macrophages, from both wild-type C57BL/6 (WT) and apolipoprotein E null mice (ApoE-/-) were used to investigate the effect of liraglutide on the inflammatory response in vitro. In parallel, ApoE-/- mice were fed a high-fat (60% calories from fat) high-cholesterol (1%) diet for 8 weeks to induce atherosclerotic disease progression with/without daily 300 µg/kg liraglutide administration for the final 6 weeks. Macrophages were analysed for MΦ1 and MΦ2 macrophage markers by Western blotting, RT-qPCR, ELISA and flow cytometry. Atherosclerotic lesions in aortae from ApoE-/- mice were analysed by en face staining and monocyte and macrophage populations from bone marrow derived cells analysed by flow cytometry. RESULTS: Liraglutide decreased atherosclerotic lesion formation in ApoE-/- mice coincident with a reduction in pro-inflammatory and increased anti-inflammatory monocyte/macrophage populations in vivo. Liraglutide decreased IL-1beta in MΦ0 THP-1 macrophages and bone marrow-derived macrophages from WT mice and induced a significant increase in the MΦ2 surface marker mannose receptor in both MΦ0 and MΦ2 macrophages. Significant reduction in total lesion development was found with once daily 300 µg/kg liraglutide treatment in ApoE-/- mice. Interestingly, liraglutide inhibited disease progression at the iliac bifurcation suggesting that it retards the initiation and development of disease. These results corresponded to attenuated MΦ1 markers (CCR7, IL-6 and TNF-alpha), augmented MΦ2 cell markers (Arg-1, IL-10 and CD163) and finally decreased MΦ1-like monocytes and macrophages from bone marrow-derived cells. CONCLUSIONS: This data supports a therapeutic role for liraglutide as an atheroprotective agent via modulating macrophage cell fate towards MΦ2 pro-resolving macrophages.

Protective Effect of let-7 miRNA Family in Regulating Inflammation in Diabetes-Associated Atherosclerosis.

The let-7 miRNA family plays a key role in modulating inflammatory responses. Vascular smooth muscle cell (SMC) proliferation and endothelial cell (EC) dysfunction are critical in the pathogenesis of atherosclerosis, including in the setting of diabetes. Here we report that let-7 levels are decreased in diabetic human carotid plaques and in a model of diabetes-associated atherosclerosis, the diabetic ApoE-/- mouse. In vitro platelet-derived growth factor (PDGF)- and tumor necrosis factor-α (TNF-α)-induced vascular SMC and EC activation was associated with reduced let-7 miRNA expression via Lin28b, a negative regulator of let-7 biogenesis. Ectopic overexpression of let-7 in SMCs inhibited inflammatory responses including proliferation, migration, monocyte adhesion, and nuclear factor-κB activation. The therapeutic potential of restoring let-7 levels using a let-7 mimic was tested: in vitro in SMCs using an endogenous anti-inflammatory lipid (lipoxin A4), ex vivo in murine aortas, and in vivo via tail vein injection in a 24-h murine model. Furthermore, we delivered let-7 mimic to human carotid plaque ex vivo and observed significant changes to the secretome in response to let-7 therapy. Restoration of let-7 expression could provide a new target for an anti-inflammatory approach in diabetic vascular disease.

NR4A Receptors Differentially Regulate NF-κB Signaling in Myeloid Cells.

Dysregulation of inflammatory responses is a hallmark of multiple diseases such as atherosclerosis and rheumatoid arthritis. As constitutively active transcription factors, NR4A nuclear receptors function to control the magnitude of inflammatory responses and in chronic inflammatory disease can be protective or pathogenic. Within this study, we demonstrate that TLR4 stimulation using the endotoxin lipopolysaccharide (LPS) rapidly enhances NR4A1-3 expression in human and murine, primary and immortalized myeloid cells with concomitant gene transcription and protein secretion of MIP-3α, a central chemokine implicated in numerous pathologies. Deficiency of NR4A2 and NR4A3 in human and murine myeloid cells reveals that both receptors function as positive regulators of enhanced MIP-3α expression. In contrast, within the same cell types and conditions, altered NR4A activity leads to suppression of LPS-induced MCP-1 gene and protein expression. An equivalent pattern of inflammatory gene regulation is replicated in TNFα-treated myeloid cells. We show that NF-κB is the critical regulator of NR4A1-3, MIP-3α, and MCP-1 during TLR4 stimulation in myeloid cells and highlight a parallel mechanism whereby NR4A activity can repress or enhance NF-κB target gene expression simultaneously. Mechanistic insight reveals that NR4A2 does not require DNA-binding capacity in order to enhance or repress NF-κB target gene expression simultaneously and establishes a role for NF-κB family member Relb as a novel NR4A target gene involved in the positive regulation of MIP-3α. Thus, our data reveal a dynamic role for NR4A receptors concurrently enhancing and repressing NF-κB activity in myeloid cells leading to altered transcription of key inflammatory mediators.

Atheroprotective effects of conjugated linoleic acid.

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 pre-established 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.

Milk-derived bioactive peptides and their health promoting effects: a potential role in atherosclerosis.

Bioactive peptides derived from milk proteins are food components that, in addition to their nutritional value, retain many biological properties and have therapeutic effects in several health disorders, including cardiovascular disease. Amongst these, atherosclerosis is the underlying cause of heart attack and strokes. It is a progressive dyslipidaemic and inflammatory disease where accumulation of oxidized lipids and inflammatory cells leads to the formation of an atherosclerotic plaque in the vessel wall. Milk-derived bioactive peptides can be released during gastrointestinal digestion, food processing or by enzymatic and bacterial fermentation and are considered to promote diverse beneficial effects such as lipid lowering, antihypertensive, immnomodulating, anti-inflammatory and antithrombotic effects. In this review, an overview of the diverse biological effects of these compounds is given, particularly focusing on their beneficial properties on cardiovascular disease and proposing novel mechanisms of action responsible for their bioactivity. Attempts to prevent cardiovascular diseases target modifications of several risk factors such as high blood pressure, obesity, high blood concentrations of lipids or insulin resistance. Milk-derived bioactive peptides are a source of health-enhancing components and the potential health benefit of these compounds has a growing commercial potential. Consequently, they have been incorporated as ingredients in functional foods, as dietary supplements and as pharmaceuticals to promote health and reduce risk of chronic diseases.

M1- and M2-Type Macrophage Responses Are Predictive of Adverse Outcomes in Human Atherosclerosis.

Atherosclerosis is an inflammatory disease caused by endothelial injury, lipid deposition, and oxidative stress. This progressive disease can be converted into an acute clinical event by plaque rupture and thrombosis. In the context of atherosclerosis, the underlying cause of myocardial infarction and stroke, macrophages uniquely possess a dual functionality, regulating lipid accumulation and metabolism and sustaining the chronic inflammatory response, two of the most well-documented pathways associated with the pathogenesis of the disease. Macrophages are heterogeneous cell populations and it is hypothesized that, during the pathogenesis of atherosclerosis, macrophages in the developing plaque can switch from a pro-inflammatory (MΦ1) to an anti-inflammatory (MΦ2) phenotype and vice versa, depending on the microenvironment. The aim of this study was to identify changes in macrophage subpopulations in the progression of human atherosclerotic disease. Established atherosclerotic plaques from symptomatic and asymptomatic patients with existing coronary artery disease undergoing carotid endarterectomy were recruited to the study. Comprehensive histological and immunohistochemical analyses were performed to quantify the cellular content and macrophage subsets of atherosclerotic lesion. In parallel, expression of MΦ1 and MΦ2 macrophage markers were analyzed by real-time PCR and Western blot analysis. Gross analysis and histological staining demonstrated that symptomatic plaques presented greater hemorrhagic activity and the internal carotid was the most diseased segment, based on the predominant prevalence of fibrotic and necrotic tissue, calcifications, and hemorrhagic events. Immunohistochemical analysis showed that both MΦ1 and MΦ2 macrophages are present in human plaques. However, MΦ2 macrophages are localized to more stable locations within the lesion. Importantly, gene and protein expression analysis of MΦ1/MΦ2 markers evidenced that MΦ1 markers and Th1-associated cytokines are highly expressed in symptomatic plaques, whereas expression of the MΦ2 markers, mannose receptor (MR), and CD163 and Th2 cytokines are inversely related with disease progression. These data increase the understanding of atherosclerosis development, identifying the cellular content of lesions during disease progression, and characterizing macrophage subpopulation within human atherosclerotic plaques.

Dysregulated post-transcriptional control of COX-2 gene expression in gestational diabetic endothelial cells.

BACKGROUND AND PURPOSE: Hyperglycaemic memory describes the progression of diabetic complications during subsequent periods of improved glycaemia. We addressed the hypothesis that transient hyperglycaemia causes aberrant COX-2 expression in HUVEC in response to IL-1ß through the induction of long-lasting epigenetic changes involving microRNA-16 (miR-16), a post-transcriptional modulator of COX-2 expression. EXPERIMENTAL APPROACH: Studies were performed on HUVEC collected from women with gestational diabetes mellitus (GDM) (dHUVEC) and normal women (nHUVEC). KEY RESULTS: In dHUVEC treated with IL-1ß, the expression of COX-2 mRNA and protein was enhanced and generation of prostanoids increased (the most abundant was the promitogenic PGF2α ). COX-2 mRNA was more stable in dHUVEC and this was associated with miR-16 down-regulation and c-Myc induction (a suppressor of miR expression). dHUVEC showed increased proliferation in response to IL-1ß, which was prevented by a COX-2 inhibitor and PGF2α receptor antagonist. Comparable changes in COX-2 mRNA, miR-16 and c-Myc detected in dHUVEC were produced in nHUVEC exposed to transient high glucose and then stimulated with IL-1ß under physiological glucose levels; superoxide anion production was enhanced under these experimental conditions. CONCLUSIONS AND IMPLICATIONS: Our results describe a possible mechanism operating in GDM that links the enhanced superoxide anion production and epigenetic changes, associated with hyperglycaemic memory, to endothelial dysfunction through dysregulated post-transcriptional control of COX-2 gene expression in response to inflammatory stimuli. The association of conventional therapy for glycaemic control with agents affecting inflammatory responses and oxidative stress might lead to a more effective prevention of the complications associated with GDM.

Dysregulation of gene expression in human fetal endothelial cells from gestational diabetes in response to TGF-ß1.

Enhanced biosynthesis of several cytokines, such as, transforming growth factor-ß1 (TGF-ß1), is detected in gestational diabetes mellitus (GDM). In this study, we addressed the question of whether the exposure to the abnormal milieu of GDM in vivo affects gene expression pattern of human umbilical vein endothelial cells (HUVEC) in response to TGF-ß1. We found that HUVEC isolated from GDM (dHUVEC) had reduced migratory capacity versus those of healthy women (nHUVEC) and this quiescent phenotype was associated with higher expression levels of the TGF-ßtype I receptor ALK5 and a slight increase in the endogenous production of TGF-ß1 (mainly in its latent form). Moreover, we performed transcriptome analysis, using microarray technology, of dHUVEC versus nHUVEC, after 3h treatment with exogenous TGF-ß1 (10 ng/ml). The treatment of dHUVEC with TGF-ß1 caused downregulation of the transcription of multiple genes involved in development, cell movement and migration of cells versus TGF-ß1-treated nHUVEC. These changes in transcriptome profile might contribute to GDM-dependent alterations in cardiac morphogenesis and placental development.

Conjugated linoleic acid induces an atheroprotective macrophage MΦ2 phenotype and limits foam cell formation.

BACKGROUND: Atherosclerosis, the underlying cause of heart attack and strokes, is a progresive dyslipidemic 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 group of isomers of linoleic acid that differ in the position and/or geometry of their double bonds. We have previously shown that a specific CLA blend (80:20 cis-9,trans-11:trans-10,cis-12-CLA) induces regression of pre-established atherosclerosis in vivo, via modulation of monocyte/macrophage function. However, the exact mechanisms through which CLA mediates this effect remain to be elucidated. METHODS: Here, we address if CLA primes monocytes towards an anti-inflammatory MΦ2 macrophage and examine the effect of individual CLA isomers and the atheroprotective blend on monocyte-macrophage differentiation, cytokine generation, foam cell formation and cholesterol metabolism in human peripheral blood monocyte (HPBMC)-derived macrophages. RESULTS: cis-9,trans-11-CLA and the atheroprotective 80:20 CLA blend regulates expression of pro-inflammatory mediators and modulates the inflammatory cytokine profile of macrophages and foam cells. In addition, cis-9,trans-11-CLA and CLA blend primes HPBMCs towards an anti-inflammatory MΦ2 phenotype, characterised by increased scavenger receptor (CD36) and efflux protein (ABCA-1) expression. Furthermore, this altered macrophage phenotype impacts on foam cell formation, inhibiting ox-LDL accumulation and promoting cholesterol efflux via both PPARγ and LXRα dependent pathways. CONCLUSION: The data increases the understanding of the pathways regulated by CLA in atheroprotection, namely, inhibiting the progressive acquisition of a pro-inflammatory macrophage phenotype.

Milk-derived bioactive peptides inhibit human endothelial-monocyte interactions via PPAR-γ dependent regulation of NF-κB.

BACKGROUND: Milk-derived bioactive peptides retain many biological properties and have therapeutic effects in cardiovascular disorders such as atherosclerosis. Under inflammatory conditions the expression of endothelial cells adhesion molecules is induced, increasing monocyte adhesion to human vessel wall, a critical step in the pathogenesis of atherosclerosis. In the present work we explored the effects of milk-derived bioactive peptides on the expression of the inflammatory phenotype of human endothelial cells and their effects on monocyte adherence to endothelial cells. RESULTS: Treatment of endothelial cells with milk-derived hydrolysate inhibited their production of inflammatory proteins MCP-1 and IL-8 and expression of VCAM-1, ICAM-1 and E-selectin. Milk derived hydrolysate also attenuated the adhesion of human monocytes to activated endothelial cells. The effect was similar to that obtained in endothelial cells treated with troglitazone, a ligand of peroxisome proliferators-activator receptor-gamma (PPAR-γ). PPAR-γ is a transcription factor which when activated antagonises the pro-inflammatory capability of nuclear factor κB (NF-κB). We further examined whether the effects of milk-derived hydrolysates on endothelial cells may be mediated through NF-κB activation via a PPAR-γ dependent mechanism. The specific PPAR-γ inhibitor, GW9662 blocked the effects of the hydrolysate on the NF-κB-mediated chemokines and adhesion molecules expression in endothelial cells. CONCLUSIONS: These results suggest that milk-derived bioactive peptides work as anti-atherogenic agents through the inhibition of endothelial-dependent adhesive interactions with monocytes by inhibiting the NF-κB pathway through a PPAR-γ dependent mechanism.

Conjugated linoleic acid targets ß2 integrin expression to suppress monocyte adhesion.

Chronic recruitment of monocytes and their subsequent migration through the activated endothelium contribute to atherosclerotic plaque development. Integrin-mediated leukocyte adhesion is central to this process. Conjugated linoleic acid (CLA) has the unique property of inducing regression of pre-established murine atherosclerosis via modulation of monocyte/macrophage function. Understanding the mechanisms through which CLA mediates its atheroprotective effect may help to identify novel pathways that limit or reverse atherosclerosis. In this study, we identified a novel mechanism through which CLA alters monocyte function. We show that CLA inhibits human peripheral blood monocyte cell adhesion to activated endothelial cells via loss of CD18 expression, the ß2 chain of LFA-1 and Mac-1 integrins. In addition, using a static-adhesion assay, we provide evidence that CLA prevents monocytes from binding to ICAM-1 and subsequently reduces the capacity of these cells to polarize. CXCL12-CXCR4 interactions induce a conformational change in ß2 integrins, facilitating leukocyte adhesion. In this study, we demonstrate that CLA inhibits CXCR4 expression, resulting in a failure of monocytes to directionally migrate toward CXCL12. Finally, using intravital microscopy, we show that, during CLA-induced regression of pre-established atherosclerosis in ApoE(-/-) mice, there is reduced leukocyte adhesion and decreased CD18 expression on Gr1(+)/CD115(+) proinflammatory monocytes. In summary, the data presented describe a novel functional role for CLA in the regulation of monocyte adhesion, polarization, and migration.