New Insights into the Role of Ferroptosis in Cardiovascular Diseases.

Cardiovascular diseases (CVDs) are the principal cause of disease burden and death worldwide. Ferroptosis is a new form of regulated cell death mainly characterized by altered iron metabolism, increased polyunsaturated fatty acid peroxidation by reactive oxygen species, depletion of glutathione and inactivation of glutathione peroxidase 4. Recently, a series of studies have indicated that ferroptosis is involved in the death of cardiac and vascular cells and has a key impact on the mechanisms leading to CVDs such as ischemic heart disease, ischemia/reperfusion injury, cardiomyopathies, and heart failure. In this article, we reviewed the molecular mechanism of ferroptosis and the current understanding of the pathophysiological role of ferroptosis in ischemic heart disease and in some cardiomyopathies. Moreover, the comprehension of the machinery governing ferroptosis in vascular cells and cardiomyocytes may provide new insights into preventive and therapeutic strategies in CVDs.

Intracellular Polyphenol Wine Metabolites Oppose Oxidative Stress and Upregulate Nrf2/ARE Pathway.

Moderate wine consumption has been associated with several benefits to human health due to its high polyphenol content. In this study, we investigated whether polyphenols contained in a particular red wine, rich in polyphenols, can pass the cell membrane and switch the oxidant/antioxidant balance toward an antioxidant pattern of THP-1 cells and human cardiomyocytes through a gene regulatory system. First, we identified which metabolite polyphenols present in red wine extract cross cell membranes and may be responsible for antioxidant effects. The results showed that the wine metabolites in treated cells belonged mainly to stilbenes, flavan-3-ols derivatives, and flavonoids. Other metabolites present in cells were not typical wine metabolites. Then, we found that red wine extract dose-dependently lowered reactive oxygen species (ROS) induced by tert-butyl hydroperoxide (TBHP) up to 50 ± 7% in both cell lines (p < 0.01). Furthermore, wine extract increased nuclear Nrf2 of about 35 ± 5% in both cell lines (p < 0.01) and counteracted its reduction induced by TBHP (p < 0.01). The rise in Nrf2 was paralleled by the increase in hemeoxygenase-1 and glutamate-cysteine ligase catalytic subunit gene expression (both mRNA and protein) (p < 0.01). These results could help explain the healthful activity of wine polyphenols within cells.

Red Blood Cell Morphologic Abnormalities in Patients Hospitalized for COVID-19.

Peripheral blood smear is a simple laboratory tool, which remains of invaluable help for diagnosing primary and secondary abnormalities of blood cells despite advances in automated and molecular techniques. Red blood cells (RBCs) abnormalities are known to occur in many viral infections, typically in the form of mild normo-microcytic anemia. While several hematological alterations at automated complete blood count (including neutrophilia, lymphopenia, and increased red cell distribution width-RDW) have been consistently associated with severity of COVID-19, there is scarce information on RBCs morphological abnormalities, mainly as case-reports or small series of patients, which are hardly comparable due to heterogeneity in sampling times and definition of illness severity. We report here a systematic evaluation of RBCs morphology at peripheral blood smear in COVID-19 patients within the first 72 h from hospital admission. One hundred and fifteen patients were included, with detailed collection of other clinical variables and follow-up. A certain degree of abnormalities in RBCs morphology was observed in 75 (65%) patients. Heterogenous alterations were noted, with spiculated cells being the more frequent morphology. The group with >10% RBCs abnormalities had more consistent lymphopenia and thrombocytopenia compared to those without abnormalities or <10% RBCs abnormalities (p < 0.018, and p < 0.021, respectively), thus underpinning a possible association with an overall more sustained immune-inflammatory "stress" hematopoiesis. Follow-up analysis showed a different mortality rate across groups, with the highest rate in those with more frequent RBCs morphological alterations compared to those with <10% or no abnormalities (41.9%, vs. 20.5%, vs. 12.5%, respectively, p = 0.012). Despite the inherent limitations of such simple association, our results point out towards further studies on erythropoiesis alterations in the pathophysiology of COVID-19.

Is Ferroptosis a Key Component of the Process Leading to Multiorgan Damage in COVID-19?

Even though COVID-19 is mostly well-known for affecting respiratory pathology, it can also result in several extrapulmonary manifestations, leading to multiorgan damage. A recent reported case of SARS-CoV-2 myocarditis with cardiogenic shock showed a signature of myocardial and kidney ferroptosis, a novel, iron-dependent programmed cell death. The term ferroptosis was coined in the last decade to describe the form of cell death induced by the small molecule erastin. As a specific inducer of ferroptosis, erastin inhibits cystine-glutamate antiporter system Xc-, blocking transportation into the cytoplasm of cystine, a precursor of glutathione (GSH) in exchange with glutamate and the consequent malfunction of GPX4. Ferroptosis is also promoted by intracellular iron overload and by the iron-dependent accumulation of polyunsaturated fatty acids (PUFA)-derived lipid peroxides. Since depletion of GSH, inactivation of GPX4, altered iron metabolism, and upregulation of PUFA peroxidation by reactive oxygen species are peculiar signs of COVID-19, there is the possibility that SARS-CoV-2 may trigger ferroptosis in the cells of multiple organs, thus contributing to multiorgan damage. Here, we review the molecular mechanisms of ferroptosis and its possible relationship with SARS-CoV-2 infection and multiorgan damage. Finally, we analyze the potential interventions that may combat ferroptosis and, therefore, reduce multiorgan damage.

Potential Role of Antioxidant and Anti-Inflammatory Therapies to Prevent Severe SARS-Cov-2 Complications.

The coronavirus disease 2019 (COVID-19) pandemic is caused by a novel severe acute respiratory syndrome (SARS)-like coronavirus (SARS-CoV-2). Here, we review the molecular pathogenesis of SARS-CoV-2 and its relationship with oxidative stress (OS) and inflammation. Furthermore, we analyze the potential role of antioxidant and anti-inflammatory therapies to prevent severe complications. OS has a potential key role in the COVID-19 pathogenesis by triggering the NOD-like receptor family pyrin domain containing 3 inflammasome and nuclear factor-kB (NF-kB). While exposure to many pro-oxidants usually induces nuclear factor erythroid 2 p45-related factor2 (NRF2) activation and upregulation of antioxidant related elements expression, respiratory viral infections often inhibit NRF2 and/or activate NF-kB pathways, resulting in inflammation and oxidative injury. Hence, the use of radical scavengers like N-acetylcysteine and vitamin C, as well as of steroids and inflammasome inhibitors, has been proposed. The NRF2 pathway has been shown to be suppressed in severe SARS-CoV-2 patients. Pharmacological NRF2 inducers have been reported to inhibit SARS-CoV-2 replication, the inflammatory response, and transmembrane protease serine 2 activation, which for the entry of SARS-CoV-2 into the host cells through the angiotensin converting enzyme 2 receptor. Thus, NRF2 activation may represent a potential path out of the woods in COVID-19 pandemic.

Ezetimibe Prevents Ischemia/Reperfusion-Induced Oxidative Stress and Up-Regulates Nrf2/ARE and UPR Signaling Pathways.

BACKGROUND: While reperfusion is crucial for survival after an episode of ischemia, it also causes oxidative stress. Nuclear factor-E2-related factor 2 (Nrf2) and unfolded protein response (UPR) are protective against oxidative stress and endoplasmic reticulum (ER) stress. Ezetimibe, a cholesterol absorption inhibitor, has been shown to activate the AMP-activated protein kinase (AMPK)/Nrf2 pathway. In this study we evaluated whether Ezetimibe affects oxidative stress and Nrf2 and UPR gene expression in cellular models of ischemia-reperfusion (IR). METHODS: Cultured cells were subjected to simulated IR with or without Ezetimibe. RESULTS: IR significantly increased reactive oxygen species (ROS) production and the percentage of apoptotic cells without the up-regulation of Nrf2, of the related antioxidant response element (ARE) gene expression or of the pro-survival UPR activating transcription factor 6 (ATF6) gene, whereas it significantly increased the pro-apoptotic CCAAT-enhancer-binding protein homologous protein (CHOP). Ezetimibe significantly decreased the cellular ROS formation and apoptosis induced by IR. These effects were paralleled by the up-regulation of Nrf2/ARE and ATF6 gene expression and by a down-regulation of CHOP. We also found that Nrf2 activation was dependent on AMPK, since Compound C, a pan inhibitor of p-AMPK, blunted the activation of Nrf2. CONCLUSIONS: Ezetimibe counteracts IR-induced oxidative stress and induces Nrf2 and UPR pathway activation.

Nuclear factor kappa B in patients with a history of unstable angina: case re-opened.

This study aims at assessing NF-kB activity in unstable angina (UA) patients free of symptoms after a 1 year follow-up (1YFU). Plasma oxidized low-density lipoproteins (oxLDL), circulating NF-kB, Interleukin 6 (IL-6) and Interleukin 1ß (IL-1ß), high-sensitivity C-reactive protein (hs-CRP), as markers of oxidative stress and inflammation and plasma double-stranded DNA (ds-DNA), as marker of Neutrophil Extracellular Traps (NETs), were measured in 23 of the previously enrolled 27 UA patients. These measurements were compared to the UA data at baseline, and then compared to the data derived from the stable angina (SA) and controls (C) enrolled in our previous study (we demonstrated that UA had higher levels of NF-kB compared to SA and C). After a 1YFU, UA patients show a significant decrease in NF-kB, IL-6, hs-CRP, oxLDL, and ds-DNA plasma levels (p < 0.001) and in IL-1ß and White Blood Cells (WBC) (p < 0.005), without differences in lipid and glucose assessment. If compared to SA and C, UA after a 1YFU have higher levels of NF-kB, IL-6, ds-DNA, WBC, and oxLDL compared to C (p < 0.001), but only IL-6 is higher than SA (p < 0.001). No differences are found in lipid and glucose assessment. After a 1YFU, patients with a history of UA improve their oxidative and inflammatory status, such as the levels of circulating ds-DNA, without achieving the status of C. They become comparable to SA subjects. This study provides new insight on the multiple and apparently contradictory facets of NF-kB in UA and on its possible role as mediator in NETs' formation.

Physical Exercise Reduces Cytotoxicity and Up-Regulates Nrf2 and UPR Expression in Circulating Cells of Peripheral Artery Disease Patients: An Hypoxic Adaptation?

AIM: Ischemia-reperfusion (I-R) produces reactive oxygen species (ROS) that damage cells and favour cytotoxicity and apoptosis in peripheral artery disease (PAD) patients. Since brief episodes of I-R (ischemic conditioning) protect cells against ischemic harms, we evaluated whether a short-course of supervised treadmill training, characterized by repeated episodes of I-R, makes peripheral blood mononuclear cells (PBMCs) from PAD patients with intermittent claudication more resistant to I-R injuries by reducing oxidative stress and by inducing an adaptative response of unfolded protein response (UPR) and nuclear factor-E2-related factor (Nrf2) pathway expression. METHODS: 24 PAD patients underwent 21 sessions of treadmill training and a treadmill test as indicator of acute response to I-R. RESULTS: Maximal and pain free walking distance improved (p＜0.01), whereas LDH leakage and apoptosis of PBMCs decreased (p＜0.01); plasma malondialdehyde and ROS generation in PBMCs declined, while plasma glutathione augmented (p＜0.01). Moreover we demonstrated an up-regulation of UPR and Nrf2 expression in PBMCs (p＜0.01). To understand whether treadmill training may act as a trigger of ischemic conditioning, we examined the effect of repeated episodes of I-R on adaptative response in PBMCs derived from the patients. We showed an up-regulation of UPR and Nrf2 gene expression (p＜0.01), while oxidative stress and cytotoxicity, after an initial increase, declined (p＜0.01). This positive effect on cytotoxicity was reduced after inhibition of UPR and Nrf2 pathways. CONCLUSIONS: Treadmill training in PAD patients through UPR and Nrf2 up-regulation may trigger hypoxic adaptation similar to conditioning, thus modifying cell survival.

Nrf2 expression is increased in peripheral blood mononuclear cells derived from mild-moderate ex-smoker COPD patients with persistent oxidative stress.

Inadequacy of antioxidant nuclear factor-E2-related factor 2 (Nrf2) and endoplasmic reticulum stress-mediated unfolded protein response has been implicated in severe chronic obstructive pulmonary disease (COPD) and cigarette smoking-induced emphysema. As evidence suggests that the ability to upregulate Nrf2 expression may influence the progression of COPD and no data exist up to now in ex-smokers with mild-moderate COPD, this study was first aimed to evaluate Nrf2 and unfolded protein response expression in peripheral blood mononuclear cells (PBMC) of mild-moderate ex-smokers with COPD compared to smoking habit-matched non-COPD subjects. Then, we tested whether oxidative stress persists after cigarette smoking cessation and whether the concentrations of oxidized phospholipids (oxidation products of the phospholipid 1-palmitoyl-2-arachidonyl-sn-glycero-3-phosphorylcholine [oxPAPC]) in the PBMC of the same subjects may have a causative role in determining the upregulation of Nrf2. The expression (mRNA and protein) of Nrf2 and of its related gene heme oxygenase-1 was significantly increased in COPD group without differences in the unfolded protein response. Plasma malondialdehyde, the circulating marker of oxidative stress, and oxPAPC in PBMC were significantly higher in COPD than in non-COPD subjects. The fact that the expression of p47phox, a subunit of NADPH oxidase, was increased in PBMC of COPD patients and that it was directly correlated with oxPAPC may indicate that oxPAPC may be one of the determinants of oxidative stress-induced Nrf2 upregulation. Finally, we also demonstrated that lung function inversely correlated with plasma malondialdehyde and with Nrf2 and heme oxygenase-1 mRNA expression in all subjects. Our results indicate that mild-moderate ex-smokers with COPD may be able to counteract oxidative stress by increasing the expression of Nrf2/antioxidant-response elements. Because Nrf2 failure significantly contributes to the development of COPD, our findings suggest that the possibility to prevent Nrf2 reduction may open a new scenario in helping to prevent the oxidative stress-associated lung function decline.

Fast method for skeletal tissue gene expression analysis.

Several chronic diseases have been associated with bone alteration in the last few years. Despite the wealth of information provided by the analysis of the transcriptome in affected tissues, only a limited number of studies evaluated gene expression in bone tissue due to the difficulty to obtain high quality RNA. Therefore, skeletal pathologies have been often associated to a defective maturation process that occurs during recruitment of progenitor stem cells. In order to explore the possibility of analysing the gene expression during osteogenic differentiation in skeletal tissue, a single-step method to extract well-preserved RNA from bone specimens was performed. A comparison between this technique and a traditional method was made by analysing the quality and yield of RNA obtained. In addition, RNAs were assayed by reverse transcription-quantitative polymerase chain reaction to analyse the expression levels of the bone genes associated with the differentiation process in a mouse model. The present data showed that good quality RNA can be obtained from bone tissue by a simple single-step method allowing the expression analysis of the genes encoded by skeletal tissue. In conclusion, the present study allows the possibility to easily obtain good quality RNA from bone tissue that is suitable for gene expression studies of bone diseases.

Are they in or out? The elusive interaction between Qtracker ® 800 vascular labels and brain endothelial cells.

AIM: Qtracker(®)800 Vascular labels (Qtracker(®)800) are promising biomedical tools for high-resolution vasculature imaging; their effects on mouse and human endothelia, however, are still unknown. MATERIALS & METHODS: Qtracker(®)800 were injected in Balb/c mice, and brain endothelium uptake was investigated by transmission electron microscopy 3-h post injection. We then investigated, in vitro, the effects of Qtracker(®)800 exposure on mouse and human endothelial cells by calcium imaging. RESULTS: Transmission electron microscopy images showed nanoparticle accumulation in mouse brain endothelia. A subset of mouse and human endothelial cells generated intracellular calcium transients in response to Qtracker(®)800. CONCLUSION: Qtracker(®)800 nanoparticles elicit endothelial functional responses, which prompts biomedical safety evaluations and may bias the interpretation of experimental studies involving vascular imaging.

Oxygenator Is the Main Responsible for Leukocyte Activation in Experimental Model of Extracorporeal Circulation: A Cautionary Tale.

In order to assess mechanisms underlying inflammatory activation during extracorporeal circulation (ECC), several small animal models of ECC have been proposed recently. The majority of them are based on home-made, nonstandardized, and hardly reproducible oxygenators. The present study has generated fundamental information on the role of oxygenator of ECC in activating inflammatory signaling pathways on leukocytes, leading to systemic inflammatory response, and organ dysfunction. The present results suggest that experimental animal models of ECC used in translational research on inflammatory response should be based on standardized, reproducible oxygenators with clinical characteristics.

Endoplasmic reticulum stress and Nrf2 signaling in cardiovascular diseases.

Various cellular perturbations implicated in the pathophysiology of human diseases, including cardiovascular and neurodegenerative diseases, diabetes mellitus, obesity, and liver diseases, can alter endoplasmic reticulum (ER) function and lead to the abnormal accumulation of misfolded proteins. This situation configures the so-called ER stress, a form of intracellular stress that occurs whenever the protein-folding capacity of the ER is overwhelmed. Reduction in blood flow as a result of atherosclerotic coronary artery disease causes tissue hypoxia, a condition that induces protein misfolding and ER stress. In addition, ER stress has an important role in cardiac hypertrophy mainly in the transition to heart failure (HF). ER transmembrane sensors detect the accumulation of unfolded proteins and activate transcriptional and translational pathways that deal with unfolded and misfolded proteins, known as the unfolded protein response (UPR). Once the UPR fails to control the level of unfolded and misfolded proteins in the ER, ER-initiated apoptotic signaling is induced. Furthermore, there is considerable evidence that implicates the presence of oxidative stress and subsequent related cellular damage as an initial cause of injury to the myocardium after ischemia/reperfusion (I/R) and in cardiac hypertrophy secondary to pressure overload. Oxidative stress is counterbalanced by complex antioxidant defense systems regulated by a series of multiple pathways, including the UPR, to ensure that the response to oxidants is adequate. Nuclear factor-E2-related factor (Nrf2) is an emerging regulator of cellular resistance to oxidants; Nrf2 is strictly interrelated with the UPR sensor called pancreatic endoplasmic reticulum kinase. A series of studies has shown that interventions against ER stress and Nrf2 activation reduce myocardial infarct size and cardiac hypertrophy in the transition to HF in animals exposed to I/R injury and pressure overload, respectively. Finally, recent data showed that Nrf2/antioxidant-response element pathway activation may be of importance also in ischemic preconditioning, a phenomenon in which the heart is subjected to one or more episodes of nonlethal myocardial I/R before the sustained coronary artery occlusion.

The atherosclerotic plaque vulnerability: focus on the oxidative and endoplasmic reticulum stress in orchestrating the macrophage apoptosis in the formation of the necrotic core.

Although the understanding the pathophysiology of atherogenesis and atherosclerosis progression has been one of the major goals of cardiovascular research during the last decades, the precise mechanisms underlying plaque destabilization are still unknown. The disruption of the plaque and the thrombosis in the lumen that are mostly determined by the expansion of the necrotic core (NC) are driven by various mechanisms, including accelerated macrophage apoptosis and defective phagocytic clearance (defective efferocytosis). Oxidative stress is implicated in the expansion of the NC: in fact, many oxidized compounds and processes contribute to the macrophage apoptosis; in addition, the oxidized derivatives of polyunsatured fatty acids promote defective efferocytosis, with the final result of NC expansion. In the last years the role of the endoplasmic reticulum (ER) stress is under investigation to better define its possible contribution in affecting the NC expansion. The abnormal amount of apoptotic cells in the vulnerable plaque has been demonstrated to be related both to the sustained ER stress and to the expression of survival and protective genes, such as the unfolded protein response or/and the nuclear erytroid- related factor 2. In this review the authors focus on the promising results of the oxidative and ER stress in contributing to triggering and orchestrating the atherosclerotic plaque vulnerability.

Do oxidized polyunsaturated Fatty acids affect endoplasmic reticulum stress-induced apoptosis in human carotid plaques?

Macrophage apoptosis is involved in atherosclerotic plaque development. The aim of this study was to evaluate the interrelationship between macrophage apoptosis and the endoplasmic reticulum (ER) stress in the tissue around the necrotic core (TANC) and in the periphery (P) of the same carotid plaques derived from patients undergoing carotid endarterectomy. Apoptosis was significantly higher in TANC than in P (p<0.001). mRNA and protein expression of the protein kinase-like ER kinase (Perk) and the nuclear erythroid-related factor 2 (Nrf2)-related survival genes was significantly higher in P than in TANC (p<0.01), while CCAAT/enhancer-binding protein homologous protein (Chop) and the apoptosis-related genes were higher in TANC than in P (p<0.01). The TANC extract was characterized by significantly higher concentrations of oxidized derivatives of polyunsaturated fatty acids (PUFAs) than the P extract (p<0.01). When THP-1 cells were incubated with P or TANC extracts there was a dose-dependent increase of Perk and Nrf2 or of Chop and of the apoptosis-related genes, respectively (p<0.01). Our observations lead to the hypothesis that ER stress induced by oxidized derivatives of PUFAs may promote macrophage apoptosis in TANC and favor the expansion of the necrotic core of the plaques, a major feature responsible for its disruption and acute luminal thrombosis.

Increased endoplasmic reticulum stress and Nrf2 repression in peripheral blood mononuclear cells of patients with stable coronary artery disease.

Endoplasmic reticulum (ER) stress is involved in the pathophysiology of atherosclerosis. Insults interfering with ER function lead to the accumulation of unfolded and misfolded proteins in the ER that initiates the unfolded protein response (UPR). When the UPR fails to control the level of unfolded and misfolded proteins, ER-initiated apoptotic signaling is induced. We evaluated: (1) the UPR and ER-initiated apoptotic signaling in peripheral blood mononuclear cells (PBMCs) of stable coronary artery disease (CAD) patients; (2) PBMC content of oxidation products of phospholipid 1-palmitoyl-2-arachidonyl-sn-glycero-3-phosphorylcholine (oxPAPC); (3) the possible origin of oxPAPC in PBMCs; and (4) the expression of nuclear erythroid-related factor 2 (Nrf2)/antioxidant-related element (ARE), a cellular defense mechanism. Twenty-nine CAD patients and 28 matched controls were enrolled. Expression of glucose-regulated protein 78kDa (GRP78/BiP), as a representative of the UPR, and of C/EBP homologous protein (CHOP), as a representative of ER apoptosis, was significantly higher in CAD than in controls (p<0.01). Concentrations of oxPAPC in PBMCs, in plasma, and in low-density lipoprotein (LDL) were significantly higher in CAD compared to controls (p<0.01). The oxPAPC in PBMCs may derive from circulating ox-LDL. Nrf2/ARE gene expression and circulating and cellular glutathione were significantly lower in CAD compared to controls (p<0.01). In in vitro studies, increasing amounts of oxPAPC induced a dose-dependent increase in CHOP and apoptosis-related protein expression (p<0.01) and a progressive decrease in Nrf2/ARE gene expression (p<0.01). In PBMCs of CAD patients there is an activation of the UPR and ER-initiated apoptotic signaling, possibly related to an abnormal concentration of oxPAPC in PBMCs.

Lysophosphatidylcholine and carotid intima-media thickness in young smokers: a role for oxidized LDL-induced expression of PBMC lipoprotein-associated phospholipase A2?

BACKGROUND: Although cigarette smoking has been associated with carotid intima-media thickness (CIMT) the mechanisms are yet not completely known. Lysophosphatidylcholine (lysoPC), a main product of lipoprotein-associated phospholipase A2 (Lp-PLA2) activity, appears to be a major determinant of the pro-atherogenic properties of oxidized LDL (oxLDL) and to induce proteoglycan synthesis, a main player in intimal thickening. In this study we assessed whether cigarette smoking-induced oxidative stress may influence plasma Lp-PLA2 and lysoPC and Lp-PLA2 expression in peripheral blood mononuclear cells (PBMC), as well as the relationship between lysoPC and CIMT. METHODS/RESULTS: 45 healthy smokers and 45 age and sex-matched subjects participated in this study. Smokers, compared to non-smokers, showed increased plasma concentrations of oxLDL, Lp-PLA2 and lysoPC together with up-regulation of Lp-PLA2 (mRNA and protein) expression in PBMC (P<0.001). Plasma Lp-PLA2 positively correlated with both lysoPC (r=0.639, P<0.001) and PBMC mRNA Lp-PLA2 (r=0.484, P<0.001) in all subjects. Moreover CIMT that was higher in smokers (P<0.001), positively correlated with lysoPC (r=0.55, P<0.001). Then in in vitro study we demonstrated that both oxLDL (at concentrations similar to those found in smoker's serum) and oxidized phospholipids contained in oxLDL, were able to up-regulate mRNA Lp-PLA2 in PBMC. This effect was likely due, at least in part, to the enrichment in oxidized phospholipids found in PBMC after exposure to oxLDL. Our results also showed that in human aortic smooth muscle cells lysoPC, at concentrations similar to those found in smokers, increased the expression of biglycan and versican, two main proteoglycans. CONCLUSIONS: In smokers a further effect of raised oxidative stress is the up-regulation of Lp-PLA2 expression in PBMC with subsequent increase of plasma Lp-PLA2 and lysoPC. Moreover the correlation between lysoPC and CIMT together with the finding that lysoPC up-regulates proteoglycan synthesis suggests that lysoPC may be a link between smoking and intimal thickening.

Expansion of necrotic core and shedding of Mertk receptor in human carotid plaques: a role for oxidized polyunsaturated fatty acids?

AIMS: Expansion of necrotic core (NC), a major feature responsible for plaque disruption, is likely the consequence of accelerated macrophage apoptosis coupled with defective phagocytic clearance (efferocytosis). The cleavage of the extracellular domain of Mer tyrosine kinase (Mertk) by metallopeptidase domain17 (Adam17) has been shown to produce a soluble Mertk protein (sMer), which can inhibit efferocytosis. Herein, we analysed the expression and localization of Mertk and Adam17 in the tissue around the necrotic core (TANC) and in the periphery (P) of human carotid plaques. Then we studied the mechanisms of NC expansion by evaluating which components of TANC induce Adam17 and the related cleavage of the extracellular domain of Mertk. METHODS AND RESULTS: We studied 97 human carotid plaques. The expression of Mertk and Adam17 was found to be higher in TANC than in P (P < 0.001). By immunohistochemistry, Mertk was higher than Adam17 in the area of TANC near to the lumen (P < 0.01) but much lower in the area close to NC (P < 0.01). The extract of this portion of TANC increased the expression (mRNA) of Adam17 and Mertk (P < 0.01) in macrophage-like THP-1 cells but it also induced the cleavage of the extracellular domain of Mertk, generating sMer in the medium (P < 0.01). This effect of TANC extract was most evoked by its content in F(2)-isoprostanes, hydroxyoctadecadienoic acids, and hydroxytetraenoic acids. CONCLUSION: Some oxidized derivatives of polyunsaturated fatty acids contained in TANC of human carotid plaques are strong inducers of Adam17, which in turn leads to the generation of sMer, which can inhibit efferocytosis.

Runx2 mRNA expression in the tissue, serum, and circulating non-hematopoietic cells of patients with thyroid cancer.

CONTEXT: Runx2, a master gene of osteogenic differentiation, is also expressed in nonosseous cancer cells. Microcalcifications are characteristic of papillary thyroid carcinoma and represent a useful find for diagnosis. However, the molecular expression of osteogenic differentiation transcription factor Runx2 has been poorly investigated in this tumor. OBJECTIVE: The aim of this study was to investigate Runx2 mRNA expression in normal and pathological thyroid tissue, serum, and circulating non-hematopoietic cells. SETTING: The study was performed in the Endocrine Unit of Internal Medicine of "Azienda Ospedaliera Universitaria Integrata of Verona" (Verona, Italy). PATIENTS: We enrolled 12 patients with a papillary thyroid carcinoma (PTC), who had undergone total thyroidectomy performed by the same surgeon. The results, obtained by real-time RT-PCR, were compared with biological samples obtained from 13 sex- and age-matched normal donors. RESULTS: Our data demonstrated that Runx2 mRNA is overexpressed (7.81-fold expression) in pathological thyroid tissue than in normal tissue (P < 0.05). Runx2 mRNA overexpression was also observed in serum and circulating non-hematopoietic cells of PTC patients with respect to normal donors (5.91-fold expression, P < 0.001; 3.82-fold expression, P < 0.05, respectively). We also observed that patients with microcalcifications expressed significantly higher levels of Runx2 mRNA in serum with respect to patients without microcalcifications (P < 0.05). CONCLUSION: This study can open up new research perspectives in the diagnosis and follow-up of PTC, even if further and larger cohort studies will be necessary to validate the Runx2 expression as biomarkers in thyroid cancer.

Serum oxidative stress-induced repression of Nrf2 and GSH depletion: a mechanism potentially involved in endothelial dysfunction of young smokers.

BACKGROUND: Although oxidative stress plays a major role in endothelial dysfunction (ED), the role of glutathione (GSH), of nuclear erythroid-related factor 2 (Nrf2) and of related antioxidant genes (ARE) are yet unknown. In this study we combined an in vivo with an in vitro model to assess whether cigarette smoking affects flow-mediated vasodilation (FMD), GSH concentrations and the Nrf2/ARE pathway in human umbilical vein endothelial cells (HUVECs). METHODS AND RESULTS: 52 healthy subjects (26 non-smokers and 26 heavy smokers) were enrolled in this study. In smokers we demonstrated increased oxidative stress, i.e., reduced concentrations of GSH and increased concentrations of oxidation products of the phospholipid 1-palmitoyl-2-arachidonyl-sn-glycero-3-phosphorylcholine (oxPAPC) in serum and in peripheral blood mononuclear cells (PBMC), used as in vivo surrogates of endothelial cells. Moreover we showed impairment of FMD in smokers and a positive correlation with the concentration of GSH in PBMC of all subjects. In HUVECs exposed to smokers' serum but not to non-smokers' serum we found that oxidative stress increased, whereas nitric oxide and GSH concentrations decreased; interestingly the expression of Nrf2, of heme oxygenase-1 (HO-1) and of glutamate-cysteine ligase catalytic (GCLC) subunit, the rate-limiting step of synthesis of GSH, was decreased. To test the hypothesis that the increased oxidative stress in smokers may have a causal role in the repression of Nrf2/ARE pathway, we exposed HUVECs to increasing concentrations of oxPAPC and found that at the highest concentration (similar to that found in smokers' serum) the expression of Nrf2/ARE pathway was reduced. The knockdown of Nrf2 was associated to a significant reduction of HO-1 and GCLC expression induced by oxPAPC in ECs. CONCLUSIONS: In young smokers with ED a novel further consequence of increased oxidative stress is a repression of Nrf2/ARE pathway leading to GSH depletion.