Circulating perturbation of phosphatidylcholine (PC) and phosphatidylethanolamine (PE) is associated to cardiac remodeling and NLRP3 inflammasome in cardiovascular patients with insulin resistance risk.

Lipidome perturbation occurring during meta-inflammation is associated to left ventricle (LV) remodeling though the activation of the NLRP3 inflammasome, a key regulator of chronic inflammation in obesity-related disorders. Little is known about phosphatidylcholine (PC) and phosphatidylethanolamine (PE) as DAMP-induced NLRP3 inflammasome. Our study is aimed to evaluate if a systemic reduction of PC/PE molar ratio can affect NLRP3 plasma levels in cardiovascular disease (CVD) patients with insulin resistance (IR) risk. Forty patients from IRCCS Policlinico San Donato were enrolled, and their blood samples were drawn before heart surgery. LV geometry measurements were evaluated by echocardiography and clinical data associated to IR risk were collected. PC and PE were quantified by ESI-MS/MS. Circulating NLRP3 was quantified by an ELISA assay. Our results have shown that CVD patients with IR risk presented systemic lipid impairment of PC and PE species and their ratio in plasma was inversely associated to NLRP3 levels. Interestingly, CVD patients with IR risk presented LV changes directly associated to increased levels of NLRP3 and a decrease in PC/PE ratio in plasma, highlighting the systemic effect of meta-inflammation in cardiac response. In summary, PC and PE can be considered bioactive mediators associated to both the NLRP3 and LV changes in CVD patients with IR risk.

Unveiling IL-33/ST2 Pathway Unbalance in Cardiac Remodeling Due to Obesity in Zucker Fatty Rats.

Obesity is an epidemic condition linked to cardiovascular disease severity and mortality. Fat localization and type represent cardiovascular risk estimators. Importantly, visceral fat secretes adipokines known to promote low-grade inflammation that, in turn, modulate its secretome and cardiac metabolism. In this regard, IL-33 regulates the functions of various immune cells through ST2 binding and-following its role as an immune sensor to infection and stress-is involved in the pro-fibrotic remodeling of the myocardium. Here we further investigated the IL-33/ST2 effects on cardiac remodeling in obesity, focusing on molecular pathways linking adipose-derived IL-33 to the development of fibrosis or hypertrophy. We analyzed the Zucker Fatty rat model, and we developed in vitro models to mimic the adipose and myocardial relationship. We demonstrated a dysregulation of IL-33/ST2 signaling in both adipose and cardiac tissue, where they affected Epac proteins and myocardial gene expression, linked to pro-fibrotic signatures. In Zucker rats, pro-fibrotic effects were counteracted by ghrelin-induced IL-33 secretion, whose release influenced transcription factor expression and ST2 isoforms balance regulation. Finally, the effect of IL-33 signaling is dependent on several factors, such as cell types' origin and the balancing of ST2 isoforms. Noteworthy, it is reasonable to state that considering IL-33 to have a unique protective role should be considered over-simplistic.

PCSK9 Expression in Epicardial Adipose Tissue: Molecular Association with Local Tissue Inflammation.

Epicardial adipose tissue (EAT) has the unique property to release mediators that nourish the heart in healthy conditions, an effect that becomes detrimental when volume expands and proinflammatory cytokines start to be produced. Proprotein convertase subtilisin/kexin type 9 (PCSK9), a proinflammatory mediator involved in atherosclerosis, is also produced by visceral fat. Due to the correlation of inflammation with PCSK9 and EAT enlargement, we evaluated whether PCSK9 was expressed in EAT and associated with EAT inflammation and volume. EAT samples were isolated during surgery. EAT thickness was measured by echocardiography. A microarray was used to explore EAT transcriptoma. The PCSK9 protein levels were measured by Western Blot in EAT and ELISA in plasma. PCSK9 was expressed at both the gene and protein levels in EAT. We found a positive association with EAT thickness and local proinflammatory mediators, in particular, chemokines for monocytes and lymphocytes. No association was found with the circulating PCSK9 level. The expression of PCSK9 in EAT argues that PCSK9 is part of the EAT secretome and EAT inflammation is associated with local PCSK9 expression, regardless of circulating PCSK9 levels. Whether reducing EAT inflammation or PCSK9 local levels may have beneficial effects on EAT metabolism and cardiovascular risk needs further investigations.

Osteopontin: The Molecular Bridge between Fat and Cardiac-Renal Disorders.

Osteopontin (OPN) is a multifaceted matricellular protein, with well-recognized roles in both the physiological and pathological processes in the body. OPN is expressed in the main organs and cell types, in which it induces different biological actions. During physiological conditioning, OPN acts as both an intracellular protein and soluble excreted cytokine, regulating tissue remodeling and immune-infiltrate in adipose tissue the heart and the kidney. In contrast, the increased expression of OPN has been correlated with the severity of the cardiovascular and renal outcomes associated with obesity. Indeed, OPN expression is at the "cross roads" of visceral fat extension, cardiovascular diseases (CVDs) and renal disorders, in which OPN orchestrates the molecular interactions, leading to chronic low-grade inflammation. The common factor associated with OPN overexpression in adipose, cardiac and renal tissues seems attributable to the concomitant increase in visceral fat size and the increase in infiltrated OPN+ macrophages. This review underlines the current knowledge on the molecular interactions between obesity and the cardiac-renal disorders ruled by OPN.

Correlative Study on Impaired Prostaglandin E2 Regulation in Epicardial Adipose Tissue and its Role in Maladaptive Cardiac Remodeling via EPAC2 and ST2 Signaling in Overweight Cardiovascular Disease Subjects.

There is recent evidence that the dysfunctional responses of a peculiar visceral fat deposit known as epicardial adipose tissue (EAT) can directly promote cardiac enlargement in the case of obesity. Here, we observed a newer molecular pattern associated with LV dysfunction mediated by prostaglandin E2 (PGE2) deregulation in EAT in a cardiovascular disease (CVD) population. A series of 33 overweight CVD males were enrolled and their EAT thickness, LV mass, and volumes were measured by echocardiography. Blood, plasma, EAT, and SAT biopsies were collected for molecular and proteomic assays. Our data show that PGE2 biosynthetic enzyme (PTGES-2) correlates with echocardiographic parameters of LV enlargement: LV diameters, LV end diastolic volume, and LV masses. Moreover, PTGES-2 is directly associated with EPAC2 gene (r = 0.70, p < 0.0001), known as a molecular inducer of ST2/IL-33 mediators involved in maladaptive heart remodelling. Furthermore, PGE2 receptor 3 (PTEGER3) results are downregulated and its expression is inversely associated with ST2/IL-33 expression. Contrarily, PGE2 receptor 4 (PTGER4) is upregulated in EAT and directly correlates with ST2 molecular expression. Our data suggest that excessive body fatness can shift the EAT transcriptome to a pro-tissue remodelling profile, may be driven by PGE2 deregulation, with consequent promotion of EPAC2 and ST2 signalling.

Correlational study on altered epicardial adipose tissue as a stratification risk factor for valve disease progression through IL-13 signaling.

AIMS: Genetic and environmental factors all interact in the risk of progression of valvular dysfunctions. Previous studies reported a relation between valve diseases and epicardial adipose tissue (EAT) thickness. The aim of this study was to verify the possible relationship between the molecular pattern of EAT related to IL-13 fibrogenic cytokine expression and valve dysfunction. METHODS AND RESULTS: A valvular heart disease (VHD) population was stratified according to their median EAT thickness (7 mm). The molecular expression of IL-13 in EAT is directly related to the molecular expression of genes associated with extracellular matrix (ECM) turnover, macrophage infiltration and promotion of the formation of ectopic calcific nodules involved in aorta coarctation and calcification. CONCLUSION: IL-13 gene expression in altered EAT is directly related to the expression of genes involved in ECM turnover and the formation of ectopic calcific nodules, suggesting measurements of EAT as a stratification risk factor for valve instability in the VHD patients.

Transglutaminase 2 Mediates the Cytotoxicity of Resveratrol in a Human Cholangiocarcinoma and Gallbladder Cancer Cell Lines.

Resveratrol is a polyphenolic compound extracted from plants and is also a constituent of red wine. Our aim was to evaluate if the cytotoxic effect of resveratrol (RES) on cholangiocarcinoma (CC) and gallbladder cancer (GBC) cell lines could be abolished by TG2 inhibition. Human CC and GBC cell lines (SK-ChA-1 and MZ-ChA-1), grown in a three-dimensional cell culture system (MCTS, multicellular tumor spheroids), were treated for 72 h with RES (32, 64 µM) alone or combined with different TG2 inhibitors (Cystamine, B003, T101). We investigated: cells viability; cell morphology with light microscopy (LM) and transmission electron microscopy (TEM); immunoreactivity with immunohistochemistry; Q-Banding karyotype analysis; TG2 activity; Western blotting. RES treatment induced a significant inhibition of cell growth, ranging from 24% to 76% in both cell lines. The inhibitors successfully reduced TG2 activity without any variation of protein quantity as demonstrated by immunohistochemistry and Western blot. TG2 inhibition resulted in cell growth normalization. In addition, morphologic analysis by light and transmission electron microscopy confirmed the cytotoxic effect of RES and its reduction consequent to TG2 inhibition. Our data demonstrated a connection between the cytotoxic effect of RES in SK-ChA-1 and MZ-ChA-1 and TG2 activity.

α-Defensin point-of-care test for diagnosis of prosthetic joint infections: neglected role of laboratory and clinical pathologists.

Periprosthetic joint infection (PJI) is a serious complication that may occur after native joint replacement leading to a severe health and economic burden. Currently, due to several confounding factors, PJI is difficult to diagnose. Today, a multidisciplinary approach is indispensable to correctly define a periprosthetic joint infection; indeed, tissue histology, microbiology cultures and clinical findings are used together to achieve this goal. Analysis of α-defensin is commonly used for PJI diagnosis, as it allows the rapid detection of α-defensin present in the synovial fluid following a microbial infection. Currently, a point-of-care testing (POCT) assay able to detect the presence of human α-defensins 1-3 in synovial fluid of patients is aimed directly at orthopedic surgeons. However, many orthopedic surgeons lack experience and training in quality laboratory practices, often failing to appreciate the significance of quality control and proper documentation when using POCT assays. To guarantee the highest quality diagnostic services, the α-defensin test should be used together with other biochemical and microbiological criteria commonly used for PJI diagnosis. Additionally, the close cooperation and communication between laboratory, pathologists and physicians is of fundamental importance in the correct diagnosis of PJI.

Vitamin D Deficiency Is Associated with Increased Osteocalcin Levels in Acute Aortic Dissection: A Pilot Study on Elderly Patients.

An imbalance between degradation and reconstruction of the aortic wall is one of the leading causes of acute aortic dissection (AAD). Vitamin D seems an intriguing molecule to explore in the field of AAD since it improves endothelial function and protects smooth muscle cells from inflammation-induced remodeling, calcification, and loss of function, all events which are strongly related to the aging process. We quantified 25-hydroxy vitamin D, calcium, parathormone, bone alkaline phosphatase, and osteocalcin levels in 24 elderly AAD patients to identify a potential pathological implication of these molecules in AAD. Median 25-hydroxy vitamin D (10.75 ng/mL, 25th-75th percentiles: 6.86-19.23 ng/mL) and calcium levels (8.70 mg/dL, 25th-75th percentiles: 7.30-8.80 mg/dL) suggested hypovitaminosis D and a moderate hypocalcemia. Thirty-eight percent of AAD patients had severe (<10 ng/mL), 38% moderate (10-20 ng/mL), and 24% mild 25-hydroxy vitamin D deficiency (20-30 ng/mL). A significant inverse correlation was observed between 25OHD and osteocalcin levels. All the other molecules were unchanged. A condition of hypovitaminosis D associated to an increase in osteocalcin levels is present in AAD patients. The identification of these molecules as new factors involved in AAD may be helpful to identify individuals at high risk as well to study preventing strategies.

Matrix metalloproteinases as biomarkers of disease: updates and new insights.

Matrix metalloproteinases (MMPs) play a pivotal role in remodeling the extracellular matrix (ECM) and are therefore of interest for new diagnostic tools for the clinical management of diseases involving ECM disruption. This setting ranges from the classical areas of MMP studies, such as vascular disease, cancer progression or bone disorders, to new emerging fields of application, such as neurodegenerative disease or sepsis. Increasing the knowledge about the role of MMPs in the pathogenesis of diseases where a clear diagnostic panel is still lacking could provide new insight and improve the identification and the clinical treatment of these human diseases. This review focuses on the latest descriptions of the clinical use of MMP as biomarkers in the diagnosis, prognosis and monitoring of different diseases, such as diabetes, cardiovascular diseases, cancer and metastasis, neurodegenerative disorders and sepsis.

Hypertension in adult Fabry's disease: is cardiotrophin-1 a diagnostic biomarker?

BACKGROUND: Cardiotrophin-1 (CT-1), a cytokine produced by cardiomyocytes and non-cardiomyocytes in conditions of stress, can be used as a biomarker of left ventricular hypertrophy and dysfunction in hypertensive patients. Hypertension is one of the main adverse events in the third and last phase of Fabry's disease (FD). We measured CT-1 in order to examine its correlation with the vascular and cardiac alterations at different ages and assess its potential for use as a biomarker of hypertension in FD. FINDINGS: The level of CT-1 was clearly higher in hypertensive adults than in adult FD patients. FD patients show a small, non-significant decrease in plasma CT-1 with age, while in hypertensive patients CT-1 in plasma rises strongly and highly significantly with age. CONCLUSIONS: CT-1 can be considered a good biomarker of the progression of hypertension with age, but particular care is needed when following hypertension in FD patients, since CT-1 does not correlate the same way with this disease.

Adenosine A(2)A receptor but not HIF-1 mediates Tyrosine hydroxylase induction in hypoxic PC12 cells.

Tyrosine hydroxylase (TH) is the rate-limiting enzyme in the biosynthesis of catecholamines released by oxygen-sensitive cells in response to hypoxic conditions. Adenosine is released in response to hypoxia in the central nervous system and CGS21680, an adenosine A(2)A receptor agonist, induces TH transcription. As we have previously demonstrated the A(2)A receptor-mediated induction of HIF-1 in macrophages and hepatocytes, we investigated the involvement of HIF-1 in the adenosine-mediated activation of TH expression. Exposure to adenosine or CGS21680 increased TH mRNA and protein levels in PC12 cells. Transcription of a reporter gene under the control of the wild type rat TH promoter was induced 3.5-fold in CGS21680-treated cells, but neither the mutation of the hypoxia responsive element in the TH promoter nor the co-transfection of a dominant negative of the HIF-1 beta subunit prevented the increase in transcription; furthermore, CGS21680 increased CREB binding activity but did not induce HIF-1 DNA binding activity or protein levels. To investigate whether HIF-1 was involved in the hypoxia-mediated induction of TH, PC12 cells were exposed to hypoxia in the presence of the A(2)A receptor antagonist ZM241385, which prevented hypoxia-dependent TH induction despite HIF-1 activation; in line with this finding, the inhibition of HIF-1 did not abolish TH induction in hypoxic PC12 cells. These results indicate that, under hypoxic conditions, TH (a key factor in systemic adaptation to reduced oxygen availability) is not regulated by HIF-1, the primary modulator of the response to hypoxia, but by the adenosine A(2)A receptor-mediated signalling pathway.

Advanced Glycation End Products (AGE) and Soluble Forms of AGE Receptor: Emerging Role as Mortality Risk Factors in CKD.

Advanced glycation end-products (AGE) can promote chronic kidney disease (CKD) progression and CKD-related morbidities. The soluble receptor for AGE (sRAGE) is a potential biomarker of inflammation and oxidative stress. Here, we explored the role of AGE, glycated albumin, sRAGE and its different forms, cRAGE and esRAGE, as prognostic factors for mortality in 111 advanced CKD patients. The median follow-up time was 39 months. AGE were quantified by fluorescence, sRAGE and its forms by ELISA. Malnutrition was screened by the Malnutrition Inflammation Score (MIS). The Cox proportional hazards regression model was used to assess the association of variables with all-cause mortality. Mean levels of sRAGE, esRAGE and cRAGE were 2318 ± 1224, 649 ± 454 and 1669 ± 901 pg/mL. The mean value of cRAGE/esRAGE was 2.82 ± 0.96. AGE were 3026 ± 766 AU and MIS 6.0 ± 4.7. eGFR correlated negatively with AGE, sRAGE, esRAGE and cRAGE, but not with cRAGE/esRAGE. Twenty-eight patients died. No difference was observed between diabetic and non-diabetic patients. Starting dialysis was not associated with enhanced risk of death. AGE, esRAGE and cRAGE/esRAGE were independently associated with all-cause mortality. AGE, esRAGE and cRAGE/esRAGE may help to stratify overall mortality risk. Implementing the clinical evaluation of CKD patients by quantifying these biomarkers can help to improve patient outcomes.

Author Correction: Dysfunctional EAT thickness may promote maladaptive heart remodeling in CVD patients through the ST2-IL33 system, directly related to EPAC protein expression.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Circulating Irisin and esRAGE as Early Biomarkers of Decline of Metabolic Health.

A decline in metabolic health may take place before observing any alteration in the levels of the traditional metabolic markers. New indicators of metabolic derangement are therefore compelling. Irisin is a myokine with important metabolic functions. The role of irisin as a metabolic biomarker in humans has not been fully established yet. We quantified plasma irisin and esRAGE in 106 apparently healthy individuals and we performed a cluster analysis to evaluate their associations with metabolic profile. Plasma levels of various traditional markers of metabolic risk (i.e., glucose and lipid levels) were all within the ranges of normality. We identified two clusters of individuals. Compared to cluster 2, individuals in cluster 1 had higher irisin levels, a metabolic profile shifted toward the limits of the reference ranges and lower esRAGE levels. The traditional metabolic blood tests seem not to be enough to identify a metabolic decline early. Irisin increase and esRAGE decrease may reflect a metabolic derangement at the beginning of its development. The role of these molecules as early biomarkers of decline of metabolic health seems an interesting topic to be further explored.

Adenosine-dependent activation of hypoxia-inducible factor-1 induces late preconditioning in liver cells.

UNLABELLED: The cellular mechanisms by which ischemic preconditioning increases liver tolerance to ischemia/reperfusion injury are still poorly understood. This study investigated the role of the hypoxia-inducible factor-1 (HIF-1) in the protection associated with the late phase of liver preconditioning. Late preconditioning was induced in primary cultured rat hepatocytes by a transient (10 minute) hypoxic stress or by 15 minutes incubation with the adenosine A(2A) receptors agonist CGS21680 24 hours before exposure to 90 minutes of hypoxia in a serum-free medium. Late preconditioning induced the nuclear translocation of HIF-1 and the expression of carbonic anhydrase IX (CAIX), a HIF-1-regulated transmembrane enzyme that catalyzes bicarbonate production. Such effects were associated with prevention of hepatocyte killing by hypoxia and the amelioration of intracellular acidosis and Na+ accumulation. The inhibition of PKC-mediated and PI3-kinase-mediated signals with, respectively, chelerythrine and wortmannin abolished HIF-1 activation and blocked both CAIX expression and the protective action of late preconditioning. CAIX expression was also prevented by interfering with the transcriptional activity of HIF-1 using a dominant negative HIF-1beta subunit. The inhibition of CAIX with acetazolamide or the block of bicarbonate influx with disodium-4-acetamido-4'-isothiocyanato-stilben-2,2'-disulfonate also reverted the protective effects of late preconditioning on intracellular acidosis and Na+ accumulation. CONCLUSION: The stimulation of adenosine A(2A) receptors induced late preconditioning in liver cells through the activation of HIF-1. HIF-1-induced expression of CAIX increases hepatocyte tolerance to ischemia by maintaining intracellular Na+ homeostasis. These observations along with the importance of HIF-1 in regulating cell survival indicates HIF-1 activation as a possible key event in liver protection by late preconditioning.

ST2/IL-33 signaling in cardiac fibrosis.

Cardiac fibrosis is a significant global health problem associated with nearly all forms of heart disease. In the heart interstitial fibrosis may be reparative, replacing areas damaged by myocyte loss after acute infarction, or compensative, responding to cardiac overload. However, after injury in chronic cases activated myofibroblasts contribute to the tissue imbalance of the newer molecules associated with cardiac fibrosis, interleukin (IL-33), and suppression of tumorigenicity 2 (ST2). Physiological stretching causes myofibroblasts to release IL-33 which binds the ST2 receptor (ST2L) on the cardiomyocyte membrane, promoting cell survival and integrity. But in chronic conditions, local and neighboring cells can increase the release of IL-33's decoy, soluble ST2 (sST2), which blocks IL-33/ST2L binding, promoting tissue fibrosis. We review recent studies that have illustrated novel aspects of ST2/IL-33 signaling mediating cardiac fibrosis, and some newer biomolecular targets for the prevention and treatment of maladaptive remodeling.

Dysfunctional EAT thickness may promote maladaptive heart remodeling in CVD patients through the ST2-IL33 system, directly related to EPAC protein expression.

Dysfunctional epicardial adipose tissue (EAT) secretome can influence the heart's stretch response. However, the molecular mechanisms are still poorly understood. The aim of this study was to clarify how dysfunctional EAT promotes maladaptive heart remodeling in cardiovascular disease (CVD) through ST2 production associated with exchange protein directly activated by cAMP (EPAC) proteins. A series of 55 CVD males were enrolled and their EAT thickness, LV mass and volumes were measured by echocardiography. Blood, plasma and EAT biopsies were collected for molecular and proteomic assays. Taking EAT thickness as a continuous variable there was a direct correlation between the ST2 cardiac stretch mediator and EAT thickness (r = 0.54, p < 0.01) and an inverse relation between the ST2 gene and IL-33 expression (r -0.50, p < 0.01). In the CVD population EPAC2 expression directly correlated with the ST2 gene (r = 0.74, p < 0.0001) causing an ST2/IL-33 system local (p < 0.001) and systemic (sST2 = 57.33 ± 3.22 and IL-33 = 0.53 ± 017 pg/mL; p < 0.0001) protein imbalance associated with maladaptive remodeling. This indicated that dysfunctional EAT is a source of both EPAC and ST2 protein and an EPAC2 isoform seems involved in ST2 production in adipose tissue. Both EPAC2 and ST2 expression were directly related to maladaptive heart remodeling indices, suggesting EAT measurements could be useful in the early assessment of CVD complications.

Epicardial adipose tissue GLP-1 receptor is associated with genes involved in fatty acid oxidation and white-to-brown fat differentiation: A target to modulate cardiovascular risk?

BACKGROUND: Epicardial adipose tissue (EAT) is a risk factor for cardiovascular diseases. Glucagon-like peptide 1 analogs (GLP-1A) may have beneficial cardiovascular effects and reduce EAT, possibly throughout targeting GLP-1 receptor (GLP-1R). Nevertheless, the role of EAT GLP-1R, GLP-2R and their interplay with EAT genes involved in adipogenesis and fatty acid (FA) metabolism are unknown. We analyzed whether EAT transcriptome is related to GLP-1R/GLP-2R gene expression, and GLP-1/GLP-2 plasma levels in coronary artery disease patients (CAD). METHODS: EAT was collected from 17 CAD patients undergoing CABG for microarray analysis of GLP-1R, GLP-2R and genes involved in FA metabolism and adipogenesis. EAT thickness was measured by echocardiography. GLP-1 and GLP-2 levels were quantified by ELISA in CAD and healthy subjects (CTR). RESULTS: EAT GLP-1R was directly correlated with genes promoting beta-oxidation and white-to-brown adipocyte differentiation, and inversely with pro-adipogenic genes. GLP-2R was positively correlated with genes involved in adipogenesis and lipid synthesis, and inversely with genes promoting beta-oxidation. GLP-1 and GLP-2 levels were higher in CAD than CTR and in patients with greater EAT thickness. CONCLUSIONS: GLP-1 analogs may target EAT GLP-1R and therefore reduce local adipogenesis, improve fat utilization and induce brown fat differentiation. As EAT lies in direct contiguity to myocardium and coronary arteries, the beneficial effects of GLP-1 activation may extent to the heart. The increased levels of circulating GLP-1 and GLP-2 and EAT GLP-2R may be compensatory mechanisms related to CAD and also EAT expansion, but the meaning of these observations needs to be further investigated.