Luca Liberale

The Role of Inflammation in Cardiovascular Outcome

Purpose of ReviewThe aim of this review is to update the pathophysiological role of innate immune response in the cardiovascular (CV) disease outcomes, particularly focusing on coronary atherosclerosis and heart failure.Recent FindingsInflammatory processes comprised with the innate immunity reaction are believed to actively trigger CV disease development and final clinical events. For instance, by releasing proteases and neutrophil extracellular traps, neutrophil recruitment and activation might strongly influence atherosclerotic plaque stability. Similarly, neutrophils drive the early inflammatory response following a myocardial infarction. However, these cells contribute themselves to infarct healing by orchestrating monocyte/macrophage recruitment and polarization within the ischemic myocardium. Given their heterogeneity and plasticity, the balance between recruitment, proliferation, and polarization of monocyte/macrophage is a further leading determinant of advanced plaque maturation. Moreover, timely shift from a pro-inflammatory to a resolving macrophage phenotype may influence cardiac remodeling as well as development of heart failure (HF). Alongside macrophage recruitment and activation into the remote, non-ischemic myocardium also contributes to cardiac remodeling and HF development.SummaryInnate immune response is a tightly regulated process where a timely modulation of the balance between damaging and resolving properties critically impacts on CV outcome. Further progress may improve the determination of the prognostic relevance of inflammatory biomarkers on clinical CV outcome.

Update on Inflammatory Biomarkers and Treatments in Ischemic Stroke.

After an acute ischemic stroke (AIS), inflammatory processes are able to concomitantly induce both beneficial and detrimental effects. In this narrative review, we updated evidence on the inflammatory pathways and mediators that are investigated as promising therapeutic targets. We searched for papers on PubMed and MEDLINE up to August 2016. The terms searched alone or in combination were: ischemic stroke, inflammation, oxidative stress, ischemia reperfusion, innate immunity, adaptive immunity, autoimmunity. Inflammation in AIS is characterized by a storm of cytokines, chemokines, and Damage-Associated Molecular Patterns (DAMPs) released by several cells contributing to exacerbate the tissue injury both in the acute and reparative phases. Interestingly, many biomarkers have been studied, but none of these reflected the complexity of systemic immune response. Reperfusion therapies showed a good efficacy in the recovery after an AIS. New therapies appear promising both in pre-clinical and clinical studies, but still need more detailed studies to be translated in the ordinary clinical practice. In spite of clinical progresses, no beneficial long-term interventions targeting inflammation are currently available. Our knowledge about cells, biomarkers, and inflammatory markers is growing and is hoped to better evaluate the impact of new treatments, such as monoclonal antibodies and cell-based therapies.

The Role of Adipocytokines in Coronary Atherosclerosis

Purpose of ReviewThe aim of this review is to overview the pathophysiological role of adipocytokines in atherogenesis, focusing on their potential role as biomarkers of coronary disease.Recent FindingsSeveral lines of evidence indicated adipose tissue not only as depot but rather as an endocrine organ. In this context, the balance between pro- and anti-inflammatory adipocytokines has been shown to critically regulate vascular homeostasis in both physiological and pathophysiological conditions. Overweight and obesity are characterized by dysfunctional adipose tissue and then the prevalence of pro-inflammatory mediators, with a detrimental effect on vascular health. As opposite to adiponectin, pro-inflammatory adipocytokines, such as leptin and resistin, promote endothelial dysfunction and inflammatory processes involved in atherosclerotic plaque progression and vulnerability. Therefore, many adipocytokines have been investigated as potential biomarkers of cardiovascular (CV) risk, but their role has not yet been clearly established. Furthermore, the perivascular adipose tissue recently emerged as a critical modulator of atherosclerotic processes, due to the close interaction with the underlying vascular tissue.SummaryThe ongoing discovery of new adipocytokines and the complex pathophysiological role of the different adipose tissue depots strongly contribute to define the complexity of adipocytokines network. Understanding those complex interactions may allow determining new potential biomarkers of CV risk and potential therapeutic targets.

Pathophysiological relevance of macrophage subsets in atherogenesis

Macrophages are highly heterogeneous and plastic cells. They were shown to play a critical role in all stages of atherogenesis, from the initiation to the necrotic core formation and plaque rupture. Lesional macrophages primarily derive from blood monocyte, but local macrophage proliferation as well as differentiation from smooth muscle cells have also been described. Within atherosclerotic plaques, macrophages rapidly respond to changes in the microenvironment, shifting between pro- (M1) or anti-inflammatory (M2) functional phenotypes. Furthermore, different stimuli have been associated with differentiation of newly discovered M2 subtypes: IL-4/IL-13 (M2a), immune-complex (M2b), IL-10/glucocorticoids (M2c), and adenosine receptor agonist (M2d). More recently, additional intraplaque macrophage phenotypes were also recognized in response to CXCL4 (M4), oxidized phospholipids (Mox), haemoglobin/haptoglobin complexes (HA-mac/M(Hb)), and heme (Mhem). Such macrophage polarization was described as a progression among multiple phenotypes, which reflect the activity of different transcriptional factors and the cross-talk between intracellular signalling. Finally, the distribution of macrophage subsets within different plaque areas was markedly associated with cardiovascular (CV) vulnerability. The aim of this review is to update the current knowledge on the role of macrophage subsets in atherogenesis. In addition, the molecular mechanisms underlying macrophage phenotypic shift will be summarised and discussed. Finally, the role of intraplaque macrophages as predictors of CV events and the therapeutic potential of these cells will be discussed.

The Pathophysiological Role of Neutrophil Extracellular Traps in Inflammatory Diseases

Neutrophil pathogen-killing mechanism termed neutrophil extracellular traps (NETs) has been recently identified. NETs consist of chromatin and histones along with serine proteases and myeloperoxidase and are induced by a great variety of infectious and non-infectious stimuli. NETosis is a kind of programmed neutrophil death characterized by chromatin decondensation and release of nuclear granular contents, mainly driven by peptidylarginine deiminase 4 citrullination of histones. Although classically related to the protection against infectious pathogens, nowadays NETs have been described as a player of several pathophysiological processes. Neutrophil dysregulation has been demonstrated in the pathogenesis of most representative vascular diseases, such as acute coronary syndrome, stroke and venous thrombosis. Indeed, NETs have been identified within atherosclerotic lesions and arterial thrombi in both human beings and animal models. Moreover, an imbalance in this mechanism has been proposed as a critical source of modified and/or externalized autoantigens in autoimmune and inflammatory diseases. Finally, an update on the role of NETs in the pathogenesis of cancer has been included. In the present review, based on papers released on PubMed and MEDLINE up to July 2017, we point to update the knowledge on NETs, from their structure to their roles in infectious diseases as well as in cardiovascular diseases, autoimmunity, metabolic disorders and cancer, with a look to future perspectives and therapeutic opportunities.

Aging: the next cardiovascular disease?

Cardiovascular (CV) and cerebrovascular (CBV) diseases are recognized age-related conditions. Current guidelines underline this feature assigning to age the highest weight among any other known CV risk factor. Contributing to the ‘perfect storm’, a combination of reduced mortality and decreased fertility is supporting a demographic shift causing a gradual aging of the population worldwide. According to the World Health Organization, the population in Europe is aging rapidly; indeed, its median age is the highest worldwide, and the proportion of people aged 65 or over is set to increase from 14% in 2010 to 25% in 2050. Moreover, the number of individuals aged 80 or over is expected to raise to 40 million in 2050. Meanwhile life expectancy at birth is expected to go beyond 90 years in several European countries before 2070 (Figure 1). Recently published data from the European Heart Journal indicate that CV and CBV diseases in Europe account for more than 4 million deaths each year (45% of total death) with large differences depending on gender and age. • Coronary heart disease accounts for 19% of total death in men and 20% in women, • While stroke-related deaths are 9 and 14%, respectively. • More than 60% of all CV deaths occur in people aged 75 years or more. • The average annual rate of first CV or CBV events rises from 3 per 1000 for men between 35 and 44 years of age to 74 per 1000 for those between 85 and 94 years.

Regulation and function of extracellular nicotinamide phosphoribosyltransferase/visfatin

Nicotinamide phosphoribosyltransferase (NAMPT) is an adipokine-enzyme, which was described as to play bioactivities both in the intracellular and in the extracellular environment. However, while the functions of intracellular NAMPT (iNAMPT) are well known, much less is known on extracellular NAMPT (eNAMPT), also called visfatin or pre-B cell colony-enhancing factor. iNAMPT catalyzes the rate-limiting step in the NAD+ biosynthesis pathway from nicotinamide. Its inhibition severely reduces intracellular NAD+ levels, achieving anti-inflammatory and anti-cancer effects. eNAMPT can be detected in the human circulation and in many extracellular environments. Studies show that eNAMPT can act as a growth factor, as an enzyme, and as a cytokine, but its true mechanism of secretion and its physiological functions are still debated. Increased levels of eNAMPT have been associated with different metabolic disorders and cancers. eNAMPT was demonstrated to modulate the pathways involved in the pathophysiology of obesity, diabetes, atherosclerosis, and cardiovascular events by regulating the oxidative stress response, apoptosis, and inflammation. In cancer, eNAMPT was shown to play a pivotal role in modulating cancer cell metabolism, in promoting epithelial-to-mesenchymal transition and in shaping the tumor microenvironment. In line with these functions, circulating eNAMPT levels are frequently increased in cancer patients. Given these pleiotropic roles of eNAMPT in human disease, this protein has attracted attention as a therapeutic target. In this narrative review, we will discuss recent evidence on eNAMPT-driven signalling, highlighting the emerging pathophysiological roles of this protein in different disorders and the potential therapeutic opportunities linked to its targeting.

Sirtuin 5 as a novel target to blunt blood–brain barrier damage induced by cerebral ischemia/reperfusion injury

BACKGROUND In acute ischemic stroke (AIS) patients, impaired blood-brain barrier (BBB) integrity is associated with hemorrhagic transformation and worsened outcome. Yet, the mechanisms underlying these relationships are poorly understood and consequently therapeutic strategies are lacking. This study sought to determine whether SIRT5 contributes to BBB damage following I/R brain injury. METHODS AND RESULTS SIRT5 knockout (SIRT5-/-) and wild type (WT) mice underwent transient middle cerebral artery (MCA) occlusion (tMCAO) followed by 48h of reperfusion. Genetic deletion of SIRT5 decreased infarct size, improved neurological function and blunted systemic inflammation following stroke. Similar effects were also achieved by in vivo SIRT5 silencing. Immunohistochemical analysis revealed decreased BBB leakage and degradation of the tight junction protein occludin in SIRT5-/- mice exposed to tMCAO as compared to WT. In primary human brain microvascular endothelial cells (HBMVECs) exposed to hypoxia/reoxygenation (H/R), SIRT5 silencing decreased endothelial permeability and upregulated occludin and claudin-5; this effect was prevented by the PI3K inhibitor wortmannin. Lastly, SIRT5 gene expression was increased in peripheral blood monocytes (PBMCs) of AIS patients at 6h after onset of stroke compared to sex- and age-matched healthy controls. CONCLUSION SIRT5 is upregulated in PBMCs of AIS patients and in the MCA of WT mice exposed to tMCAO; SIRT5 mediates I/R-induced brain damage by increasing BBB permeability through degradation of occludin. This effect was reproduced in HBMVECs exposed to H/R, mediated by the PI3K/Akt pathway. Our findings shed new light on the mechanisms of I/R-dependent brain damage and suggest SIRT5 as a novel therapeutic target.

Serum levels of osteopontin predict major adverse cardiovascular events in patients with severe carotid artery stenosis

BACKGROUND Inflammatory mediators in the blood stream and within plaques are key determinants in atherogenesis. Here, we investigated serum osteopontin (OPN) as a potential predictor of poor outcome in patients with severe carotid atherosclerosis. METHODS Carotid plaques and serum were collected from patients asymptomatic (n=185) or symptomatic (n=40) for ischemic stroke. Plaques were stained for lipids, smooth muscle cells, neutrophils, M1 and M2 macrophage subsets and matrix metallopropteinase-9 (MMP-9). Serum levels of OPN and interleukin-6 (IL-6) were determined by colorimetric enzyme-linked immunosorbent assays. RESULTS Symptomatic patients showed a two-fold increase in serum OPN levels. In both symptomatic and asymptomatic patients, OPN levels positively correlated with intraplaque count of neutrophils, total macrophages, and MMP-9 content. In asymptomatic patients, OPN levels also positively correlated with lipids and M1 macrophage subsets. Receiver operating characteristic curve analysis identified serum OPN concentration of 70ng/ml as the best cut-off value to predict major adverse cardiovascular events (MACEs). Patients with high OPN levels had more vulnerable plaque phenotype and reduced levels of HDL-cholesterol and IL-6 as compared to low OPN levels. Kaplan-Meier curve confirmed that patients with OPN levels >70ng/ml had more MACEs at a 24-month follow-up. In the multivariate survival analysis, OPN levels >70ng/ml predicted MACEs, independently of age, gender, and symptomatic status. CONCLUSION High circulating OPN levels were strongly correlated with vulnerability parameters within plaques and predict MACEs in patients with severe carotid artery stenosis. Although confirmation is needed from larger trials, OPN could be a promising clinical tool to assess atherosclerotic outcomes.

Plasma palmitoylethanolamide (PEA) as a potential biomarker for impaired coronary function

BACKGROUND Among endocannabinoid (EC)-related mediators, Oleoyl-ethanolamide (OEA) and Palmitoyl-ethanolamide (PEA), two endogenous PPARα agonists with lipolytic and anti-inflammatory action, respectively, are being actively investigated. Here, we assessed the potential association between plasma levels of PEA and OEA and coronary function in a cohort including normal, overweight, obese, and morbidly obese (MOB) individuals. METHODS Myocardial perfusion and endothelium-related myocardial blood flow (MBF) responses to cold pressor test (CPT) and during pharmacological vasodilation with dipyridamole were measured with 13N-ammonia positron emission tomography/computed tomography. OEA and PEA were extracted from human plasma by liquid-liquid extraction, separated by liquid chromatography and quantified by mass spectrometry. Serum levels of intercellular adhesion molecule-1 and vascular cell adhesion molecule-1 (VCAM-1) were measured by colorimetric enzyme-linked immunosorbent assay. RESULTS Circulating levels of PEA and VCAM-1 were increased in MOB as compared to normal weight subjects. Circulating levels of OEA and PEA were associated with body mass index, but not with adhesion molecules. Increases of PEA levels were associated with and predictive of worsened coronary function in MOB and the overall cohort studied. CONCLUSION Plasma levels of PEA are increased in MOB patients and associated with coronary dysfunction as a functional precursor of CAD process. Larger trials are needed to confirm PEA as a potential circulating biomarker of coronary dysfunction in both MOB patients and the general population.