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Methods and results The internal mammary artery (IMA) is the conduit of choice to bypass the left anterior descending (LAD) coronary artery in patients eligible for coronary artery bypass grafting (CABG). In case of graft occlusion, it is still not clear whether it is better to revascularize the graft or the recipient native vessel, on top of optimal medical therapy. A 55-year-old man, with prior CABG, underwent urgent coronary angiography for an acute coronary syndrome. LAD CTO, severe proximal circumflex artery stenosis, and mild atherosclerosis of the right coronary artery were found. Moreover, a proximal occlusion of the venous graft to the obtuse marginal branch and LIMA CTO within the body of the graft were shown as well. Surprisingly, many well-developed bridging collaterals, arising close to the proximal cap of the LIMA occlusion, supplied the bloodstream circulation distal to the occlusion within the LIMA. At that time, a left main–proximal circumflex artery stenting was performed (3.5×24-mm Xience Sierra, Abbott) because that was judged the culprit lesion. One month later the patient still complained of effort angina and dyspnoea despite optimized antianginal medical therapy. The patient was scheduled for elective antegrade LIMA CTO PCI, considered the most appropriate treatment strategy. Indeed, the presence of a prior stent implanted in the left main– circumflex artery would have made difficult a proximal cap negotiation and its penetration by guidewires in case of antegrade LAD CTO PCI. Thus, the LIMA CTO negotiation was initially attempted with a Fielder FC guidewire (Asahi Intecc) and a supporting Corsair Pro (Asahi Intecc) microcatheter. The polymeric soft wire prolapsed preferentially into the bridging collaterals without the chance to engage the CTO proximal cap. Finally, a moderate-weight hydrophilic GAIA second (Asahi Intecc) succeeded in cap penetration and final CTO crossing. After lesion predilatation with a 2.0×15-mm balloon (Emerge, Boston Scientific), 2 overlapped stents, respectively 2.75×44 mm and 3.0×18 mm (VIVO ISAR, Translumina) were deployed and postdilated by a 3.0×15-mm non-compliant balloon (Accuforce, Terumo). The final result was deemed very satisfactory with a TIMI 3 flow. Conclusions Many studies showed that IMA has active biological functions, providing a sort of protection against atherosclerosis and self-reparative properties. These biological properties might justify the IMA high long-term patency rates as CABG conduits. The development of bridging collaterals in this patient, represents a clear proof of the LIMA self-reparative properties. Whereas bridging collaterals are a common feature of coronary CTO, their development in a LIMA bypass graft represents a unique finding which requires further investigation.

Leadless pacemaker (LPs) is a safe device and the implantation rates of this device is increasing. The device extraction and replacement are today a challenging procedures especially in case of infections, fragile and older patients or in unfavorable venous anatomy; LPs can be a valid alternative strategy in these cases. We report a case of management of a patient with multiple previous device replacements and extractions, with malfunction of transvenous pacemaker and with a fibrous membrane between the walls of the ventricular lead and the superior vena cava (SVC), who underwent a successful LP implantation.

Atherosclerosis is a degenerative disease characterized by lesions that develop in the wall of large- and medium-sized arteries due to the accumulation of low-density lipoproteins (LDLs) in the intima. A growing bulk of evidence suggests that cholesterol oxidation products, known as oxysterols, and the aldehyde 4-hydroxy-2-nonenal (HNE), the major pro-atherogenic components of oxidized LDLs, significantly contribute to atherosclerotic plaque progression and destabilization, with eventual plaque rupture. The involvement of certain members of the protein convertase subtilisin/kexin proteases (PCSKs) in atherosclerosis has been recently hypothesized. Among them, PCSK6 has been associated with plaque instability, mainly thanks to its ability to stimulate the activity of matrix metalloproteinases (MMPs) involved in extracellular matrix remodeling and to enhance inflammation. In U937 promonocytic cells and in human umbilical vein endothelial cells, an oxysterol mixture and HNE were able to up-regulate the level and activity of PCSK6, resulting in MMP-9 activation as demonstrated by PCSK6 silencing. Inflammation, enhanced by these lipid oxidation products, plays a key role in the up-regulation of PCSK6 activity as demonstrated by cell pretreatment with NS-398, with epigallocatechin gallate or with acetylsalicylic acid, all with anti-inflammatory effects. For the first time, we demonstrated that both oxysterols and HNE, which substantially accumulate in the atherosclerotic plaque, up-regulate the activity of PCSK6. Of note, we also suggest a potential association between PCSK6 activity and MMP-9 activation, pointing out that PCSK6 could contribute to atherosclerotic plaque development.

Chronic inflammation represents a main event in the onset and progression of atherosclerosis and is closely associated with oxidative stress in a sort of vicious circle that amplifies and sustains all stages of the disease. Key players of atherosclerosis are monocytes/macrophages. According to their pro- or anti-inflammatory phenotype and biological functions, lesional macrophages can release various mediators and enzymes, which in turn contribute to plaque progression and destabilization or, alternatively, lead to its resolution. Among the factors connected to atherosclerotic disease, lipid species carried by low density lipoproteins and pro-oxidant stimuli strongly promote inflammatory events in the vasculature, also by modulating the macrophage phenotyping. Therapies specifically aimed to balance macrophage inflammatory state are increasingly considered as powerful tools to counteract plaque formation and destabilization. In this connection, several molecules of natural origin have been recognized to be active mediators of diverse metabolic and signaling pathways regulating lipid homeostasis, redox state, and inflammation; they are, thus, considered as promising candidates to modulate macrophage responsiveness to pro-atherogenic stimuli. The current knowledge of the capability of nutraceuticals to target macrophage polarization and to counteract atherosclerotic lesion progression, based mainly on in vitro investigation, is summarized in the present review.

Aims The purpose of this study was to evaluate whether microvascular dysfunction is more present in patients with metabolic syndrome (MetS) compared to diabetics and hypertensive patients with two angiographic imaging methods, to evaluate the degree of microcirculation dysfunction, the TIMI frame count and Myocardial Blush grade. Both techniques of rapid use and relatively cheap, and allow us to have a good degree of evaluation referred to the function of the coronary microcirculation. Methods and results The study included 445 patients allocated into three groups, 157 in the MetS group, 128 in the diabetics group, and 160 patients in the hypertensive group. All patients accessed to the emergency room for anginal chest pain, all were hospitalized in the cardiac intensive care unit from 2015 to 2020. Inclusion criteria were the presences of chest pain and/or their positive stress test, and epicardial coronary arteries free from stenosis at coronary angiography. We compared the results obtained from the angiographic techniques (TIMI Frame Count and Myocardial Blush Grade) in the two subgroups: MetS vs. hypertensive, and MetS vs. diabetics. In the first subgroup, we analyzed the TFCs of the three vessels in patients with hypertension and comparing them with patients with MetS, we observed that the latter have a worse perfusion condition: the three epicardial coronary vessels have a higher TFC than the hypertensive population (TFC LAD 33.1 ± 5.6 vs. 28.4 ± 5.6, P = 0.018), (TFC RCA 27.2 ± 5.2 vs. 23.1 ± 5.2, P = 0.014) (TFC CX 27.9 ± 5.4 vs. 26.9 ± 5.4, P = 0.03). That indicates slow flow in patients with MetS coronary microcirculation. Analyzing the MBG, however, in the three coronary vessels of patients with hypertension compared to patients with metabolic syndrome, no difference was found in terms of worsening of the coronary microcirculation. Finally comparing the indices that summarize the values of the individual arteries both for the TFC and MBG, was seen as the TMBS is reduced in both groups (7.1 ± 0.49 vs. 7.1 ± 0.6, P-value = 0.04). The TTFC is instead higher in patients with MetS (83.9 ± 5.8 vs. 77.8 ± 6.7, P-value =0.024). Then we performed the same type of comparison between MetS and type 2 diabetic subgroup, in this comparison we observed how by analyzing the TFCs of the three coronary vessels, MetS patients have a slower coronary flow than patients with type 2 diabetes mellitus (TFC LAD 33.1 ± 5.6 vs. 30.6 ± 6.2, P = 0.04), (TFC RCA 27.2 ± 5.2 vs. 25 ± 5.3, P = 0.02), (TFC CX 27.9 ± 5.4 vs. 27.2 ± 5.6, P = 0.05). Comparing MBG of the three coronary vessels instead, the flow is lower in diabetic patients TTFC was higher in patients with metabolic syndrome (83.9 ± 5.8 vs. 82.7 ± 8.6, P-value = 0.02). While TMBS was lower in diabetic patients than in patients with metabolic syndrome (7.1 ± 0.49 vs. 6.7 ± 0.74, P-value = 0.01). Conclusions This study shows that patients with MetS had a major coronary microvascular dysfunction using TFC imaging technique, analysis compared to diabetics or hypertensive patients, these differences resulted to be statistically significant. A clinical evaluation of this parameters using TFC such in this study, might give further information about (CMD) in this patients in order to act to develop the best treatment to this patients and to improve their clinical condition.

High amounts of cholesterol have been definitely associated with the pathogenesis of several diseases, including metabolic and neurodegenerative disorders, cardiovascular diseases, and cancer. In all these pathologies the exacerbation of pro-oxidant and inflammatory responses is a consistent feature. In this scenario, species derived from enzymatic and non-enzymatic cholesterol oxidation, namely oxysterols, are strongly suspected to play a primary role. The consideration of these bioactive lipids is therefore helpful in investigating pathological mechanisms and may also acquire clinical value for the diagnosis and treatment of diseases. For this purpose and considering that a great number of oxysterols may be present together in the body, the employment of lipidomics technology certainly represents a powerful strategy for the simultaneous detection and characterization of these compounds in biological specimens. In this review, we will discuss the applicability of the lipidomics approach in the study of the association between oxysterols and diseases.

The development of Alzheimer’s disease (AD) is influenced by several events, among which the dysregulation of cholesterol metabolism in the brain plays a major role. Maintenance of brain cholesterol homeostasis is essential for neuronal functioning and brain development. To maintain the steady-state level, excess brain cholesterol is converted into the more hydrophilic metabolite 24-S-hydroxycholesterol (24-OHC), also called cerebrosterol, by the neuron-specific enzyme CYP46A1. A growing bulk of evidence suggests that cholesterol oxidation products, named oxysterols, are the link connecting altered cholesterol metabolism to AD. It has been shown that the levels of some oxysterols, including 27-hydroxycholesterol, 7β-hydroxycholesterol and 7-ketocholesterol, significantly increase in AD brains contributing to disease progression. In contrast, 24-OHC levels decrease, likely due to neuronal loss. Among the different brain oxysterols, 24-OHC is certainly the one whose role is most controversial. It is the dominant oxysterol in the brain and evidence shows that it represents a signaling molecule of great importance for brain function. However, numerous studies highlighted the potential role of 24-OHC in favoring AD development, since it promotes neuroinflammation, amyloid β (Aβ) peptide production, oxidative stress and cell death. In parallel, 24-OHC has been shown to exert several beneficial effects against AD progression, such as preventing tau hyperphosphorylation and Aβ production. In this review we focus on the current knowledge of the controversial role of 24-OHC in AD pathogenesis, reporting a detailed overview of the findings about its levels in different AD biological samples and its noxious or neuroprotective effects in the brain. Given the relevant role of 24-OHC in AD pathophysiology, its targeting could be useful for disease prevention or slowing down its progression.

Myocardial infarction with non-obstructive coronary arteries (MINOCA) is defined by clinical evidence of myocardial infarction (MI) with normal or near-normal coronary arteries on angiography. This condition is present in about 5% to 25% of patients presenting with acute coronary syndromes. MINOCA is a working diagnosis. Current guidelines and consensus recommend identification of underlying causes of MINOCA in order to optimize treatment, improve prognosis, and promote prevention of recurrent myocardial infarction. An accurate evaluation of patient history, symptoms and use of invasive and non-invasive imaging should lead to identification of epicardial or microvascular causes of MINOCA and differentiation from non-ischemic myocardial injury due to both cardiac (e.g. myocarditis) and non-cardiac disease (e.g. pulmonary embolism). In this review, we highlight the role of coronary imaging in differential diagnosis of patients presenting with MINOCA. Intravascular ultrasound and optical coherence tomography are well known technologies used in different settings from acute to chronic coronary syndromes. In MINOCA patients, coronary imaging could help to identify pathological alterations of the epicardial vessels that are not visible by coronary angiography such as plaque disruption, coronary dissection, coronary thromboembolism, coronary spasm, and coronary artery disease in patients presenting with takotsubo syndrome. In future, the widespread use of these technologies, in the right clinical context, could lead to optimization and personalization of treatment, and to better prognosis of patients presenting with MINOCA.

Background: Among Alzheimer’s disease (AD) brain hallmarks, the presence of reactive astrocytes was demonstrated to correlate with neuronal loss and cognitive deficits. Evidence indeed supports the role of reactive astrocytes as mediators of changes in neurons, including synapses. However, the complexity and the outcomes of astrocyte reactivity are far from being completely elucidated. Another key role in AD pathogenesis is played by alterations in brain cholesterol metabolism. Oxysterols (cholesterol oxidation products) are crucial for brain cholesterol homeostasis, and we previously demonstrated that changes in the brain levels of various oxysterols correlate with AD progression. Moreover, oxysterols have been shown to contribute to various pathological mechanisms involved in AD pathogenesis.In order to deepen the role of oxysterols in AD, we investigated whether they could contribute to astrocyte reactivity, and consequently impact on neuronal health.Methods: Mouse primary astrocyte cultures were used to test the effect of two oxysterol mixtures, that represent the oxysterol composition respectively of mild or severe AD brains, on astrocyte morphology, markers of reactivity, and secretion profile. Co-culture experiments were performed to investigate the impact of oxysterol-treated astrocytes on neurons. Neuronal cultures were exposed to astrocyte conditioned media (ACM) deprived of lipocalin-2 (Lcn2) to investigate the contribution of this mediator to synaptotoxicity.Results: Results showed that oxysterols induce a clear morphological change in astrocytes, accompanied by the upregulation of some reactive astrocyte markers, including Lcn2. Moreover, ACM analysis revealed a significant increase in the release of Lcn2, cytokines, and chemokines in response to oxysterols. A significant reduction of postsynaptic density protein 95 (PSD95) and a concurrent increase in cleaved caspase-3 protein levels have been demonstrated in neurons co-cultured with oxysterol-treated astrocytes, pointing out that mediators released by astrocytes have an impact on neurons. Among these mediators, Lcn2 has been demonstrated to play a major role on synapses, affecting neurite morphology and decreasing dendritic spine density. Conclusions: These data demonstrated that oxysterols present in the AD brain promote astrocyte reactivity, determining the release of several mediators that affect neuronal health and synapses. Lcn2 has been shown to exert a key role in mediating the synaptotoxic effect of oxysterol-treated astrocytes.

Autophagy has been shown to be stimulated in advanced atherosclerotic plaques by metabolic stress, inflammation and oxidized lipids. The lack of published studies addressing the potential stimulation of pro-survival autophagy by oxysterols, a family of cholesterol oxidation products, has prompted our study. Thus, the goal of the current study is to elucidate the molecular mechanism of the autophagy induced by 27-hydroxycholesterol (27-OH), that is one of the most abundant oxysterols in advanced atherosclerotic lesions, and to assess whether the pro-oxidant effect of the oxysterol is involved in the given response. Here we showed that 27-OH, in a low micromolar range, activates a pro-survival autophagic response in terms of increased LC3 II/LC3 I ratio and Beclin 1, that depends on the up-regulation of extracellular signal-regulated kinase (ERK) and phosphoinositide 3-kinase (PI3K)/Akt pathways as a potential result of an intracellular reactive oxygen species increase provoked by the oxysterol in human promonocytic U937 cells. Moreover, 27-OH induced autophagy is dependent on the relation between nuclear factor erythroid 2 p45-related factor 2 (Nrf2)-dependent antioxidant response and p62. The data obtained highlight the involvement of cholesterol oxidation products in the pathogenesis of oxidative stress related chronic diseases like atherosclerosis. Therefore, deeply understanding the complex mechanism and generating synthetic or natural molecules targeting this survival mechanism might be very promising tools in the prevention of such diseases., Graphical abstract A hypothetical scheme for 27-hydroxycholesterol (27-OH) involvement in autophagy modulated survival signaling.fx1, Highlights • Low concentrations of 27-OH activates autophagy in human promonocytic cells. • Pharmacological inhibition of ERK or Akt pathways prevents autophagy. • Nrf2/p62 pathway affects the autophagy response induced by 27-OH.

In Alzheimer’s disease (AD), both cholesterol and glucose dysmetabolism precede the onset of memory deficit and contribute to the disease’s progression. It is indeed now believed that oxidized cholesterol in the form of oxysterols and altered glucose uptake are the main triggers in AD affecting production and clearance of Aβ, and tau phosphorylation. However, only a few studies highlight the relationship between them, suggesting the importance of further extensive studies on this topic. Recently, a molecular link was demonstrated between cholesterol oxidative metabolism and glucose uptake in the brain. In particular, 27-hydroxycholesterol, a key linker between hypercholesterolemia and the increased AD risk, is considered a biomarker for reduced glucose metabolism. In fact, its excess increases the activity of the renin-angiotensin system in the brain, thus reducing insulin-mediated glucose uptake, which has a major impact on brain functioning. Despite this important evidence regarding the role of 27-hydroxycholesterol in regulating glucose uptake by neurons, the involvement of other cholesterol oxidation products that have been clearly demonstrated to be key players in AD cannot be ruled out. This review highlights the current understanding of the potential role of cholesterol and glucose dysmetabolism in AD progression, and the bidirectional crosstalk between these two phenomena.

It is now established that cholesterol oxidation products (oxysterols) are involved in several events underlying Alzheimer's disease (AD) pathogenesis. Of note, certain oxysterols cause neuron dysfunction and degeneration but, recently, some of them have been shown also to have neuroprotective effects. The present study, which aimed to understand the potential effects of 24-hydroxycholesterol (24-OH) against the intraneuronal accumulation of hyperphosphorylated tau protein, stressed these latter effects. A beneficial effect of 24-OH was demonstrated in SK-N-BE neuroblastoma cells, and is due to its ability to modulate the deacetylase sirtuin 1 (SIRT1), which contributes to preventing the neurotoxic accumulation of the hyperphosphorylated tau protein. Unlike 24-OH, 7-ketocholesterol (7-K) did not modulate the SIRT1-dependent neuroprotective pathway. To confirm the neuroprotective role of 24-OH, in vivo experiments were run on mice that express human tau without spontaneously developing tau pathology (hTau mice), by means of the intracerebroventricular injection of 24-OH. 24-OH, unlike 7-K, was found to completely prevent the hyperphosphorylation of tau induced by amyloid β monomers. These data highlight the importance of preventing the loss of 24-OH in the brain, and of maintaining high levels of the enzyme SIRT1, in order to counteract neurodegeneration. Graphical abstract: A hypothetical scheme of the molecular mechanisms underlying the effects of 24-OH on hyperphosphorylated tau accumulation.fx1 Keywords: 24-hydroxycholesterol, Sirtuin 1, Tau, Oxysterols, Alzheimer's disease

Cholesterol oxidation products such as oxysterols are considered critical factors in the atherosclerotic plaque formation since they induce oxidative stress, inflammation and apoptotic cell death. 27-hydroxycholesterol (27-OH) is one of the most represented oxysterols in atherosclerotic lesions. We recently showed that relatively low concentrations of 27-OH generated a strong survival signaling through an early and transient increase of cellular ROS level, that enhanced MEK-ERK/PI3K-Akt phosphorylation, in turn responsible of a sustained quenching of ROS production. It remains to identify the link between ERK/Akt up-regulation and the consequent quenching effect on ROS intracellular level that efficiently and markedly delay the pro-apoptotic effect of the oxysterol. Here we report on the potent activation of Nrf2 redox-sensitive transcription factor by low micromolar amount of 27-OH added to U937 promonocytic cells. The 27-OH-exerted induction of Nrf2 and subsequently of the target genes, HO-1 and NQO-1, was proved to be: (i) dependent upon the activation of ERK and Akt pathways, (ii) directly responsible for the quenching of intracellular oxidative stress and by this way (iii) ultimately responsible for the observed oxysterol-induced pro-survival response. (C) 2015 Elsevier Inc. All rights reserved.

The ageing endothelium progressively loses its remarkable and crucial ability to maintain homeostasis of the vasculature, as it acquires a proinflammatory phenotype. Cellular and structural changes gradually accumulate in the blood vessels, and markedly in artery walls. Most changes in aged arteries are comparable to those occurring during the atherogenic process, the latter being more marked: pro-oxidant and proinflammatory molecules, mainly deriving from or triggered by oxidized low density lipoproteins (oxLDLs), are undoubtedly a major driving force of this process. Oxysterols, quantitatively relevant components of oxLDLs, are likely candidate molecules in the pathogenesis of vascular ageing, because of their marked pro-oxidant, proinflammatory and proapoptotic properties. An increasing bulk of experimental data point to the contribution of a variety of oxysterols of pathophysiological interest, also in the age-related genesis of endothelium dysfunction, intimal thickening due to lipid accumulation, and smooth muscle cell migration and arterial stiffness due to increasing collagen deposition and calcification. This review provides an updated analysis of the molecular mechanisms whereby oxysterols accumulating in the wall of ageing blood vessels may 'activate' endothelial and monocytic cells, through expression of an inflammatory phenotype, and 'convince' smooth muscle cells to proliferate, migrate and, above all, to act as fibroblast-like cells.

Atherosclerosis is currently understood to be mainly the consequence of a complicated inflammatory process at the different stages of plaque development. Among the several inflammatory molecules involved, up-regulation of the functional cyclooxygenase 2/membrane-bound prostaglandin E synthase 1 (COX-2/mPGES-1) axis plays a key role in plaque development. Excessive production of oxidized lipids, following low-density lipoprotein (LDL) oxidation, is a characteristic feature of atherosclerosis. Among the oxidized lipids of LDLs, the oxysterol 27-hydroxycholesterol (27-OH) and the aldehyde 4-hydroxynonenal (HNE) substantially accumulate in the atherosclerotic plaque, contributing to its progression and instability through a variety of processes. This study shows that 27-OH and HNE promote up-regulation of both the inducible enzymes COX-2 and mPGES-1, leading to increased production of prostaglandin (PG) E2 and inducible nitric oxide synthase, and the subsequent release of nitric oxide in human promonocytic U937 cells. The study also examined the potential involvement of the functionally coupled COX-2/mPGES-1 in enhancing the production of certain pro-inflammatory cytokines and of matrix metalloproteinase 9 by U937 cells. This enhancement is presumably due to the induction of PGE2 synthesis, as a result of the up-regulation of the COX-2/mPGES-1, stimulated by the two oxidized lipids, 27-OH and HNE. Induction of PGE2 synthesis might thus be a mechanism of plaque instability and eventual rupture, contributing to matrix metalloproteinase production by activated macrophages.

Dietary oxysterols are cholesterol auto-oxidation products widely present in cholesterol-rich foods. They are thought to affect the intestinal barrier function, playing a role in gut inflammation. This study has characterized specific cell signals that are up-regulated in differentiated CaCo-2 colonic epithelial cells by a mixture of oxysterols representative of a hyper-cholesterolemic diet. p38 MAPK activation plays a major role, while other signal branches, i.e. the JNK and ERK pathways, make minor contributions to the intestinal inflammation induced by dietary oxysterols. p38 transduction might be the missing link connecting the known NADPH oxidase activation, and the induction of NF-κB-dependent inflammatory events related to oxysterols' action in the intestine. A NOX1/p38 MAPK/NF-κB signaling axis was demonstrated by the quenched inflammation observed on blocking individual branches of this signal with specific chemical inhibitors. Furthermore, all these signaling sites were prevented when CaCo-2 cells were pre-incubated with phenolic compounds extracted from selected wines made of typical Sardinian grape varieties: red Cannonau and white Vermentino. Notably, Cannonau was more effective than Vermentino. The effect of Sardinian wine extracts on intestinal inflammation induced by dietary oxysterols might mainly be due to their phenolic content, more abundant in Cannonau than in Vermentino. Furthermore, among different phenolic components of both wines, epicatechin and caffeic acid exerted the strongest effects. These findings show a major role of the NOX1/p38 MAPK/NF-κB signaling axis in the activation of oxysterol-dependent intestinal inflammation, and confirm the concept that phenolics act as modulators at different sites of pro-oxidant and pro-inflammatory cell signals.

A growing bulk of evidence suggests that cholesterol oxidation products, known as oxysterols, and 4-hydroxy-2-nonenal (HNE), the major proatherogenic components of oxidized low density lipoproteins (oxLDLs), significantly contribute to atherosclerotic plaque progression and destabilization, with eventual plaque rupture. These oxidized lipids are involved in various key steps of this complex process, mainly thanks to their ability to induce inflammation, oxidative stress, and apoptosis. This review summarizes the current knowledge of the effects induced by these compounds on vascular cells, after their accumulation in the arterial wall and in the atherosclerotic plaque.

Cholesterol oxidation products, termed oxysterols, have been shown to be more reactive than unoxidized cholesterol, possessing marked pro-inflammatory and cytotoxic effects in a number of cells and tissues. Oxysterols, absorbed with the diet as products of cholesterol auto-oxidation, have recently been suggested to potentially interfere with homeostasis of the mucosal intestinal epithelium, by promoting and sustaining irreversible damage. However, the treatment of colon cancer cells with a diet-compatible mixture of oxysterols does not elicit the same responses than individual components added to the cells at the same concentrations at which they are present in the mixture. Sixty μM oxysterol mixture showed a slight pro-apoptotic effect on human colon cancer CaCo-2 cell line, evaluated in terms of caspase-3 and caspase-7 activation; conversely, 7α-hydroxycholesterol, 7β-hydroxycholesterol and 5α,6α-epoxycholesterol were identified to be able to induce a significant pro-apoptotic effect if added to cell culture singly; 7β-hydroxycholesterol had stronger action than other compounds. The enhanced production of reactive oxygen species through up-regulation of the colonic NADPH-oxidase isoform NOX1 appeared to be the key event in oxysterol-induced apoptosis in these colon cancer cells. As regards pro-inflammatory effects of oxysterols, IL-8 and MCP-1 were evaluated for their chemotactic activity. Only MCP-1 production was significantly induced by 7β-hydroxycholesterol, as well as by cholesterol and oxysterol mixture. However, oxysterol-induced inflammation appeared to be NOX1-independent, suggesting a secondary role of this enzyme in inducing inflammation in colon cancer cells. A selective cell death induced by specific oxysterols against colon cancer cells, mainly exploiting their ability to activate NOX1 in generating oxidative reactions, might represent a promising field of investigation in colorectal cancer, and might bring new insights on strategies in anticancer therapy.

The hypercholesterolemia-atherosclerosis association is now established; hypercholesterolemia may induce vascular-cell activation, subsequently increasing expression of adhesion molecules, cytokines, chemokines, growth factors, and other key inflammatory molecules. Among inflammatory molecules expressed by vascular cells, integrins play a critical role in regulating macrophage activation and migration to the site of inflammation, by mediating cell-cell and cell-extracellular matrix interactions. The main lipid oxidation products present in oxidized LDL that may be responsible for inflammatory processes in atherogenesis, are cholesterol oxidation products, known as oxysterols. This study demonstrates the effect of an oxysterol mixture, compatible with that detectable in human hypercholesterolemic plasma, on the expression and synthesis of β1-integrin in cells of the macrophage lineage. The molecular signaling whereby oxysterols induce β1-integrin up-regulation is also comprehensively investigated. Over-expression of β1-integrin depends on activation of classic and novel members of protein kinase C and extracellular signal-regulated kinases 1 and 2, as well as of the up-stream G-protein (Gq and G13), c-Src, and phospholipase C. In addition, the localization of β1-integrin in advanced human carotid plaques is highlighted, marking its importance in atherosclerotic plaque progression.

Alzheimer's disease (AD), the most common form of dementia, is characterized by the progressive loss of neurons and synapses, and by extracellular deposits of amyloid-β (Aβ) as senile plaques, Aβ deposits in the cerebral blood vessels, and intracellular inclusions of hyperphosphorylated tau in the form of neurofibrillary tangles. Several mechanisms contribute to AD development and progression, and increasing epidemiological and molecular evidence suggests a key role of cholesterol in its initiation and progression. Altered cholesterol metabolism and hypercholesterolemia appear to play fundamental roles in amyloid plaque formation and tau hyperphosphorylation. Over the last decade, growing evidence supports the idea that cholesterol oxidation products, known as oxysterols, may be the missing link between altered brain cholesterol metabolism and AD pathogenesis, as their involvement in neurotoxicity, mainly by interacting with Aβ peptides, is reported.

Takotsubo cardiomyopathy, or broken heart syndrome, is characterized by transient left ventricular dysfunction associated to chest pain, elevation of cardiac enzymes, and electrocardiographic changes, mimicking an acute coronary syndrome, especially in older women after a physical or emotional stress. It is extremely infrequent in children as well as after infective stress. We described a celiac 4-year-old girl, following a gluten-free diet, who developed features of cardiac failure few days after episodes of acute diarrhea with fever. The patient was treated with oral anticongestive therapy and intravenous immunoglobulins, and she had a dramatic and rapid improvement; echocardiographic features normalized in 48 hours.

All three cholesterol oxidation products implicated thus far in the pathogenesis of Alzheimer's disease, 7β-hydroxycholesterol, 24-hydroxycholesterol, and 27-hydroxycholesterol, markedly enhance the binding of amyloid-beta (Aβ) to human differentiated neuronal cell lines (SK-N-BE and NT-2) by up-regulating net expression and synthesis of CD36 and β1-integrin receptors. However, only 24-hydroxycholesterol markedly potentiates the pro-apoptotic and pro-necrogenic effects of Aβ(1-42) peptide on these cells: 7β-hydroxycholesterol and 27-hydroxycholesterol, like unoxidized cholesterol, show no potentiating effect. This peculiar behavior of 24-hydroxycholesterol at physiologic concentrations (1 μm) depends on its strong enhancement of the intracellular generation of NADPH oxidase-dependent reactive oxygen species (ROS), mainly H(2) O(2) , and the consequent impairment of neuronal cell redox equilibrium, measured in terms of the GSSG/GSH ratio. Cell incubation with antioxidants quercetin or genistein prevents 24-hydroxycholesterol's pro-oxidant effect and potentiation of Aβ-induced necrosis and apoptosis. Thus, the presence of 24-hydroxycholesterol in the close vicinity of amyloid plaques appears to enhance the adhesion of large amounts of Aβ to the plasma membrane of neurons and then to amplify the neurotoxic action of Aβ by locally increasing ROS steady-state levels. This report further supports a primary involvement of altered brain cholesterol metabolism in the complex pathogenesis of Alzheimer's disease.

Alzheimer's disease (AD), the most common neurodegenerative disorder associated with dementia, is typified by the pathological accumulation of amyloid Aβ peptides and neurofibrillary tangles (NFT) within the brain. Considerable evidence indicates that many events contribute to AD progression, including oxidative stress, inflammation, and altered cholesterol metabolism. The brain's high lipid content makes it particularly vulnerable to oxidative species, with the consequent enhancement of lipid peroxidation and cholesterol oxidation, and the subsequent formation of end products, mainly 4-hydroxynonenal and oxysterols, respectively from the two processes. The chronic inflammatory events observed in the AD brain include activation of microglia and astrocytes, together with enhancement of inflammatory molecule and free radical release. Along with glial cells, neurons themselves have been found to contribute to neuroinflammation in the AD brain, by serving as sources of inflammatory mediators. Oxidative stress is intimately associated with neuroinflammation, and a vicious circle has been found to connect oxidative stress and inflammation in AD. Alongside oxidative stress and inflammation, altered cholesterol metabolism and hypercholesterolemia also significantly contribute to neuronal damage and to progression of AD. Increasing evidence is now consolidating the hypothesis that oxidized cholesterol is the driving force behind the development of AD, and that oxysterols are the link connecting the disease to altered cholesterol metabolism in the brain and hypercholesterolemia; this is because of the ability of oxysterols, unlike cholesterol, to cross the blood brain barrier (BBB). The key role of oxysterols in AD pathogenesis has been strongly supported by research pointing to their involvement in modulating neuroinflammation, Aβ accumulation, and cell death. This review highlights the key role played by cholesterol and oxysterols in the brain in AD pathogenesis.

Objective: to evaluate the incidence of coronary anomalies (CAA) and risk factors for coronary involvement in children with KD from 2000 through 2013 in Emilia-Romagna. Design: an 11-centers retrospective study was conducted using data from 132 patients diagnosed with KD. Results: during the acute phase, CAA developed in 13/132 pts (9.8%): 2 pts had isolated ectasia of right coronary artery (RCA), 10 pts had ectasia of left anterior descending CA (LAD), 1 pt had aneurysm of LAD, 1 pt had ectasia of circonflex CA (CX). During the subacute phase 14/117 pts (11.9%) presented CAA: 3 had aneurysm of LAD, 1 had aneurysm of LAD and RCA, 1 had isolated ectasia of RCA, 9 had ectasia of LAD. During the convalescent phase CAA were detected in 18/83 pts (21%): 3 had aneurysm of LAD and RCA, 2 isolated aneurysm of LAD, 1 had aneurysm of RCA, 12 ectasia of LAD. 3 out of 6 aneurysms detected in theconvalescent phase developed in the subacute stage. Univariate analyses identified WBC and RBC in the subacute phase, and PLT in chronic phase as predictors of chronic CAA (respectively p 0.04, 0.013 and 0.002). A multivariate logistic regression analysis revealed that only PLT of the chronic stage was predictor for chronic CAA. ROC curve for PLT identified a value superior to 652000/mm3 to be the cut off value for CAA, with an 72% sensitvity and 87% specificity. Incomplete or atipical clinical presentation was correlated with CAA (p 0.02). Younger age (< 1 yr) at diagnosis, seasonal distribution, IVIG responsivness, race, elevation of ALT, percentage of neutrophil, hemoglobin, CRP and ESR, albumin and sodium were not correlated with risk for CAL. Conclusions: atipical/incomplete clinical presentation, higher WBC and RBC in the subacute phase and PLT of the convalescent phase were predictors of CAA in our region, while blood exams of the acute stage, IVIG responsivness, sex and younger age were not.

We updated our regional database for KD patients from 2011 to 2014 to investigate differences in epidemiological, clinical and outcome data between 2011-2014 pts and those diagnosed between 2000-2010 in Emilia Romagna. We had 77 KD patients during last 4 years ( vs 81 pts during 2000-2010), mean age at diagnosis 40 months+- 36,4 (vs 31,2 mths ), 10% were younger than 6 months at diagnosis (vs 14%), male were 60 % (vs 59%). Seasonality was as follows: spring 27 (% vs 33%), winter 25% (vs 28%), autumn 25% (vs 26%), summer 23% (vs 14%). Clinical presentation was complete in 69 % (vs vs 74%), incomplete in 30% (vs 26%), atipical 1 % (vs 0%). IVIG responder were 77% (vs 67%), non responders 16 % (vs 12%), late treatment 2 % (vs 12% P 0.002), not treated with IVIG 5 % (vs 9%). 18/77 pts ( vs 7 of 2000-2010,P 0.004 ) had alteration of ALT ( v> 45 U/) and AST ( v > 55 U/l) and among those with transminases alteration 8 ( vs 4 pts) had cholestasis. Coronary arteries anomalies (CAA) were present in 15% ( 9 % ectasia and 6% (vs 2 %) aneurysms ( mean age 12,6 months, 2 pts non responders to standard therapy, 1 pts with late treatment, 1 pts not treated, and 1 pts well treated. The 2 patients who developed giant aneurysms were both non responder to standard therapy, 1 pts had associated cholestasis. Conclusion: during the last four years we observed an higher number of diagnosis of KD than 2000-2010; incomplete and atipical cases, and non-responder pts were more frequent but we noted a decreased of pts not treated or delayed treated; more cases of cholestasis associated to KD and an increased number of patients with aneurysms.

It is now thought that atherosclerosis, although due to increased plasma lipids, is mainly the consequence of a complicated inflammatory process, with immune responses at the different stages of plaque development. Increasing evidence points to a significant role of Toll-like receptor 4 (TLR4), a key player in innate immunity, in the pathogenesis of atherosclerosis. This study aimed to determine the effects on TLR4 activation of two reactive oxidized lipids carried by oxidized low-density lipoproteins, the oxysterol 27-hydroxycholesterol (27-OH) and the aldehyde 4-hydroxynonenal (HNE), both of which accumulate in atherosclerotic plaques and play a key role in the pathogenesis of atherosclerosis. Secondarily, it examined their potential involvement in mediating inflammation and extracellular matrix degradation, the hallmarks of high-risk atherosclerotic unstable plaques. In human promonocytic U937 cells, both 27-OH and HNE were found to enhance cell release of IL-8, IL-1β, and TNF-α and to upregulate matrix metalloproteinase-9 (MMP-9) via TLR4/NF-κB-dependent pathway; these actions may sustain the inflammatory response and matrix degradation that lead to atherosclerotic plaque instability and to their rupture. Using specific antibodies, it was also demonstrated that these inflammatory cytokines increase MMP-9 upregulation, thus enhancing the release of this matrix-degrading enzyme by macrophage cells and contributing to plaque instability. These innovative results suggest that, by accumulating in atherosclerotic plaques, the two oxidized lipids may contribute to plaque instability and rupture. They appear to do so by sustaining the release of inflammatory molecules and MMP-9 by inflammatory and immune cells, for example, macrophages, through activation of TLR4 and its NF-κB downstream signaling.

The oxysterol 27-hydroxycholesterol (27-OH) is increasingly considered to be involved in a variety of pathophysiological processes, having been shown to modulate cell proliferation and metabolism, and also to exert proinflammatory and proapoptotic effects. This study aimed to elucidate the molecular pathways whereby 27-OH may generate survival signals in cells of the macrophage lineage, and to clarify whether its known prooxidant effect is involved in that process. A net up-regulation of survival signaling, involving the extracellular signal-regulated kinase (ERIC) and phosphoinositide 3-kinase (PI3K)/Akt phosphorylation pathways, was observed in U937 promonocytic cells cultivated over time in the presence of a low micromolar concentration of the oxysterol. Interestingly, the up-regulation of both kinases was shown to be closely dependent on an early 27-OH-induced intracellular increase of reactive oxygen species (ROS). In turn, stimulation of ERR and PI3K/Akt both significantly quenched ROS steady state and markedly phosphorylated Bad, thereby determining a marked delay of the oxysterol's proapoptotic action. The 27-OH-induced survival pathways thus appear to be redox modulated and, if they occur within or nearby inflammatory cells during progression of chronic diseases such as cancer and atherosclerosis, they could significantly impact the growth and evolution of such diseases. (C) 2014 Elsevier Inc. All rights reserved.

An abnormal accumulation of cholesterol oxidation products in the brain of patients with Alzheimer's disease (AD) would further link an impaired cholesterol metabolism in the pathogenesis of the disease. The first evidence stemming from the content of oxysterols in autopsy samples from AD and normal brains points to an increase in both 27-hydroxycholesterol (27-OH) and 24-hydroxycholesterol (24-OH) in the frontal cortex of AD brains, with a trend that appears related to the disease severity. The challenge of differentiated SK-N-BE human neuroblastoma cells with patho-physiologically relevant amounts of 27-OH and 24-OH showed that both oxysterols induce a net synthesis of Aβ1-42 by up-regulating expression levels of amyloid precursor protein and β-secretase, as well as the β-secretase activity. Interestingly, cell pretreatment with N-acetyl-cysteine (NAC) fully prevented the enhancement of β-amyloidogenesis induced by the two oxysterols. The reported findings link an impaired cholesterol oxidative metabolism to an excessive β-amyloidogenesis and point to NAC as an efficient inhibitor of oxysterols-induced Aβ toxic peptide accumulation in the brain.

Cholesterol auto-oxidation products, namely oxysterols, are widely present in cholesterol-rich foods. They are thought to potentially interfere with homeostasis of the human digestive tract, playing a role in intestinal mucosal damage. This report concerns the marked up-regulation in differentiated CaCo-2 colonic epithelial cells of two key inflammatory interleukins, IL-6 and IL-8, caused by a mixture of oxysterols representative of a high cholesterol diet. This strong pro-inflammatory effect appeared to be dependent on the net imbalance of red-ox equilibrium with the production of excessive levels of reactive oxygen species through the colonic NADPH-oxidase NOX1 activation. Induction of NOX1 was markedly while not fully inhibited by CaCo-2 cell pre-incubation with phenolic extracts obtained from well-selected wines from typical grape varieties grown in Sardinia. Oxysterol-dependent NOX1 activation, as well as interleukin synthesis, were completely prevented by Cannonau red wine extract that contains an abundant phenolic fraction, in particular phenolic acids and flavonoids. Conversely, cell pre-treatment with Vermentino white wine extract with smaller phenolic fraction showed only a partial NOX1 down-regulation and was ineffective in interleukin synthesis induced by dietary oxysterols. It is thus likely that the effects of Sardinian wine extracts against intestinal inflammation induced by dietary oxysterols are mainly due to their high phenolic content: low doses of phenolics would be responsible only for direct scavenging oxysterol-dependent ROS production. Besides this direct activity, an excess of phenolic compounds detectable in red wine, may exert an additional indirect action by blocking oxysterol-related NOX1 induction, thus totally preventing the pro-oxidant and pro-inflammatory events triggered by dietary oxysterols.

The hypercholesterolemia-atherosclerosis association is now established; hypercholesterolemia may induce vascular-cell activation, subsequently increasing expression of adhesion molecules, cytokines, chemokines, growth factors, and other key inflammatory molecules. Among inflammatory molecules expressed by vascular cells, integrins play a critical role in regulating macrophage activation and migration to the site of inflammation, by mediating cell-cell and cell-extracellular matrix interactions. The main lipid oxidation products present in oxidized LDL that may be responsible for inflammatory processes in atherogenesis, are cholesterol oxidation products, known as oxysterols. This study demonstrates the effect of an oxysterol mixture, compatible with that detectable in human hypercholesterolemic plasma, on the expression and synthesis of β(1)-integrin in cells of the macrophage lineage. The molecular signaling whereby oxysterols induce β(1)-integrin up-regulation is also comprehensively investigated. Over-expression of β(1)-integrin depends on activation of classic and novel members of protein kinase C and extracellular signal-regulated kinases 1 and 2, as well as of the up-stream G-protein (Gq and G13), c-Src, and phospholipase C. In addition, the localization of β(1)-integrin in advanced human carotid plaques is highlighted, marking its importance in atherosclerotic plaque progression.

Lipid peroxidation is generally considered as primarily implicated in the pathogenesis of Alzheimer's disease (AD); one of its more reactive end products, 4-hydroxynonenal (HNE), has been shown to cause neuron dysfunction and degeneration. HNE production in the brain is stimulated by the amyloid-β peptide (Aβ), whose excessive accumulation in specific brain areas is a hallmark of AD. Conversely, Aβ production is up-regulated by this multifunctional aldehyde. Findings reported here point to the ability of HNE and Aβ to interact, with consequent potentiation of Aβ's cytotoxicity as determined in vitro using neuron-like cells derived from human dental-pulp progenitor cells. Preincubation of cells with the aldehyde markedly up-regulated Aβ uptake and intracellular accumulation, by overexpressing two of the three components of the plasma membrane multireceptor complex CD36/CD47/β1-integrin: experimental and clinical data indicate that intraneuronal accumulation of Aβ is an early event possibly playing a primary role in AD pathogenesis. That HNE-mediated overexpression of CD36 and β1-integrin, which plays a key role in HNE's potentiating Aβ neurotoxicity, in terms of necrosis, was confirmed when this effect was prevented by specific antibodies against the two receptors.

Catheter-related central thrombosis is a rare complication of long-term central line. We describe the case of an asymptomatic boy who was diagnosed a calcified thrombus in right atrium eight years after the removal of a long-term central venous device. Although the most appropriate therapeutic approach for managing floating right heart thrombi remains to be determined, surgical removal is an effective and safe procedure for calcified long-standing thrombus and it is to be preferred in elective conditions especially in young asymptomatic patients without hemodynamic involvement, that are at low risk of surgery-related morbidity and mortality. Cardiol Res. 2011;2(4):189-192 doi: https://doi.org/10.4021/cr24w