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.

Liraglutide preserves CD34+ stem cells from dysfunction Induced by high glucose exposure.

BACKGROUND: Glucagon like peptide-1 receptor agonists (GLP-1RAs) have shown to reduce mortality and cardiovascular events in patients with type 2 diabetes mellitus (T2DM). Since the impairment in number and function of vasculotrophic circulating CD34+ hematopoietic stem progenitor cells (HSPCs) in T2D has been reported to increase cardiovascular (CV) risk, we hypothesized that one of the mechanisms whereby GLP-1 RAs exert CV protective effects may be related to the ability to improve CD34+ HSPC function. METHODS: In cord blood (CB)-derived CD34+ HSPC, the expression of GLP-1 receptor (GLP-1R) mRNA, receptor protein and intracellular signaling was evaluated by RT-qPCR and Western Blot respectively. CD34+ HSPCs were exposed to high glucose (HG) condition and GLP-1RA liraglutide (LIRA) was added before as well as after functional impairment. Proliferation, CXCR4/SDF-1α axis activity and intracellular ROS production of CD34+ HSPC were evaluated. RESULTS: CD34+ HSPCs express GLP-1R at transcriptional and protein level. LIRA treatment prevented and rescued HSPC proliferation, CXCR4/SDF-1α axis activity and metabolic imbalance from HG-induced impairment. LIRA stimulation promoted intracellular cAMP accumulation as well as ERK1/2 and AKT signaling activation. The selective GLP-1R antagonist exendin (9-39) abrogated LIRA-dependent ERK1/2 and AKT phosphorylation along with the related protective effects. CONCLUSION: We provided the first evidence that CD34+ HSPC express GLP-1R and that LIRA can favorably impact on cell dysfunction due to HG exposure. These findings open new perspectives on the favorable CV effects of GLP-1 RAs in T2DM patients.

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.

Cyclophilin A inhibition as potential treatment of human aortic valve calcification.

Aortic valve stenosis (AS) is a pathological condition that affects about 3% of the population, representing the most common valve disease. The main clinical feature of AS is represented by the impaired leaflet motility, due to calcification, which leads to the left ventricular outflow tract obstruction during systole. The formation and accumulation of calcium nodules are driven by valve interstitial cells (VICs). Unfortunately, to date, the in vitro and in vivo studies were not sufficient to fully recapitulate all the pathological pathways involved in AS development, as well as to define a specific and effective pharmacological treatment for AS patients. Cyclophilin A (CyPA), the most important immunophilin and endogenous ligand of cyclosporine A (CsA), is strongly involved in several detrimental cardiovascular processes, such as calcification. To date, there are no data on the CyPA role in VIC-mediated calcification process of AS. Here, we aimed to identify the role of CyPA in AS by studying VIC calcification, in vitro. In this study, we found that (i) CyPA is up-regulated in stenotic valves of AS patients, (ii) pro-calcifying medium promotes CyPA secretion by VICs, (iii) in vitro treatment of VICs with exogenous CyPA strongly stimulates calcium deposition, and (iv) exogenous CyPA inhibition mediated by CsA analogue MM284 abolished in vitro calcium potential. Thus, CyPA represents a biological target that may act as a novel candidate in the detrimental AS development and its inhibition may provide a novel pharmacological approach for AS treatment.

MiRNA profiling revealed enhanced susceptibility to oxidative stress of endothelial cells from bicuspid aortic valve.

AIMS: Calcific aortic valve stenosis (CAVS) is the most frequent manifestation of aortic valve disease and the third leading cause of cardiovascular disease in the Western countries associated with significant morbidity and mortality. An active biological progression involving inflammation and oxidation leading to valve endothelial damage is considered a hallmark of the early stages of valve degeneration. However, tricuspid (TAV) and bicuspid (BAV) aortic valve deterioration are considered to differ only by shear stress. We hypothesized that endothelial cells (EC) derived from BAV and TAV patients have different miRNA expression patterns and thus distinct pathways could lead to endothelial damage in BAV than TAV patients. METHODS AND RESULTS: We isolated ECs from patients with bicuspid or tricuspid aortic valve, which underwent surgery due to CAVS. MiRNA expression profile by PCR revealed eight upregulated miRNAs between BAV and TAV ECs. Functional analysis identified that BAV ECs presented altered cellular response to oxidative stress and DNA damage stimulus via p53 and alteration in the intrinsic apoptotic signaling pathway. GPX3 and SRXN1 mRNA were express at lower levels in BAV compared to TAV ECs, leading to an increment of DNA double-strand breaks. BAV ECs had a sustained apoptosis activation when compared to TAV ECs. This difference was exacerbated by oxidative stress stimulus leading to a reduced survival rate but completely reverted by miR-328-3p inhibition. CONCLUSION: The present data showed molecular differences in oxidative stress susceptibility, DNA damage magnitude, and apoptosis induction between ECs derived from BAV and TAV patients.

Cyclophilin A in Arrhythmogenic Cardiomyopathy Cardiac Remodeling.

Arrhythmogenic cardiomyopathy (ACM) is a genetic disorder characterized by the progressive substitution of functional myocardium with noncontractile fibro-fatty tissue contributing to ventricular arrhythmias and sudden cardiac death. Cyclophilin A (CyPA) is a ubiquitous protein involved in several pathological mechanisms, which also characterize ACM (i.e., fibrosis, inflammation, and adipogenesis). Nevertheless, the involvement of CyPA in ACM cardiac remodeling has not been investigated yet. Thus, we first evaluated CyPA expression levels in the right ventricle (RV) tissue specimens obtained from ACM patients and healthy controls (HC) by immunohistochemistry. Then, we took advantage of ACM- and HC-derived cardiac mesenchymal stromal cells (C-MSC) to assess CyPA modulation during adipogenic differentiation. Interestingly, CyPA was more expressed in the RV sections obtained from ACM vs. HC subjects and positively correlated with the adipose replacement extent. Moreover, CyPA was upregulated at early stages of C-MSC adipogenic differentiation and was secreted at higher level over time in ACM- derived C-MSC. Our study provides novel ex vivo and in vitro information on CyPA expression in ACM remodeling paving the way for future C-MSC-based mechanistic and therapeutic investigations.

Integrin ανß5 in vitro inhibition limits pro-fibrotic response in cardiac fibroblasts of spontaneously hypertensive rats.

BACKGROUND: To date the TGF-ß1 activation mediated by integrin ανß5 during fibrosis is well-known. This process has been shown also in the heart, where cardiac fibroblasts (CF) differentiate into α-smooth muscle actin (α-SMA)-positive myofibroblasts (MyoFB). Here, we studied the effects on CF, isolated by spontaneously hypertensive rats (SHR), of integrin ανß5 inhibition in MyoFB differentiation. METHODS: Staining and immunohistochemistry were performed on rat cardiac tissue. CF were isolated by enzymatic digestion from SHR (SHR-CF) and normotensive WKY (WKY-CF) rat hearts and then treated for in vitro evaluation. RESULTS: SHR heart tissues revealed a higher TGF-ß1 expression vs. WKY samples. SHR-CF showed an enhanced SMAD2/3 activation and an up-regulated expression of α-SMA, a typical MyoFB marker, especially after TGF-ß1 treatment. Immunostaining on cardiac tissues revealed a higher expression of integrin ανß5 in SHR vs. WKY rat hearts. In vitro results confirmed the up-regulation of integrin ανß5 expression in SHR-CF at basal condition and after TGF-ß1 treatment, in comparison with WKY-CF. Inhibition of integrin ανß5 by cilengitide treatment led a decreased expression of ανß5, collagen I, and α-SMA in SHR-CF vs. WKY-CF, resulting in a diminished differentiation of CF into MyoFB. Taking together, results suggested that SHR-CF are more susceptible to TGF-ß1, showing an up-regulated activation of SMAD2/3 signaling, and an increased ανß5, α-SMA, and collagen I expression. Hypertension stimulus promoted an up-regulation of integrin ανß5 on SHR cardiac tissue and its in vitro inhibition reverted pro-fibrotic events of SHR-CF. CONCLUSION: Inhibition of integrin ανß5 exerted by cilengitide strongly diminished SHR-CF differentiation into detrimental MyoFB. So, integrin ανß5 might be considered a novel therapeutic target and cilengitide an effective pharmacological tool to limit the progression of hypertension-induced cardiac fibrosis.

Vascular smooth muscle cells in Marfan syndrome aneurysm: the broken bricks in the aortic wall.

Marfan syndrome (MFS) is a connective tissue disorder with multiple organ manifestations. The genetic cause of this syndrome is the mutation of the FBN1 gene, encoding the extracellular matrix (ECM) protein fibrillin-1. This genetic alteration leads to the degeneration of microfibril structures and ECM integrity in the tunica media of the aorta. Indeed, thoracic aortic aneurysm and dissection represent the leading cause of death in MFS patients. To date, the most effective treatment option for this pathology is the surgical substitution of the damaged aorta. To highlight novel therapeutic targets, we review the molecular mechanisms related to MFS etiology in vascular smooth muscle cells, the foremost cellular type involved in MFS pathogenesis.

c-kit(+) cells: the tell-tale heart of cardiac regeneration?

Cardiovascular disease is the leading cause of morbidity and mortality in the developed world. Although ongoing therapeutic strategies ameliorate symptoms and prolong life for patients with cardiovascular diseases, they do not solve the critical issue related to the loss of cardiac tissue. Accordingly, stem/progenitor cell therapy has emerged as a paramount approach for cardiac repair and regeneration. In this regard, c-kit(+) cells have animated much interest and controversy. These cells are self-renewing, clonogenic, and multipotent and display a noteworthy potential to differentiate into all cardiovascular lineages. However, their functional contribution to cardiomyocyte turnover is one of the centrally debated issues concerning their regenerative potential. Regardless, plentiful preclinical and clinical studies have been conducted which provide evidence for the capacity of c-kit(+) cells to improve cardiac function. The purpose of this review is to give a comprehensive, impartial, critical description and evaluation of the literature on c-kit(+) cells from bench to bedside in order to address their true potential, benefits and controversies.

Assessing cytokines' talking patterns following experimental myocardial damage by applying Shannon's information theory.

BACKGROUND: The simultaneous measurement of multiple cytokines in parallel by using multiplex proteome arrays (MPA) is of great interest to understanding the inflammatory response following myocardial infarction; however, since cytokines are pleiotropic and redundant, increase of information throughput (IT) attained by measuring multiple cytokines remain to be determined. We aimed this study to assess the IT of an MPA system designed to assess 8 cytokines - commercially available at the time of the study - serum levels, before (control state) and after experimental myocardial cryoinjury (activated state) in rats. METHODS: By assuming that redundant information do not generally increase the IT, we derived Entropy (H) and Redundancy (R) of information by using formulas of Shannon modified accordingly, where a high IT (high H and low R) corresponds to a low level of correlation between cytokines and vice versa for a low IT. The maximum theoretical level of IT and the contribution of each cytokine were also estimated. RESULTS: In control state, no significant correlations were found between cytokines showing high IT; on the contrary, in activated state, several significant correlations were found supporting a complex cross-talk pattern between cytokines with low IT. Using as reference the maximum theoretical level of IT, in activated state, H was reduced of 67.0% and R was increased of 77.4% supporting a reduction of IT. Furthermore, the contribution of individual cytokines to H value of MPA was variable: in control state, IL-2 gave the most contribution to H value, conversely during activated state IL-10 gave most contribution. Finally during activated state, IL-1ß was the only cytokine strongly correlated with values of all other cytokines, suggesting a crucial role in the inflammatory cascade. CONCLUSIONS: Paradoxically, by analyzing an MPA system designed for redundant analytes such as cytokines, translating the Shannon's information theory from the field of communication to biology, the IT system in our model deteriorates during the activation state by increasing its redundancy, showing maximum value of entropy in the control conditions. Finally, the study of the mutual interdependence between cytokines by the contribution to the IT may allow formulating alternative models to describe the inflammatory cascade after myocardial infarction.

Exploiting the anti-fibrotic effects of statins on thoracic aortic aneurysm progression: results from a meta-analysis and experimental data.

Aims: Thoracic aortic aneurysm (TAA) that progress to acute aortic dissection is often fatal and there is no pharmacological treatment that can reduce TAA progression. We aim to evaluate statins' effects on TAA growth rate and outcomes using a meta-analysis approach. Methods and results: A detailed search related to the effects of statins on TAA was conducted according to PRISMA guidelines. The analyses of statins' effects on TAA growth rate were performed on 4 studies (n = 1850), while the impact on outcomes was evaluated on 3 studies (n = 2,867). Patients under statin treatment showed a reduced TAA growth rate (difference in means = -0.36 cm/year; 95%CI: -0.64, -0.08; p = 0.013) when compared to controls, patients not taking statins. Regarding the outcomes (death, dissection, or rupture of the aorta, and the need for operative repair), statins exhibited a protective effect reducing the number of events (log odds ratio = -0.56; 95%CI: -1.06, -0.05; p = 0.030). In vitro, the anti-fibrotic effect of atorvastatin was tested on vascular smooth muscle cells (VMSC) isolated from patients with TAA. Our results highlighted that, in transforming growth factor beta 1 (TGF-ß1) pro-fibrotic condition, VSMC expressed a significant lower amount of collagen type I alpha 1 chain (COL1A1) when treated with atorvastatin (untreated = +2.66 ± 0.23 fold-change vs. treated = +1.63 ± 0.09 fold-change; p = 0.014). Conclusion: Statins show a protective effect on TAA growth rate and adverse outcomes in patients with TAA, possibly via their anti-fibrotic properties on VSMC. Given the current lack of effective drug treatments for TAA, we believe our findings highlight the need for more in-depth research to explore the potential benefits of statins in this context.

Extracellular matrix proteins and displacement of cultured fibroblasts from duodenal biopsies in celiac patients and controls.

BACKGROUND: Celiac disease (CD) is mainly characterised by villous atrophy and mucosal architectural rearrangement. The fibroblasts (FBs) are the most abundant mesenchymal cell type in the intestinal mucosa and are responsible for both the architectural arrangement of the villi and the formation of the extracellular matrix (ECM). This study aimed at the evaluation of both the intracellular distribution of different proteins involved in ECM and FBs characterisation, and the cellular displacement of primary FBs obtained from duodenal endoscopic biopsies of healthy subjects and celiac patients. METHODS: Primary healthy and celiac duodenal FBs were evaluated by means of immuno-fluorescence assay for collagen type I and IV, fibronectin, actin, alpha-Smooth Muscle Actin (alpha-SMA), Fibroblast Surface Protein (FSP) and transglutaminase type 2 (TG2). The geometric indexes of the fluorescence signals were investigated by image analysis software (Image J, NIH). Both morphology and kinetic were evaluated during a 72 hours time course movie. TG2 medium activity was evaluated by means of ELISA. RESULTS: All the cells examined were immunopositive for FSP, alpha-SMA, actin, collagen I, collagen IV and TG2. CD cells showed a signet collagen-I and collagen-IV pattern, as compared to the controls being characterised by a spindle geometry. Moreover, the collagen signals in CD FBs showed a significantly higher circularity index (major orthogonal diameter ratio) than the controls (p<0.0001), whereas the perimeter and area ratio were significantly lower (p<0.0001). The TG2 signal had a decreased area (p<0.05), but a two-fold increased medium activity. The time course highlighted a reduction of the displacement of CD FBs. CONCLUSIONS: The isolated primary CD FBs showed a different collagen and TG2 pattern of distribution associated with a different cellular displacement. The reasons for such CD cell peculiar characteristics are yet unknown but they might represent a factor in the progression of the intestinal damage.

Multi-omics in thoracic aortic aneurysm: the complex road to the simplification.

BACKGROUND: Thoracic aortic aneurysm (TAA) is a serious condition that affects the aorta, characterized by the dilation of its first segment. The causes of TAA (e.g., age, hypertension, genetic syndromes) are heterogeneous and contribute to the weakening of the aortic wall. This complexity makes treating this life-threatening aortopathy challenging, as there are currently no etiological therapy available, and pharmacological strategies, aimed at avoiding surgical aortic replacement, are merely palliative. Recent studies on novel therapies for TAA have focused on identifying biological targets and etiological mechanisms of the disease by using advanced -omics techniques, including epigenomics, transcriptomics, proteomics, and metabolomics approaches. METHODS: This review presents the latest findings from -omics approaches and underscores the importance of integrating multi-omics data to gain more comprehensive understanding of TAA. RESULTS: Literature suggests that the alterations in TAA mediators frequently involve members of pro-fibrotic process (i.e., TGF-ß signaling pathways) or proteins associated with cell/extracellular structures (e.g., aggrecans). Further analyses often reported the importance in TAA of processes as inflammation (PCR, CD3, leukotriene compounds), oxidative stress (chromatin OXPHOS, fatty acids), mitochondrial respiration and glycolysis/gluconeogenesis (e.g., PPARs and HIF1a). Of note, more recent metabolomics studies added novel molecular markers to the list of TAA-specific detrimental mediators (proteoglycans). CONCLUSION: It is increasingly clear that integrating data from different -omics branches, along with clinical data, is essential as well as complicated both to reveal hidden relevant information and to address complex diseases such as TAA. Importantly, recent progresses in metabolomics highlighted novel potential and unprecedented marks in TAA diagnosis and therapy.

Marfan Syndrome: Enhanced Diagnostic Tools and Follow-up Management Strategies.

Marfan syndrome (MFS) is a rare inherited autosomic disorder, which encompasses a variety of systemic manifestations caused by mutations in the Fibrillin-1 encoding gene (FBN1). Cardinal clinical phenotypes of MFS are highly variable in terms of severity, and commonly involve cardiovascular, ocular, and musculoskeletal systems with a wide range of manifestations, such as ascending aorta aneurysms and dissection, mitral valve prolapse, ectopia lentis and long bone overgrowth, respectively. Of note, an accurate and prompt diagnosis is pivotal in order to provide the best treatment to the patients as early as possible. To date, the diagnosis of the syndrome has relied upon a systemic score calculation as well as DNA mutation identification. The aim of this review is to summarize the latest MFS evidence regarding the definition, differences and similarities with other connective tissue pathologies with severe systemic phenotypes (e.g., Autosomal dominant Weill-Marchesani syndrome, Loeys-Dietz syndrome, Ehlers-Danlos syndrome) and clinical assessment. In this regard, the management of MFS requires a multidisciplinary team in order to accurately control the evolution of the most severe and potentially life-threatening complications. Based on recent findings in the literature and our clinical experience, we propose a multidisciplinary approach involving specialists in different clinical fields (i.e., cardiologists, surgeons, ophthalmologists, orthopedics, pneumologists, neurologists, endocrinologists, geneticists, and psychologists) to comprehensively characterize, treat, and manage MFS patients with a personalized medicine approach.

Presence of SARS-CoV-2 Nucleoprotein in Cardiac Tissues of Donors with Negative COVID-19 Molecular Tests.

The 2019 Coronavirus disease (COVID-19) outbreak had detrimental effects on essential medical services such as organ and tissue donation. Lombardy, one of the most active Italian regions in organ/tissue procurement, has been strongly affected by the COVID-19 pandemic. To date, data concerning the risk of SARS-CoV-2 transmission after tissue transplantation are controversial. Here, we aimed to evaluate the presence/absence of SARS-CoV-2 in different cardiac tissues eligible for transplantation obtained from Lombard donors. We used cardiovascular tissues from eight donors potentially suitable for pulmonary valve transplantation. All donor subjects involved in the study returned negative results for the SARS-CoV-2 RNA molecular tests (quantitative real-time reverse-transcription PCR, qRT-PCR, and chip-based digital PCR) in nasopharyngeal swabs (NPS) or bronchoalveolar lavage (BAL). None of the eight donors included in this study revealed the presence of the SARS-CoV-2 viral genome. However, evaluation of the protein content of pulmonary vein wall (PVW) tissue revealed variable levels of SARS-CoV-2 nucleoprotein signal in all donors. Our study demonstrated for the first time, to the best of our knowledge, that viral nucleoprotein but not viral RNA was present in the examined tissue bank specimens, suggesting the need for caution and in-depth investigations on implantable tissue specimens collected during the COVID-19 pandemic period.

Evidence of SARS-CoV-2 Transcriptional Activity in Cardiomyocytes of COVID-19 Patients without Clinical Signs of Cardiac Involvement.

AIMS: A considerable proportion of patients affected by coronavirus respiratory disease (COVID-19) develop cardiac injury. The viral impact in cardiomyocytes deserves, however, further investigations, especially in asymptomatic patients. METHODS: We investigated for SARS-CoV-2 presence and activity in heart tissues of six consecutive COVID-19 patients deceased from respiratory failure showing no signs of cardiac involvement and with no history of heart disease. Cardiac autopsy samples were collected within 2 h after death, and then analysed by digital PCR, Western blot, immunohistochemistry, immunofluorescence, RNAScope, and transmission electron microscopy assays. RESULTS: The presence of SARS-CoV-2 into cardiomyocytes was invariably detected in all assays. A variable pattern of cardiomyocyte injury was observed, spanning from absence of cell death and subcellular alterations hallmarks, to intracellular oedema and sarcomere ruptures. In addition, we found active viral transcription in cardiomyocytes, by detecting both sense and antisense SARS-CoV-2 spike RNA. CONCLUSIONS: In this autopsy analysis of patients with no clinical signs of cardiac involvement, the presence of SARS-CoV-2 in cardiomyocytes has been detected, determining variable patterns of intracellular damage. These findings suggest the need for cardiologic surveillance in surviving COVID-19 patients not displaying a cardiac phenotype.

Relationship Between Plasma Osteopontin and Arginine Pathway Metabolites in Patients With Overt Coronary Artery Disease.

INTRODUCTION: Osteopontin (OPN) is involved in ectopic calcification. Its circulating form is upregulated in coronary artery disease (CAD) patients. Circulating OPN levels positively correlate with oxidative stress, one of the major triggers of endothelial dysfunction. Endothelial dysfunction is, in turn, associated with reduced nitric oxide (NO) bioavailability due to the impaired arginine pathway. The aim of this study was to better understand the correlations between OPN, oxidative stress markers, and the arginine pathway metabolites. METHODS AND RESULTS: ELISA and mass spectrometry techniques have been used to evaluate circulating OPN and arginine pathway/oxidative stress metabolites, respectively, in twenty-five control subjects and thirty-three patients with overt atherosclerosis. OPN positively correlates with 2,3-dinor-8isoPGF2a levels (p = 0.02), ornithine (p = 0.01), ADMA (p = 0.001), SDMA (p = 0.03), and citrulline (p = 0.008) levels only in CAD patients. In addition, citrulline positively correlated with ADMA (p = 0.02) levels, possibly as result of other sources of citrulline biosynthetic pathways. CONCLUSION: The association between OPN and impaired arginine/NO pathway could play a role in the inhibition of endothelial NO synthase (eNOS) and/or in the arginase activation in the context of CAD patients. However, further studies are needed to verify the cause-effect relationship between OPN, oxidative stress, and arginine/NO pathway dysregulation.

Cyclophilin A/EMMPRIN Axis Is Involved in Pro-Fibrotic Processes Associated with Thoracic Aortic Aneurysm of Marfan Syndrome Patients.

: Background: Marfan syndrome (MFS) is a genetic disease, characterized by thoracic aortic aneurysm (TAA), which treatment is to date purely surgical. Understanding of novel molecular targets is mandatory to unveil effective pharmacological approaches. Cyclophilin A (CyPA) and its receptor EMMPRIN are associated with several cardiovascular diseases, including abdominal aortic aneurysm. Here, we envisioned the contribution of CyPA/EMMPRIN axis in MFS-related TAA. METHODS: We obtained thoracic aortic samples from healthy controls (HC) and MFS patients' aortas and then isolated vascular smooth muscle cells (VSMC) from the aortic wall. RESULTS: our findings revealed that MFS aortic tissue samples isolated from the dilated zone of aorta showed higher expression levels of EMMPRIN vs. MFS non-dilated aorta and HC. Interestingly, angiotensin II significantly stimulated CyPA secretion in MFS-derived VSMC (MFS-VSMC). CyPA treatment on MFS-VSMC led to increased levels of EMMPRIN and other MFS-associated pro-fibrotic mediators, such as TGF-ß1 and collagen I. These molecules were downregulated by in vitro treatment with CyPA inhibitor MM284. Our results suggest that CyPA/EMMPRIN axis is involved in MFS-related TAA development, since EMMPRIN is upregulated in the dilated zone of MFS patients' TAA and the inhibition of its ligand, CyPA, downregulated EMMPRIN and MFS-related markers in MFS-VSMC. CONCLUSIONS: these insights suggest both a novel detrimental role for CyPA/EMMPRIN axis and its inhibition as a potential therapeutic strategy for MFS-related TAA treatment.

Unchain My Heart: Integrins at the Basis of iPSC Cardiomyocyte Differentiation.

The cellular response to the extracellular matrix (ECM) microenvironment mediated by integrin adhesion is of fundamental importance, in both developmental and pathological processes. In particular, mechanotransduction is of growing importance in groundbreaking cellular models such as induced pluripotent stem cells (iPSC), since this process may strongly influence cell fate and, thus, augment the precision of differentiation into specific cell types, e.g., cardiomyocytes. The decryption of the cellular machinery starting from ECM sensing to iPSC differentiation calls for new in vitro methods. Conveniently, engineered biomaterials activating controlled integrin-mediated responses through chemical, physical, and geometrical designs are key to resolving this issue and could foster clinical translation of optimized iPSC-based technology. This review introduces the main integrin-dependent mechanisms and signalling pathways involved in mechanotransduction. Special consideration is given to the integrin-iPSC linkage signalling chain in the cardiovascular field, focusing on biomaterial-based in vitro models to evaluate the relevance of this process in iPSC differentiation into cardiomyocytes.

Soluble EMMPRIN levels discriminate aortic ectasia in Marfan syndrome patients.

Marfan syndrome (MFS) is a rare genetic disease characterized by a matrix metalloproteases (MMPs) dysregulation that leads to extracellular matrix degradation. Consequently, MFS patients are prone to develop progressive thoracic aortic enlargement and detrimental aneurysm. Since MMPs are activated by the extracellular MMP inducer (EMMPRIN) protein, we determined whether its plasmatic soluble form (sEMMPRIN) may be considered a marker of thoracic aortic ectasia (AE). Methods: We compared plasma sEMMPRIN levels of 42 adult Caucasian MFS patients not previously subjected to aortic surgery with those of matched healthy controls (HC) by ELISA. In the MFS cohort we prospectively evaluated the relationship between plasma sEMMPRIN levels and the main MFS-related manifestations. Results: MFS patients had lower plasma sEMMPRIN levels (mean±SD: 2071±637 pg/ml) than HC (2441±642 pg/ml, p=0.009). Amongst all considered MFS-related clinical features, we found that only aortic root dilatation associated with circulating sEMMPRIN levels. Specifically, plasma sEMMPRIN levels negatively correlated with aortic Z-score (r=-0.431, p=0.004), and were significantly lower in patients with AE (Z-score≥2, 1788±510 pg/ml) compared to those without AE (Z-score<2, 2355±634 pg/ml; p=0.003). ROC curve analysis revealed that plasma sEMMPRIN levels discriminated patients with AE (AUC [95%CI]: 0.763 [0.610-0.916], p=0.003) with 85.7% sensitivity, 76.2% specificity, and 81% accuracy. We defined plasma sEMMPRIN levels ≤2246 pg/ml as the best threshold discriminating the presence of AE in MFS patients with an odds ratio [95%CI] of 19.2 [3.947-93.389] (p<0.001). Conclusions: MFS patients are characterized by lower sEMMPRIN levels than HC. Notably, plasma sEMMPRIN levels are strongly associated with thoracic AE.