Risk Stratification in Bicuspid Aortic Valve Aortopathy: Emerging Evidence and Future Perspectives.

The current management of aortic dilatation associated with congenital bicuspid aortic valve (bicuspid aortic valve aortopathy) is based on dimensional parameters (diameter of the aneurysm, growth of the diameter over time) and few other criteria. The disease is however heterogeneous in terms of natural and clinical history and risk of acute complications, ie aortic dissection. Dimensional criteria are now admitted to have limited value as predictors of such complications. Thus, novel principles for risk stratification have been recently investigated, including phenotypic criteria, flow-related metrics, and circulating biomarkers. A systematization of the typical anatomoclinical forms that the aortopathy can assume has led to the identification of the more severe root phenotype, associated with higher risk of progression of the aneurysm and possible higher aortic dissection risk. Four-dimensional-flow magnetic resonance imaging studies are searching for potentially clinically significant metrics of flow derangement, based on the recognized association of local abnormal shear stress with wall pathology. Other research initiatives are addressing the question whether circulating molecules could predict the presence or, more importantly, the future development of aortopathy. The present review summarizes the latest progresses in the knowledge on risk stratification of bicuspid aortic valve aortopathy, focusing on critical aspects and debated points.

Author Correction: Pro-inflammatory cytokines activate hypoxia-inducible factor 3α via epigenetic changes in mesenchymal stromal/stem cells.

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

Is there a role for autophagy in ascending aortopathy associated with tricuspid or bicuspid aortic valve?

Autophagy is a conserved process by which cytoplasmatic elements are sequestered in vesicles and degraded after their fusion with lysosomes, thus recycling the precursor molecules. The autophagy-mediated removal of redundant/harmful/damaged organelles and biomolecules plays not only a replenishing function, but protects against stressful conditions through an adaptive mechanism. Autophagy, known to play a role in several pathological conditions, is now gaining increasing attention also in the perspective of the identification of the pathogenetic mechanisms at the basis of ascending thoracic aortic aneurysm (TAA), a localized or diffused dilatation of the aorta with an abnormal widening greater than 50 percent of the vessel's normal diameter. TAA is less frequent than abdominal aortic aneurysm (AAA), but is encountered with a higher percentage in patients with congenital heart disease or known genetic syndromes. Several biological aspects of TAA pathophysiology remain to be elucitated and therapeutic needs are still widely unmet. One of the most controversial and epidemiologically important forms of TAA is that associated with the congenital bicuspid malformation of the aortic valve (BAV). Dysregulated autophagy in response, for example, to wall shear stress alterations, has been demonstrated to affect the phenotype of vascular cells relevant to aortopathy, with potential consequences on signaling, remodeling, and angiogenesis. The most recent findings and hypotheses concerning the multiple aspects of autophagy and of its dysregulation are summarized, both in general and in the context of the different vascular cell types and of TAA progression, with particular reference to BAV-related aortopathy.

Locally different proteome in aortas from patients with stenotic tricuspid and bicuspid aortic valves†.

OBJECTIVES: We aimed to compare the intracellular proteome of ascending aortas from patients with stenotic bicuspid (BAV) and tricuspid aortic valves (TAV) to identify BAV-specific pathogenetic mechanisms of aortopathy and to verify the previously reported asymmetric expression of BAV aortopathy [concentrated at the convexity (CVX)] in its 'ascending phenotype' form. METHODS: Samples were collected from the CVX and concavity sides of non-aneurysmal ascending aortas in 26 TAV and 26 BAV patients undergoing stenotic aortic valve replacement. Aortic lysates were subjected to cellular protein enrichment by subfractionation, and to proteome comparison by 2-dimensional fluorescence difference in-gel electrophoresis. Differentially regulated protein spots were identified by liquid chromatography-tandem mass spectrometry and analysed in silico. Selected results were verified by immunofluorescence and reverse transcription-polymerase chain reaction. RESULTS: In BAV samples, 52 protein spots were differentially regulated versus TAV samples at the CVX and 10 spots at the concavity: liquid chromatography-tandem mass spectrometry identified 35 and 10 differentially regulated proteins, respectively. Charge trains of individual proteins (e.g. annexins) suggested the presence of post-translational modifications possibly modulating their activity. At the CVX, 37 of the 52 different protein spots showed decreased expression in BAV versus TAV. The affected biological pathways included those involved in smooth muscle cell contractile phenotype, metabolism and cell stress. CONCLUSIONS: The observed differential proteomics profiles may have a significant impact on the pathogenesis of the aortopathy, pointing the way for further studies. At a preaneurysmal stage, an aorta with BAV shows more protein expression changes and potentially more post-translational modifications at the CVX of the ascending aorta than at the concavity, compared to that of TAV.

Ascending aortas from heart donors and CABG patients are not equivalent as control in aortopathy studies.

OBJECTIVES: A careful selection of reference samples in studies on the pathogenesis of thoracic ascending aorta (TAA) dilation is crucial for reliability, consistency and reproducibility of experimental results. Several studies include control TAA samples from heart donors. Others include samples harvested during coronary artery bypass graft (CABG) procedures or a mix of samples from heart donors and CABG patients. We verified the equivalence/homogeneity of TAA samples from heart donors and CABG patients in terms of basal gene expression and thus their reliability as reference groups in aortopathy studies. DESIGN: We analysed by RT-PCR and Western blot the differential expression of smoothelin, α-smooth muscle actin (α-SMA) and transforming growth factor-ß1 (TGF-ß1), selected as major players in smooth muscle cell and myofibroblast phenotype and remodelling. The mean age and comorbidities of subjects were consistent with data routinely seen in clinical practice. RESULTS: Data revealed the loss of smoothelin in samples from CABG patients, together with a significant increase of α-SMA, while TGF-ß1 dimer showed a marked increase in CABG patients versus heart donors, accompanied by a decrease of the corresponding mRNA. Differences in gene expression were maintained after adjustment for age. However, TGF-ß1 mRNA and CABG patients' age showed a positive correlation (ρ = 0.89, p < .05), while α-SMA mRNA and age showed a negative correlation (ρ = -0.85, p < .05). CONCLUSIONS: We revealed the non-equivalence of samples from heart donors and CABG patients, presumably for the presence of microscopic atherosclerotic lesions in CABG patients, suggesting the necessity of a careful selection of control groups in aortopathy studies.

Pro-inflammatory cytokines activate hypoxia-inducible factor 3α via epigenetic changes in mesenchymal stromal/stem cells.

Human mesenchymal stromal/stem cells (hMSCs) emerged as a promising therapeutic tool for ischemic disorders, due to their ability to regenerate damaged tissues, promote angiogenesis and reduce inflammation, leading to encouraging, but still limited results. The outcomes in clinical trials exploring hMSC therapy are influenced by low cell retention and survival in affected tissues, partially influenced by lesion's microenvironment, where low oxygen conditions (i.e. hypoxia) and inflammation coexist. Hypoxia and inflammation are pathophysiological stresses, sharing common activators, such as hypoxia-inducible factors (HIFs) and NF-κB. HIF1α and HIF2α respond essentially to hypoxia, activating pathways involved in tissue repair. Little is known about the regulation of HIF3α. Here we investigated the role of HIF3α in vitro and in vivo. Human MSCs expressed HIF3α, differentially regulated by pro-inflammatory cytokines in an oxygen-independent manner, a novel and still uncharacterized mechanism, where NF-κB is critical for its expression. We investigated if epigenetic modifications are involved in HIF3α expression by methylation-specific PCR and histone modifications. Robust hypermethylation of histone H3 was observed across HIF3A locus driven by pro-inflammatory cytokines. Experiments in a murine model of arteriotomy highlighted the activation of Hif3α expression in infiltrated inflammatory cells, suggesting a new role for Hif3α in inflammation in vivo.

Neural stem cells from a mouse model of Rett syndrome are prone to senescence, show reduced capacity to cope with genotoxic stress, and are impaired in the differentiation process.

Several aspects of stem cell life are governed by epigenetic variations, such as DNA methylation, histone modifications, and chromatin remodeling. Epigenetic events are also connected with the impairment of stem cell functions. For example, during senescence, there are significant changes in chromatin organization that alter transcription. The MECP2 protein can bind methylated cytosines and contribute to regulating gene expression at one of the highest hierarchical levels. Researchers are particularly interested in this protein, as up to 90% of Rett syndrome patients have an MECP2 gene mutation. Nevertheless, the role of MECP2 in this disease remains poorly understood. We used a mouse model of Rett syndrome to evaluate whether residual MECP2 activity in neural stem cells (NSCs) induced the senescence phenomena that could affect stem cell function. Our study clearly demonstrated that the reduced expression of MECP2 is connected with an increase in senescence, an impairment in proliferation capacity, and an accumulation of unrepaired DNA foci. Mecp2 +/- NSCs did not cope with genotoxic stress in the same way as the control cells did. Indeed, after treatment with different DNA-damaging agents, the NSCs from mice with mutated Mecp2 accumulated more DNA damage foci (γ-H2AX+) and were more prone to cell death than the controls. Senescence in Mecp2 +/- NSCs decreased the number of stem cells and progenitors and gave rise to a high percentage of cells that expressed neither stem/progenitor nor differentiation markers. These cells could be senescent and dysfunctional.

Polyamine concentration is increased in thoracic ascending aorta of patients with bicuspid aortic valve.

Polyamines are cationic molecules synthesized via a highly regulated pathway, obtained from the diet or produced by the gut microbiota. They are involved in general molecular and cellular phenomena that play a role also in vascular disease. Bicuspid aortic valve (BAV) is a congenital malformation associated to a greater risk of thoracic ascending aorta (TAA) aneurysm, whose pathogenesis is not yet well understood. We focused on differential analysis of key members of polyamine pathway and on polyamine concentration in non-dilated TAA samples from patients with either stenotic tricuspid aortic valve (TAV) or BAV (diameter ≤ 45 mm), vs. normal aortas from organ donors, with the aim of revealing a potential involvement of polyamines in early aortopathy. Changes of gene expression in TAA samples were evaluated by RT-PCR. Changes of ornithine decarboxylase 1 (ODC1), a key enzyme in polyamine formation, and cationic amino acid transporter 1 (SLC7A1/CAT-1) expression were analyzed also by Western blot. ODC1 subcellular localization was assessed by immunohistochemistry. Polyamine concentration in TAA samples was evaluated by HPLC. BAV TAA samples showed an increased concentration of putrescine and spermidine vs. TAV and donor samples, together with a decreased mRNA level of polyamine anabolic enzymes and of the putative polyamine transporter SLC7A1/CAT-1. The catabolic enzyme spermidine/spermine N1-acetyltransferase 1 showed a significant mRNA increase in TAV samples only, together with a decreased concentration of spermine. The decreased expression of SLC7A1/CAT-1 and ODC1 mRNAs in BAV corresponded to increased or unchanged expression of the respective proteins. ODC was located mainly in smooth muscle cell (SMC) nucleus in TAV and donor samples, while it was present also in SMC cytoplasm in BAV samples, suggesting its activation. In conclusion, BAV, but not TAV non-dilated samples show increased polyamine concentration, accompanied by the activation of a regulatory negative feedback mechanism.

Misidentified Human Gene Functions with Mouse Models: The Case of the Retinoblastoma Gene Family in Senescence.

Although mice models rank among the most widely used tools for understanding human genetics, biology, and diseases, differences between orthologous genes among species as close as mammals are possible, particularly in orthologous gene pairs in which one or more paralogous (i.e., duplicated) genes appear in the genomes of the species. Duplicated genes can possess overlapping functions and compensate for each other. The retinoblastoma gene family demonstrates typical composite functionality in its three member genes (i.e., RB1, RB2/P130, and P107), all of which participate in controlling the cell cycle and associated phenomena, including proliferation, quiescence, apoptosis, senescence, and cell differentiation. We analyzed the role of the retinoblastoma gene family in regulating senescence in mice and humans. Silencing experiments with each member of the gene family in mesenchymal stromal cells (MSCs) and fibroblasts from mouse and human tissues demonstrated that RB1 may be indispensable for senescence in mouse cells, but not in human ones, as an example of species specificity. Furthermore, although RB2/P130 seems to be implicated in maintaining human cell senescence, the function of RB1 within any given species might differ by cell type, as an example of cell specificity. For instance, silencing RB1 in mouse fibroblasts induced a reduced senescence not observed in mouse MSCs. Our findings could be useful as a general paradigm of cautions to take when inferring the role of human genes analyzed in animal studies and when examining the role of the retinoblastoma gene family in detail.

A Possible Early Biomarker for Bicuspid Aortopathy: Circulating Transforming Growth Factor ß-1 to Soluble Endoglin Ratio.

RATIONALE: The pathogenesis of bicuspid aortic valve (BAV)-associated aortopathy is poorly understood, and no prognostic biomarker is currently available. OBJECTIVE: We aimed to identify putative circulating biomarkers pathogenetically and prognostically linked to bicuspid aortopathy. METHODS AND RESULTS: By reverse transcription polymerase chain reaction, we evaluated gene expression variations (versus normal aorta) of transforming growth factor-ß1 (TGF-ß1), connective tissue growth factor, matrix metalloproteinase-2 (MMP-2), MMP-14, endoglin (ENG), and superoxide dismutase 3 in ascending aorta samples from 50 tricuspid and 70 patients with BAV undergoing surgery for aortic stenosis (aorta diameter ≤45 mm: BAVnon-dil or >45 mm: BAVdil). Expression changes of the TGF-ß1 active dimer and ENG were analyzed also by Western blot in ascending aorta samples from other 10 tricuspid aortic valve, 10 BAVnon-dil, and 10 BAVdil patients. The serum concentration of study targets was assessed through ELISA and the ratio of serum TGF-ß1/ENG (T/E) was evaluated. All BAVnon-dil patients underwent follow-up echocardiography to assess aortic growth rate. In BAVnon-dil patients, TGF-ß1 and MMP-2 gene expression increased significantly, whereas MMP-14 and ENG expression decreased versus controls. Expression changes were confirmed at protein level for TGF-ß1 and ENG. TGF-ß1 serum concentration significantly decreased in tricuspid aortic valve and BAVnon-dil patients versus healthy subjects. ENG serum concentration decreased in all patients, more markedly in BAVdil. A significant increase of the T/E ratio versus healthy subjects was unique of patients with BAV. In BAVnon-dil patients, a T/E ≥9 was independently associated in multivariable analysis with higher MMP-2 and lower superoxide dismutase 3 gene expression, independent of age and aortic diameter. A significant correlation was observed between baseline T/E ratio and aortic diameter growth rate in BAVnon-dil patients (r=0.66, P<0.001). CONCLUSIONS: The novel evidence of a possible value of the T/E ratio as a biomarker of BAV aortopathy was presented: further validation studies are warranted.

Patients with bicuspid and tricuspid aortic valve exhibit distinct regional microrna signatures in mildly dilated ascending aorta.

MicroRNAs are able to modulate gene expression in a range of diseases. We focused on microRNAs as potential contributors to the pathogenesis of ascending aorta (AA) dilatation in patients with stenotic tricuspid (TAV) or bicuspid aortic valve (BAV). Aortic specimens were collected from the 'concavity' and the 'convexity' of mildly dilated AAs and of normal AAs from heart transplant donors. Aortic RNA was analyzed through PCR arrays, profiling the expression of 84 microRNAs involved in cardiovascular disease. An in silico analysis identified the potential microRNA-mRNA interactions and the enriched KEGG pathways potentially affected by microRNA changes in dilated AAs. Distinct signatures of differentially expressed microRNAs are evident in TAV and BAV patients vs. donors, as well as differences between aortic concavity and convexity in patients only. MicroRNA changes suggest a switch of SMC phenotype, with particular reference to TAV concavity. MicroRNA changes potentially affecting mechanotransduction pathways exhibit a higher prevalence in BAV convexity and in TAV concavity, with particular reference to TGF-ß1, Hippo, and PI3K/Akt/FoxO pathways. Actin cytoskeleton emerges as potentially affected by microRNA changes in BAV convexity only. MicroRNAs could play distinct roles in BAV and TAV aortopathy, with possible implications in diagnosis and therapy.

Impact of lysosomal storage disorders on biology of mesenchymal stem cells: Evidences from in vitro silencing of glucocerebrosidase (GBA) and alpha-galactosidase A (GLA) enzymes.

Lysosomal storage disorders (LDS) comprise a group of rare multisystemic diseases resulting from inherited gene mutations that impair lysosomal homeostasis. The most common LSDs, Gaucher disease (GD), and Fabry disease (FD) are caused by deficiencies in the lysosomal glucocerebrosidase (GBA) and alpha-galactosidase A (GLA) enzymes, respectively. Given the systemic nature of enzyme deficiency, we hypothesized that the stem cell compartment of GD and FD patients might be also affected. Among stem cells, mesenchymal stem cells (MSCs) are a commonly investigated population given their role in hematopoiesis and the homeostatic maintenance of many organs and tissues. Since the impairment of MSC functions could pose profound consequences on body physiology, we evaluated whether GBA and GLA silencing could affect the biology of MSCs isolated from bone marrow and amniotic fluid. Those cell populations were chosen given the former's key role in organ physiology and the latter's intriguing potential as an alternative stem cell model for human genetic disease. Our results revealed that GBA and GLA deficiencies prompted cell cycle arrest along with the impairment of autophagic flux and an increase of apoptotic and senescent cell percentages. Moreover, an increase in ataxia-telangiectasia-mutated staining 1 hr after oxidative stress induction and a return to basal level at 48 hr, along with persistent gamma-H2AX staining, indicated that MSCs properly activated DNA repair signaling, though some damages remained unrepaired. Our data therefore suggest that MSCs with reduced GBA or GLA activity are prone to apoptosis and senescence due to impaired autophagy and DNA repair capacity.

Mesenchymal stromal cells having inactivated RB1 survive following low irradiation and accumulate damaged DNA: Hints for side effects following radiotherapy.

Following radiotherapy, bone sarcomas account for a significant percentage of recurring tumors. This risk is further increased in patients with hereditary retinoblastoma that undergo radiotherapy. We analyzed the effect of low and medium dose radiation on mesenchymal stromal cells (MSCs) with inactivated RB1 gene to gain insights on the molecular mechanisms that can induce second malignant neoplasm in cancer survivors. MSC cultures contain subpopulations of mesenchymal stem cells and committed progenitors that can differentiate into mesodermal derivatives: adipocytes, chondrocytes, and osteocytes. These stem cells and committed osteoblast precursors are the cell of origin in osteosarcoma, and RB1 gene mutations have a strong role in its pathogenesis. Following 40 and 2000 mGy X-ray exposure, MSCs with inactivated RB1 do not proliferate and accumulate high levels of unrepaired DNA as detected by persistence of gamma-H2AX foci. In samples with inactivated RB1 the radiation treatment did not increase apoptosis, necrosis or senescence versus untreated cells. Following radiation, CFU analysis showed a discrete number of cells with clonogenic capacity in cultures with silenced RB1. We extended our analysis to the other members of retinoblastoma gene family: RB2/P130 and P107. Also in the MSCs with silenced RB2/P130 and P107 we detected the presence of cells with unrepaired DNA following X-ray irradiation. Cells with unrepaired DNA may represent a reservoir of cells that may undergo neoplastic transformation. Our study suggests that, following radiotherapy, cancer patients with mutations of retinoblastoma genes may be under strict controls to evaluate onset of secondary neoplasms following radiotherapy.

Epigenetic regulation of TGF-ß1 signalling in dilative aortopathy of the thoracic ascending aorta.

The term 'epigenetics' refers to heritable, reversible DNA or histone modifications that affect gene expression without modifying the DNA sequence. Epigenetic modulation of gene expression also includes the RNA interference mechanism. Epigenetic regulation of gene expression is fundamental during development and throughout life, also playing a central role in disease progression. The transforming growth factor ß1 (TGF-ß1) and its downstream effectors are key players in tissue repair and fibrosis, extracellular matrix remodelling, inflammation, cell proliferation and migration. TGF-ß1 can also induce cell switch in epithelial-to-mesenchymal transition, leading to myofibroblast transdifferentiation. Cellular pathways triggered by TGF-ß1 in thoracic ascending aorta dilatation have relevant roles to play in remodelling of the vascular wall by virtue of their association with monogenic syndromes that implicate an aortic aneurysm, including Loeys-Dietz and Marfan's syndromes. Several studies and reviews have focused on the progression of aneurysms in the abdominal aorta, but research efforts are now increasingly being focused on pathogenic mechanisms of thoracic ascending aorta dilatation. The present review summarizes the most recent findings concerning the epigenetic regulation of effectors of TGF-ß1 pathways, triggered by sporadic dilative aortopathy of the thoracic ascending aorta in the presence of a tricuspid or bicuspid aortic valve, a congenital malformation occurring in 0.5-2% of the general population. A more in-depth comprehension of the epigenetic alterations associated with TGF-ß1 canonical and non-canonical pathways in dilatation of the ascending aorta could be helpful to clarify its pathogenesis, identify early potential biomarkers of disease, and, possibly, develop preventive and therapeutic strategies.

G-CSF contributes at the healing of tunica media of arteriotomy-injured rat carotids by promoting differentiation of vascular smooth muscle cells.

Restenosis is a complex pathophysiological disease whose causative mechanisms are not fully understood. Previous studies allowed us to demonstrate the efficacy of bone marrow mesenchymal stromal cells (MSCs) transplantation in limiting the pathophysiological remodeling in a model of arteriotomy-induced (re) stenosis. In the current research we studied the effectiveness of G-CSF treatment on male rate rats that were subjected carotid arteriotomy in order to evaluate a potentially effective non-invasive strategy that recapitulates the MSC-mediated recovery of injured vessels. WKY male rats were subjected carotid arteriotomy and given a nine day treatment (3 days pre- to 6 days post-arteriotomy) with G-CSF or saline. Carotids were harvested 7 and 30 days following arteriotomy (early- and late-phase, respectively). Although morphometrical analysis did not reveal differences in lumen narrowing between G-CSF- and PBS-carotids 30 days following arteriotomy, we detected a noticeable conservative effect of G-CSF treatment on vascular wall morphology. Histological and molecular analysis revealed an increase in cellularity within the tunica media with a concomitant increase of the VSMCs differentiation markers both at early- and late-phases of (re) stenotic response in G-CSF-treated carotids (Sm22-alpha, Myocd, and Smtn). These findings were accompanied by the downregulation of oxidative stress-related genes in G-CSF-injured rats. The effect exerted by G-CSF in our model of arteriotomy-induced (re) stenosis seemed support the recovery of the architecture of the tunica media of injured vessels by: (i) inducing VSMCs differentiation; and (ii) limiting the oxidative-stress response induced by arteriotomy.

Changes in autophagy, proteasome activity and metabolism to determine a specific signature for acute and chronic senescent mesenchymal stromal cells.

A sharp definition of what a senescent cell is still lacking since we do not have in depth understanding of mechanisms that induce cellular senescence. In addition, senescent cells are heterogeneous, in that not all of them express the same genes and present the same phenotype. To further clarify the classification of senescent cells, hints may be derived by the study of cellular metabolism, autophagy and proteasome activity. In this scenario, we decided to study these biological features in senescence of Mesenchymal Stromal Cells (MSC). These cells contain a subpopulation of stem cells that are able to differentiate in mesodermal derivatives (adipocytes, chondrocytes, osteocytes). In addition, they can also contribute to the homeostatic maintenance of many organs, hence, their senescence could be very deleterious for human body functions. We induced MSC senescence by oxidative stress, doxorubicin treatment, X-ray irradiation and replicative exhaustion. The first three are considered inducers of acute senescence while extensive proliferation triggers replicative senescence also named as chronic senescence. In all conditions, but replicative and high IR dose senescence, we detected a reduction of the autophagic flux, while proteasome activity was impaired in peroxide-treated and irradiated cells. Differences were observed also in metabolic status. In general, all senescent cells evidenced metabolic inflexibility and prefer to use glucose as energy fuel. Irradiated cells with low dose of X-ray and replicative senescent cells show a residual capacity to use fatty acids and glutamine as alternative fuels, respectively. Our study may be useful to discriminate among different senescent phenotypes.

De-regulated expression of the BRG1 chromatin remodeling factor in bone marrow mesenchymal stromal cells induces senescence associated with the silencing of NANOG and changes in the levels of chromatin proteins.

Stem cells have a peculiar chromatin architecture that contributes to their unique properties, including uncommitted status, multi/pluripotency and self-renewal. We analyzed the effect of the de-regulation of the SWI/SNF chromatin remodeling complex in mesenchymal stromal cells (MSC) through the silencing and up-regulation of BRG1, which is the ATPase subunit of the complex. The altered expression of BRG1 promoted the senescence of MSC with suppression of the NANOG transcription, which is part of the transcriptional circuitry governing stem cell functions. To gain insight on the way NANOG was silenced, we evaluated how the de-regulated BRG1 expression affect the binding of activators and repressors on the NANOG promoter. We found 4 E2F binding motifs on NANOG promoter, which can be occupied by RB1 and RB2/P130. These are members of the retinoblastoma gene family. In MSC with a silenced BRG1, the relative binding of the 2 retinoblastoma proteins increased, and this was associated with the recruitment of DNMT1. This induced the methylation of CpG on the NANOG promoter. Opposingly, when a high level of BRG1 was present, the same E2F binding motifs were docking sites for BRG1, which induced chromatin compaction without CpG methylation but with increased histone deacetylation, associated with the presence of HDAC1 on E2F binding sites. Besides the sharp regulation of the NANOG expression, we evidenced, through proteomic analysis, that the de-regulation of the SWI/SNF function affected the expression of histones and other nuclear proteins involved in "nuclear architecture," suggesting that BRG1 may act as global regulator of gene expression.

Low dose radiation induced senescence of human mesenchymal stromal cells and impaired the autophagy process.

Low doses of radiation may have profound effects on cellular function. Individuals may be exposed to low doses of radiation either intentionally for medical purposes or accidentally, such as those exposed to radiological terrorism or those who live near illegal radioactive waste dumpsites.We studied the effects of low dose radiation on human bone marrow mesenchymal stromal cells (MSC), which contain a subpopulation of stem cells able to differentiate in bone, cartilage, and fat; support hematopoiesis; and contribute to body's homeostasis.The main outcome of low radiation exposure, besides reduction of cell cycling, is the triggering of senescence, while the contribution to apoptosis is minimal. We also showed that low radiation affected the autophagic flux. We hypothesize that the autophagy prevented radiation deteriorative processes, and its decline contributed to senescence.An increase in ATM staining one and six hours post-irradiation and return to basal level at 48 hours, along with persistent gamma-H2AX staining, indicated that MSC properly activated the DNA repair signaling, though some damages remained unrepaired, mainly in non-cycling cells. This suggested that the impaired DNA repair capacity of irradiated MSC seemed mainly related to the reduced activity of a non-homologous end-joining (NHEJ) system rather than HR (homologous recombination).

Novel potential targets for prevention of arterial restenosis: insights from the pre-clinical research.

Restenosis is the pathophysiological process occurring in 10-15% of patients submitted to revascularization procedures of coronary, carotid and peripheral arteries. It can be considered as an excessive healing reaction of the vascular wall subjected to arterial/venous bypass graft interposition, endarterectomy or angioplasty. The advent of bare metal stents, drug-eluting stents and of the more recent drug-eluting balloons, have significantly reduced, but not eliminated, the incidence of restenosis, which remains a clinically relevant problem. Biomedical research in pre-clinical animal models of (re)stenosis, despite its limitations, has contributed enormously to the identification of processes involved in restenosis progression, going well beyond the initial dogma of a primarily proliferative disease. Although the main molecular and cellular mechanisms underlying restenosis have been well described, new signalling molecules and cell types controlling the progress of restenosis are continuously being discovered. In particular, microRNAs and vascular progenitor cells have recently been shown to play a key role in this pathophysiological process. In addition, the advanced highly sensitive high-throughput analyses of molecular alterations at the transcriptome, proteome and metabolome levels occurring in injured vessels in animal models of disease and in human specimens serve as a basis to identify novel potential therapeutic targets for restenosis. Molecular analyses are also contributing to the identification of reliable circulating biomarkers predictive of post-interventional restenosis in patients, which could be potentially helpful in the establishment of an early diagnosis and therapy. The present review summarizes the most recent and promising therapeutic strategies identified in experimental models of (re)stenosis and potentially translatable to patients subjected to revascularization procedures.

Sera of overweight people promote in vitro adipocyte differentiation of bone marrow stromal cells.

INTRODUCTION: Overweight status should not be considered merely an aesthetic concern; rather, it can incur health risks since it may trigger a cascade of events that produce further fat tissue through altered levels of circulating signaling molecules. METHODS: We decided to investigate the influence of overweight individuals' sera on in vitro MSC proliferation and differentiation. RESULTS: We observed that in vitro incubation of bone marrow stromal cells with the sera of overweight individuals promotes the adipogenic differentiation of MSCs while partially impairing proper osteogenesis. CONCLUSIONS: These results, which represent a pilot study, might suggest that becoming overweight triggers further weight gains by promoting a bias in the differentiation potential of MSCs toward adipogenesis. The circulating factors involved in this phenomenon remain to be determined, since the great majority of the well known pro-inflammatory cytokines and adipocyte-secreted factors we investigated did not show relevant modifications in overweight serum samples compared with controls.