Regulation of autophagosome biogenesis by OFD1-mediated selective autophagy.

Autophagy is a lysosome-dependent degradation pathway essential to maintain cellular homeostasis. Therefore, either defective or excessive autophagy may be detrimental for cells and tissues. The past decade was characterized by significant advances in molecular dissection of stimulatory autophagy inputs; however, our understanding of the mechanisms that restrain autophagy is far from complete. Here, we describe a negative feedback mechanism that limits autophagosome biogenesis based on the selective autophagy-mediated degradation of ATG13, a component of the ULK1 autophagy initiation complex. We demonstrate that the centrosomal protein OFD1 acts as bona fide autophagy receptor for ATG13 via direct interaction with the Atg8/LC3/GABARAP family of proteins. We also show that patients with Oral-Facial-Digital type I syndrome, caused by mutations in the OFD1 gene, display excessive autophagy and that genetic inhibition of autophagy in a mouse model of the disease, significantly ameliorates polycystic kidney, a clinical manifestation of the disorder. Collectively, our data report the discovery of an autophagy self-regulated mechanism and implicate dysregulated autophagy in the pathogenesis of renal cystic disease in mammals.

Omics Analyses of Stromal Cells from ACM Patients Reveal Alterations in Chromatin Organization and Mitochondrial Homeostasis.

Arrhythmogenic cardiomyopathy (ACM) is a genetic disorder characterized by ventricular arrhythmias, contractile dysfunctions and fibro-adipose replacement of myocardium. Cardiac mesenchymal stromal cells (CMSCs) participate in disease pathogenesis by differentiating towards adipocytes and myofibroblasts. Some altered pathways in ACM are known, but many are yet to be discovered. We aimed to enrich the understanding of ACM pathogenesis by comparing epigenetic and gene expression profiles of ACM-CMSCs with healthy control (HC)-CMSCs. Methylome analysis identified 74 differentially methylated nucleotides, most of them located on the mitochondrial genome. Transcriptome analysis revealed 327 genes that were more expressed and 202 genes that were less expressed in ACM- vs. HC-CMSCs. Among these, genes implicated in mitochondrial respiration and in epithelial-to-mesenchymal transition were more expressed, and cell cycle genes were less expressed in ACM- vs. HC-CMSCs. Through enrichment and gene network analyses, we identified differentially regulated pathways, some of which never associated with ACM, including mitochondrial functioning and chromatin organization, both in line with methylome results. Functional validations confirmed that ACM-CMSCs exhibited higher amounts of active mitochondria and ROS production, a lower proliferation rate and a more pronounced epicardial-to-mesenchymal transition compared to the controls. In conclusion, ACM-CMSC-omics revealed some additional altered molecular pathways, relevant in disease pathogenesis, which may constitute novel targets for specific therapies.

Cardiac Biomarkers and Autoantibodies in Endurance Athletes: Potential Similarities with Arrhythmogenic Cardiomyopathy Pathogenic Mechanisms.

The "Extreme Exercise Hypothesis" states that when individuals perform training beyond the ideal exercise dose, a decline in the beneficial effects of physical activity occurs. This is due to significant changes in myocardial structure and function, such as hemodynamic alterations, cardiac chamber enlargement and hypertrophy, myocardial inflammation, oxidative stress, fibrosis, and conduction changes. In addition, an increased amount of circulating biomarkers of exercise-induced damage has been reported. Although these changes are often reversible, long-lasting cardiac damage may develop after years of intense physical exercise. Since several features of the athlete's heart overlap with arrhythmogenic cardiomyopathy (ACM), the syndrome of "exercise-induced ACM" has been postulated. Thus, the distinction between ACM and the athlete's heart may be challenging. Recently, an autoimmune mechanism has been discovered in ACM patients linked to their characteristic junctional impairment. Since cardiac junctions are similarly impaired by intense physical activity due to the strong myocardial stretching, we propose in the present work the novel hypothesis of an autoimmune response in endurance athletes. This investigation may deepen the knowledge about the pathological remodeling and relative activated mechanisms induced by intense endurance exercise, potentially improving the early recognition of whom is actually at risk.

Excess TGF-ß1 Drives Cardiac Mesenchymal Stromal Cells to a Pro-Fibrotic Commitment in Arrhythmogenic Cardiomyopathy.

Arrhythmogenic Cardiomyopathy (ACM) is characterized by the replacement of the myocardium with fibrotic or fibro-fatty tissue and inflammatory infiltrates in the heart. To date, while ACM adipogenesis is a well-investigated differentiation program, ACM-related fibrosis remains a scientific gap of knowledge. In this study, we analyze the fibrotic process occurring during ACM pathogenesis focusing on the role of cardiac mesenchymal stromal cells (C-MSC) as a source of myofibroblasts. We performed the ex vivo studies on plasma and right ventricular endomyocardial bioptic samples collected from ACM patients and healthy control donors (HC). In vitro studies were performed on C-MSC isolated from endomyocardial biopsies of both groups. Our results revealed that circulating TGF-ß1 levels are significantly higher in the ACM cohort than in HC. Accordingly, fibrotic markers are increased in ACM patient-derived cardiac biopsies compared to HC ones. This difference is not evident in isolated C-MSC. Nevertheless, ACM C-MSC are more responsive than HC ones to TGF-ß1 treatment, in terms of pro-fibrotic differentiation and higher activation of the SMAD2/3 signaling pathway. These results provide the novel evidence that C-MSC are a source of myofibroblasts and participate in ACM fibrotic remodeling, being highly responsive to ACM-characteristic excess TGF-ß1.

CaMKII Activity in the Inflammatory Response of Cardiac Diseases.

Inflammation is a physiological process by which the body responds to external insults and stress conditions, and it is characterized by the production of pro-inflammatory mediators such as cytokines. The acute inflammatory response is solved by removing the threat. Conversely, a chronic inflammatory state is established due to a prolonged inflammatory response and may lead to tissue damage. Based on the evidence of a reciprocal regulation between inflammation process and calcium unbalance, here we described the involvement of a calcium sensor in cardiac diseases with inflammatory drift. Indeed, the Ca2+/calmodulin-dependent protein kinase II (CaMKII) is activated in several diseases with an inflammatory component, such as myocardial infarction, ischemia/reperfusion injury, pressure overload/hypertrophy, and arrhythmic syndromes, in which it actively regulates pro-inflammatory signaling, among which includes nuclear factor kappa-B (NF-κB), thus contributing to pathological cardiac remodeling. Thus, CaMKII may represent a key target to modulate the severity of the inflammatory-driven degeneration.

Calcium as a Key Player in Arrhythmogenic Cardiomyopathy: Adhesion Disorder or Intracellular Alteration?

Arrhythmogenic cardiomyopathy (ACM) is an inherited heart disease characterized by sudden death in young people and featured by fibro-adipose myocardium replacement, malignant arrhythmias, and heart failure. To date, no etiological therapies are available. Mutations in desmosomal genes cause abnormal mechanical coupling, trigger pro-apoptotic signaling pathways, and induce fibro-adipose replacement. Here, we discuss the hypothesis that the ACM causative mechanism involves a defect in the expression and/or activity of the cardiac Ca2+ handling machinery, focusing on the available data supporting this hypothesis. The Ca2+ toolkit is heavily remodeled in cardiomyocytes derived from a mouse model of ACM defective of the desmosomal protein plakophilin-2. Furthermore, ACM-related mutations were found in genes encoding for proteins involved in excitation‒contraction coupling, e.g., type 2 ryanodine receptor and phospholamban. As a consequence, the sarcoplasmic reticulum becomes more eager to release Ca2+, thereby inducing delayed afterdepolarizations and impairing cardiac contractility. These data are supported by preliminary observations from patient induced pluripotent stem-cell-derived cardiomyocytes. Assessing the involvement of Ca2+ signaling in the pathogenesis of ACM could be beneficial in the treatment of this life-threatening disease.

The Amino-Terminal Domain of GRK5 Inhibits Cardiac Hypertrophy through the Regulation of Calcium-Calmodulin Dependent Transcription Factors.

We have recently demonstrated that the amino-terminal domain of G protein coupled receptor kinase (GRK) type 5, (GRK5-NT) inhibits NFκB activity in cardiac cells leading to a significant amelioration of LVH. Since GRK5-NT is known to bind calmodulin, this study aimed to evaluate the functional role of GRK5-NT in the regulation of calcium-calmodulin-dependent transcription factors. We found that the overexpression of GRK5-NT in cardiomyoblasts significantly reduced the activation and the nuclear translocation of NFAT and its cofactor GATA-4 in response to phenylephrine (PE). These results were confirmed in vivo in spontaneously hypertensive rats (SHR), in which intramyocardial adenovirus-mediated gene transfer of GRK5-NT reduced both wall thickness and ventricular mass by modulating NFAT and GATA-4 activity. To further verify in vitro the contribution of calmodulin in linking GRK5-NT to the NFAT/GATA-4 pathway, we examined the effects of a mutant of GRK5 (GRK5-NTPB), which is not able to bind calmodulin. When compared to GRK5-NT, GRK5-NTPB did not modify PE-induced NFAT and GATA-4 activation. In conclusion, this study identifies a double effect of GRK5-NT in the inhibition of LVH that is based on the regulation of multiple transcription factors through means of different mechanisms and proposes the amino-terminal sequence of GRK5 as a useful prototype for therapeutic purposes.

Fibrotic extracellular matrix impacts cardiomyocyte phenotype and function in an iPSC-derived isogenic model of cardiac fibrosis.

Cardiac fibrosis occurs following insults to the myocardium and is characterized by the abnormal accumulation of non-compliant extracellular matrix (ECM), which compromises cardiomyocyte contractile activity and eventually leads to heart failure. This phenomenon is driven by the activation of cardiac fibroblasts (cFbs) to myofibroblasts and results in changes in ECM biochemical, structural and mechanical properties. The lack of predictive in vitro models of heart fibrosis has so far hampered the search for innovative treatments, as most of the cellular-based in vitro reductionist models do not take into account the leading role of ECM cues in driving the progression of the pathology. Here, we devised a single-step decellularization protocol to obtain and thoroughly characterize the biochemical and micro-mechanical properties of the ECM secreted by activated cFbs differentiated from human induced pluripotent stem cells (iPSCs). We activated iPSC-derived cFbs to the myofibroblast phenotype by tuning basic fibroblast growth factor (bFGF) and transforming growth factor beta 1 (TGF-ß1) signalling and confirmed that activated cells acquired key features of myofibroblast phenotype, like SMAD2/3 nuclear shuttling, the formation of aligned alpha-smooth muscle actin (α-SMA)-rich stress fibres and increased focal adhesions (FAs) assembly. Next, we used Mass Spectrometry, nanoindentation, scanning electron and confocal microscopy to unveil the characteristic composition and the visco-elastic properties of the abundant, collagen-rich ECM deposited by cardiac myofibroblasts in vitro. Finally, we demonstrated that the fibrotic ECM activates mechanosensitive pathways in iPSC-derived cardiomyocytes, impacting on their shape, sarcomere assembly, phenotype, and calcium handling properties. We thus propose human bio-inspired decellularized matrices as animal-free, isogenic cardiomyocyte culture substrates recapitulating key pathophysiological changes occurring at the cellular level during cardiac fibrosis.

Patient-specific primary and pluripotent stem cell-derived stromal cells recapitulate key aspects of arrhythmogenic cardiomyopathy.

Primary cardiac mesenchymal stromal cells (C-MSCs) can promote the aberrant remodeling of cardiac tissue that characterizes arrhythmogenic cardiomyopathy (ACM) by differentiating into adipocytes and myofibroblasts. These cells' limitations, including restricted access to primary material and its manipulation have been overcome by the advancement of human induced pluripotent stem cells (hiPSCs), and their ability to differentiate towards the cardiac stromal population. C-MSCs derived from hiPSCs make it possible to work with virtually unlimited numbers of cells that are genetically identical to the cells of origin. We performed in vitro experiments on primary stromal cells (Primary) and hiPSC-derived stromal cells (hiPSC-D) to compare them as tools to model ACM. Both Primary and hiPSC-D cells expressed mesenchymal surface markers and possessed typical MSC differentiation potentials. hiPSC-D expressed desmosomal genes and proteins and shared a similar transcriptomic profile with Primary cells. Furthermore, ACM hiPSC-D exhibited higher propensity to accumulate lipid droplets and collagen compared to healthy control cells, similar to their primary counterparts. Therefore, both Primary and hiPSC-D cardiac stromal cells obtained from ACM patients can be used to model aspects of the disease. The choice of the most suitable model will depend on experimental needs and on the availability of human source samples.

Nicotinic Acid Adenine Dinucleotide Phosphate Induces Intracellular Ca2+ Signalling and Stimulates Proliferation in Human Cardiac Mesenchymal Stromal Cells.

Nicotinic acid adenine dinucleotide phosphate (NAADP) is a newly discovered second messenger that gates two pore channels 1 (TPC1) and 2 (TPC2) to elicit endo-lysosomal (EL) Ca2+ release. NAADP-induced lysosomal Ca2+ release may be amplified by the endoplasmic reticulum (ER) through the Ca2+-induced Ca2+ release (CICR) mechanism. NAADP-induced intracellular Ca2+ signals were shown to modulate a growing number of functions in the cardiovascular system, but their occurrence and role in cardiac mesenchymal stromal cells (C-MSCs) is still unknown. Herein, we found that exogenous delivery of NAADP-AM induced a robust Ca2+ signal that was abolished by disrupting the lysosomal Ca2+ store with Gly-Phe ß-naphthylamide, nigericin, and bafilomycin A1, and blocking TPC1 and TPC2, that are both expressed at protein level in C-MSCs. Furthermore, NAADP-induced EL Ca2+ release resulted in the Ca2+-dependent recruitment of ER-embedded InsP3Rs and SOCE activation. Transmission electron microscopy revealed clearly visible membrane contact sites between lysosome and ER membranes, which are predicted to provide the sub-cellular framework for lysosomal Ca2+ to recruit ER-embedded InsP3Rs through CICR. NAADP-induced EL Ca2+ mobilization via EL TPC was found to trigger the intracellular Ca2+ signals whereby Fetal Bovine Serum (FBS) induces C-MSC proliferation. Furthermore, NAADP-evoked Ca2+ release was required to mediate FBS-induced extracellular signal-regulated kinase (ERK), but not Akt, phosphorylation in C-MSCs. These finding support the notion that NAADP-induced TPC activation could be targeted to boost proliferation in C-MSCs and pave the way for future studies assessing whether aberrant NAADP signaling in C-MSCs could be involved in cardiac disorders.

Pressure Overload Activates DNA-Damage Response in Cardiac Stromal Cells: A Novel Mechanism Behind Heart Failure With Preserved Ejection Fraction?

Heart failure with preserved ejection fraction (HFpEF) is a heterogeneous syndrome characterized by impaired left ventricular (LV) diastolic function, with normal LV ejection fraction. Aortic valve stenosis can cause an HFpEF-like syndrome by inducing sustained pressure overload (PO) and cardiac remodeling, as cardiomyocyte (CM) hypertrophy and fibrotic matrix deposition. Recently, in vivo studies linked PO maladaptive myocardial changes and DNA damage response (DDR) activation: DDR-persistent activation contributes to mouse CM hypertrophy and inflammation, promoting tissue remodeling, and HF. Despite the wide acknowledgment of the pivotal role of the stromal compartment in the fibrotic response to PO, the possible effects of DDR-persistent activation in cardiac stromal cell (C-MSC) are still unknown. Finally, this novel mechanism was not verified in human samples. This study aims to unravel the effects of PO-induced DDR on human C-MSC phenotypes. Human LV septum samples collected from severe aortic stenosis with HFpEF-like syndrome patients undergoing aortic valve surgery and healthy controls (HCs) were used both for histological tissue analyses and C-MSC isolation. PO-induced mechanical stimuli were simulated in vitro by cyclic unidirectional stretch. Interestingly, HFpEF tissue samples revealed DNA damage both in CM and C-MSC. DDR-activation markers γH2AX, pCHK1, and pCHK2 were expressed at higher levels in HFpEF total tissue than in HC. Primary C-MSC isolated from HFpEF and HC subjects and expanded in vitro confirmed the increased γH2AX and phosphorylated checkpoint protein expression, suggesting a persistent DDR response, in parallel with a higher expression of pro-fibrotic and pro-inflammatory factors respect to HC cells, hinting to a DDR-driven remodeling of HFpEF C-MSC. Pressure overload was simulated in vitro, and persistent activation of the CHK1 axis was induced in response to in vitro mechanical stretching, which also increased C-MSC secreted pro-inflammatory and pro-fibrotic molecules. Finally, fibrosis markers were reverted by the treatment with a CHK1/ATR pathway inhibitor, confirming a cause-effect relationship. In conclusion we demonstrated that, in severe aortic stenosis with HFpEF-like syndrome patients, PO induces DDR-persistent activation not only in CM but also in C-MSC. In C-MSC, DDR activation leads to inflammation and fibrosis, which can be prevented by specific DDR targeting.

Modeling Cardiomyopathies in a Dish: State-of-the-Art and Novel Perspectives on hiPSC-Derived Cardiomyocytes Maturation.

The stem cell technology and the induced pluripotent stem cells (iPSCs) production represent an excellent alternative tool to study cardiomyopathies, which overcome the limitations associated with primary cardiomyocytes (CMs) access and manipulation. CMs from human iPSCs (hiPSC-CMs) are genetically identical to patient primary cells of origin, with the main electrophysiological and mechanical features of CMs. The key issue to be solved is to achieve a degree of structural and functional maturity typical of adult CMs. In this perspective, we will focus on the main differences between fetal-like hiPSC-CMs and adult CMs. A viewpoint is given on the different approaches used to improve hiPSC-CMs maturity, spanning from long-term culture to complex engineered heart tissue. Further, we outline limitations and future developments needed in cardiomyopathy disease modeling.

Fibrosis in Arrhythmogenic Cardiomyopathy: The Phantom Thread in the Fibro-Adipose Tissue.

Arrhythmogenic cardiomyopathy (ACM) is an inherited heart disorder, predisposing to malignant ventricular arrhythmias leading to sudden cardiac death, particularly in young and athletic patients. Pathological features include a progressive loss of myocardium with fibrous or fibro-fatty substitution. During the last few decades, different clinical aspects of ACM have been well investigated but still little is known about the molecular mechanisms that underlie ACM pathogenesis, leading to these phenotypes. In about 50% of ACM patients, a genetic mutation, predominantly in genes that encode for desmosomal proteins, has been identified. However, the mutation-associated mechanisms, causing the observed cardiac phenotype are not always clear. Until now, the attention has been principally focused on the study of molecular mechanisms that lead to a prominent myocardium adipose substitution, an uncommon marker for a cardiac disease, thus often recognized as hallmark of ACM. Nonetheless, based on Task Force Criteria for the diagnosis of ACM, cardiomyocytes death associated with fibrous replacement of the ventricular free wall must be considered the main tissue feature in ACM patients. For this reason, it urges to investigate ACM cardiac fibrosis. In this review, we give an overview on the cellular effectors, possible triggers, and molecular mechanisms that could be responsible for the ventricular fibrotic remodeling in ACM patients.

Generation of human induced pluripotent stem cell line LUMCi027-A and its isogenic gene-corrected line from a patient affected by arrhythmogenic cardiomyopathy and carrying the c.2013delC PKP2 mutation.

Arrhythmogenic Cardiomyopathy (ACM) is a rare inherited heart muscle disease characterised by progressive fibro-fatty replacement of the ventricular myocardium leading to life-threatening arrhythmias. We generated human induced pluripotent stem cells (hiPSCs) from a patient affected by ACM and carrying the heterozygous c.2013delC (p.K672Rfs) PKP2 mutation and then corrected the mutation using CRISPR/Cas9 technology. Both hiPSC lines expressed pluripotency markers, maintained a normal karyotype, and differentiated into derivatives of the three germ layers. This isogenic hiPSC pair represents a genetically controlled system to study the role of the c.2013delC PKP2 mutation in vitro.

Clinical and Molecular Data Define a Diagnosis of Arrhythmogenic Cardiomyopathy in a Carrier of a Brugada-Syndrome-Associated PKP2 Mutation.

Plakophilin-2 (PKP2) is the most frequently mutated desmosomal gene in arrhythmogenic cardiomyopathy (ACM), a disease characterized by structural and electrical alterations predominantly affecting the right ventricular myocardium. Notably, ACM cases without overt structural alterations are frequently reported, mainly in the early phases of the disease. Recently, the PKP2 p.S183N mutation was found in a patient affected by Brugada syndrome (BS), an inherited arrhythmic channelopathy most commonly caused by sodium channel gene mutations. We here describe a case of a patient carrier of the same BS-related PKP2 p.S183N mutation but with a clear diagnosis of ACM. Specifically, we report how clinical and molecular investigations can be integrated for diagnostic purposes, distinguishing between ACM and BS, which are increasingly recognized as syndromes with clinical and genetic overlaps. This observation is fundamentally relevant in redefining the role of genetics in the approach to the arrhythmic patient, progressing beyond the concept of "one mutation, one disease", and raising concerns about the most appropriate approach to patients affected by structural/electrical cardiomyopathy. The merging of genetics, electroanatomical mapping, and tissue and cell characterization summarized in our patient seems to be the most complete diagnostic algorithm, favoring a reliable diagnosis.

Arrhythmogenic cardiomyopathy: what blood can reveal?

Blood, serum and plasma represent accessible sources of data about physiological and pathologic status. In arrhythmogenic cardiomyopathy (ACM), circulating nucleated cells are routinely used for detection of germinal genetic mutations. In addition, different biomarkers have been proposed for diagnostic purposes and for monitoring disease progression, including inflammatory cytokines, markers of myocardial dysfunction and damage, and microRNAs. This review summarizes the current information that can be retrieved from the blood of ACM patients and considers the future prospects. Improvements in current knowledge of circulating factors may provide noninvasive means to simplify and improve the diagnosis, prognosis prediction, and management of ACM patients.

Isolation and Characterization of Cardiac Mesenchymal Stromal Cells from Endomyocardial Bioptic Samples of Arrhythmogenic Cardiomyopathy Patients.

A normal adult heart is composed of several different cell types, among which cardiac mesenchymal stromal cells represent an abundant population. The isolation of these cells offers the possibility of studying their involvement in cardiac diseases, and, in addition, provides a useful primary cell model to investigate biological mechanisms. Here, the method for the isolation of C-MSC from arrhythmogenic cardiomyopathy patients' bioptic samples is described. The endomyocardial biopsy sampling is guided in the right ventricular areas adjacent to the scar visualized by electro-anatomical mapping. The digestion of the biopsies in collagenase and their plating on a plastic dish in culture medium to allow C-MSC growth is described. The isolated cells can be expanded in culture for several passages. To confirm their mesenchymal phenotype, the description of immuno-phenotypical characterization is provided. C-MSC are able to differentiate into several cell types like adipocytes, chondrocytes, and osteoblasts: in the context of ACM, characterized by adipocyte deposits in patients' hearts, the protocols for the adipogenic differentiation of C-MSC and the characterization of lipid droplet accumulation are described.

Cellular subtype expression and activation of CaMKII regulate the fate of atherosclerotic plaque.

BACKGROUND AND AIMS: Atherosclerosis is a degenerative process of the arterial wall implicating activation of macrophages and proliferation of vascular smooth muscle cells. Calcium-calmodulin dependent kinase type II (CaMKII) in vascular smooth muscle cells (VSMCs) regulates proliferation, while in macrophages, this kinase governs diapedesis, infiltration and release of extracellular matrix enzymes. We aimed at understanding the possible role of CaMKII in atherosclerosis plaques to regulate plaque evolution towards stability or instability. METHODS: Clinically defined stable and unstable plaques obtained from patients undergoing carotid end arteriectomy were processed for evaluation of CaMKs protein expression, activity and localization. RESULTS: The larger content of CaMKII was found in CD14+myeloid cells that were more abundant in unstable rather than stable plaques. To test the biological effect of activated CD14+myeloid cells, VSMCs were exposed to the conditioned medium (CM) of macrophages extracted from carotid plaques. CM induced attenuation of CaMKs expression and activity in VSMCs, leading to the reduction of VSMCs proliferation. This appears to be due to the CaMKII dependent release of cytokines. CONCLUSIONS: These results indicate a pivotal role of CaMKs in atherosclerosis by regulating activated myeloid cells on VSMCs activity. CaMKII could represent a possible target for therapeutic strategies based on macrophages specific inhibition for the stabilization of arteriosclerotic lesions.

Oxidative Stress Mediates the Antiproliferative Effects of Nelfinavir in Breast Cancer Cells.

The discovery of the anti-proliferative activity of nelfinavir in HIV-free models has encouraged its investigation as anticancer drug. Although the molecular mechanism by which nelfinavir exerts antitumor activity is still unknown, its effects have been related to Akt inhibition. Here we tested the effects of nelfinavir on cell proliferation, viability and death in two human breast cancer cell lines and in human normal primary breast cells. To identify the mechanism of action of nelfinavir in breast cancer, we evaluated the involvement of the Akt pathway as well as the effects of nelfinavir on reactive oxygen species (ROS) production and ROS-related enzymes activities. Nelfinavir reduced breast cancer cell viability by inducing apoptosis and necrosis, without affecting primary normal breast cells. The antitumor activity of nelfinavir was related to alterations of the cell redox state, coupled with an increase of intracellular ROS production limited to cancer cells. Nelfinavir treated tumor cells also displayed a downregulation of the Akt pathway due to disruption of the Akt-HSP90 complex, and subsequent degradation of Akt. These effects resulted to be ROS dependent, suggesting that ROS production is the primary step of nelfinavir anticancer activity. The analysis of ROS-producers and ROS-detoxifying enzymes revealed that nelfinavir-mediated ROS production was strictly linked to flavoenzymes activation. We demonstrated that ROS enhancement represents the main molecular mechanism required to induce cell death by nelfinavir in breast cancer cells, thus supporting the development of new and more potent oxidizing molecules for breast cancer therapy.

Metabolic Syndrome and Aging: Calcium Signaling as Common Regulator.

Aging is one of the most important societal challenges that western societies face, as a result of longer life expectancy and reduced natality rates. Aging is a success story of our health and social systems, but raises sustainability issues that are linked to the increased need for services of older adults, due to the reduction of their independence and to the co-existence of multiple chronic diseases. The metabolic syndrome can be considered an age-related disease, since its prevalence increases with age. Current demographic trends in the population highlight aging-related dysfunctions that contribute to the onset of several metabolic diseases, and the need for innovative, effective and sustainable approaches. This review describes the correlation between the metabolic syndrome and aging, and the underlying common molecular mechanisms, focusing on calcium signaling and its crosstalks.