Fibrosis: Sirtuins at the checkpoints of myofibroblast differentiation and profibrotic activity.

Fibrotic diseases are still a serious concern for public health, due to their high prevalence, complex etiology and lack of successful treatments. Fibrosis consists of excessive accumulation of extracellular matrix components. As a result, the structure and function of tissues are impaired, thus potentially leading to organ failure and death in several chronic diseases. Myofibroblasts represent the principal cellular mediators of fibrosis, due to their extracellular matrix producing activity, and originate from different types of precursor cells, such as mesenchymal cells, epithelial cells and fibroblasts. Profibrotic activation of myofibroblasts can be triggered by a variety of mechanisms, including the transforming growth factor-ß signalling pathway, which is a major factor driving fibrosis. Interestingly, preclinical and clinical studies showed that fibrotic degeneration can stop and even reverse by using specific antifibrotic treatments. Increasing scientific evidence is being accumulated about the role of sirtuins in modulating the molecular pathways responsible for the onset and development of fibrotic diseases. Sirtuins are NAD+ -dependent protein deacetylases that play a crucial role in several molecular pathways within the cells, many of which at the crossroad between health and disease. In this context, we will report the current knowledge supporting the role of sirtuins in the balance between healthy and diseased myofibroblast activity. In particular, we will address the signalling pathways and the molecular targets that trigger the differentiation and profibrotic activation of myofibroblasts and can be modulated by sirtuins.

Sweeteners modulate bioactivity of endothelial progenitor cells but not induce detrimental effects both on inflammation and behavioural changes.

This study sought to determine the possible detrimental effects of several low- or non-caloric sweeteners on endothelial progenitor cells (EPCs), inflammation and behavioural changes in mice. C57BL/6 male mice received low and high dose of natural and artificial sweeteners for 4 weeks. EPCs, physical and biochemical variables, inflammation and behavioural changes were evaluated. A significant reduction of about 25% of EPCs was found when mice received a moderate amount of all sweeteners (p < .05). This reduction was more strongly significant when a double dose of glucose, aspartame, rebaudioside A and cyclamate (p < .005) in comparison to fructose and sucrose (p < .05) was administered. During inflammation carrageenan-induced, all sweeteners produced a significant increase of EPCs compared to the control group (p < .05). Consumption of glucose and sugar substitutes affect mouse EPC number according to the absence or presence of an inflammatory status, but does not induce detrimental effects on inflammation and behavioural changes.

Sirtuins as Mediator of the Anti-Ageing Effects of Calorie Restriction in Skeletal and Cardiac Muscle.

Fighting diseases and controlling the signs of ageing are the major goals of biomedicine. Sirtuins, enzymes with mainly deacetylating activity, could be pivotal targets of novel preventive and therapeutic strategies to reach such aims. Scientific proofs are accumulating in experimental models, but, to a minor extent, also in humans, that the ancient practice of calorie restriction could prove an effective way to prevent several degenerative diseases and to postpone the detrimental signs of ageing. In the present review, we summarize the evidence about the central role of sirtuins in mediating the beneficial effects of calorie restriction in skeletal and cardiac muscle since these tissues are greatly damaged by diseases and advancing years. Moreover, we entertain the possibility that the identification of sirtuin activators that mimic calorie restriction could provide the benefits without the inconvenience of this dietary style.

Genetic and Epigenetic Control of CDKN1C Expression: Importance in Cell Commitment and Differentiation, Tissue Homeostasis and Human Diseases.

The CDKN1C gene encodes the p57Kip2 protein which has been identified as the third member of the CIP/Kip family, also including p27Kip1 and p21Cip1. In analogy with these proteins, p57Kip2 is able to bind tightly and inhibit cyclin/cyclin-dependent kinase complexes and, in turn, modulate cell division cycle progression. For a long time, the main function of p57Kip2 has been associated only to correct embryogenesis, since CDKN1C-ablated mice are not vital. Accordingly, it has been demonstrated that CDKN1C alterations cause three human hereditary syndromes, characterized by altered growth rate. Subsequently, the p57Kip2 role in several cell phenotypes has been clearly assessed as well as its down-regulation in human cancers. CDKN1C lies in a genetic locus, 11p15.5, characterized by a remarkable regional imprinting that results in the transcription of only the maternal allele. The control of CDKN1C transcription is also linked to additional mechanisms, including DNA methylation and specific histone methylation/acetylation. Finally, long non-coding RNAs and miRNAs appear to play important roles in controlling p57Kip2 levels. This review mostly represents an appraisal of the available data regarding the control of CDKN1C gene expression. In addition, the structure and function of p57Kip2 protein are briefly described and correlated to human physiology and diseases.

Osteosarcoma cells induce endothelial cell proliferation during neo-angiogenesis.

Understanding the mechanisms inducing endothelial cell (EC) proliferation following tumor microenvironment stimuli may be important for the development of antiangiogenic therapies. Here, we show that cyclin-dependent kinase 2 and 5 (Cdk2, Cdk5) are important mediators of neoangiogenesis in in vitro and in vivo systems. Furthermore, we demonstrate that a specific Yin Yang 1 (YY1) protein-dependent signal from osteosarcoma (SaOS) cells determines proliferation of human aortic endothelial cells (HAECs). Following tumor cell stimuli, HAECs overexpress Cdk2 and Cdk5, display increased Cdk2 activity, undergo enhanced proliferation, and form capillary-like structures. Moreover, Roscovitine, an inhibitor of Cdks, blunted overexpression of Cdk2 and Cdk5 and Cdk2 activity induced by the YY1-dependent signal secreted by SaOS cells. Furthermore, Roscovitine decreased HAEC proliferation and angiogenesis (the latter by 70% in in vitro and 50% in in vivo systems; P < 0.01 vs. control). Finally, the finding that Roscovitine triggers apoptosis in SaOS cells as well as in HAECs by activating caspase-3/7 indicates multiple mechanisms for the potential antitumoral effect of Roscovitine. Present work suggests that Cdk2 and Cdk5 might be pharmacologically accessible targets for both antiangiogenic and antitumor therapy.

Recent advances in proteomic technologies applied to cardiovascular disease.

In recent years, the diagnosis of cardiovascular disease (CVD) has increased its potential, also thanks to mass spectrometry (MS) proteomics. Modern MS proteomics tools permit analyzing a variety of biological samples, ranging from single cells to tissues and body fluids, like plasma and urine. This approach enhances the search for informative biomarkers in biological samples from apparently healthy individuals or patients, thus allowing an earlier and more precise diagnosis and a deeper comprehension of pathogenesis, development and outcome of CVD to further reduce the enormous burden of this disease on public health. In fact, many differences in protein expression between CVD-affected and healthy subjects have been detected, but only a few of them have been useful to establish clinical biomarkers because they did not pass the verification and validation tests. For a concrete clinical support of MS proteomics to CVD, it is, therefore, necessary to: ameliorate the resolution, sensitivity, specificity, throughput, precision, and accuracy of MS platform components; standardize procedures for sample collection, preparation, and analysis; lower the costs of the analyses; reduce the time of biomarker verification and validation. At the same time, it will be fundamental, for the future perspectives of proteomics in clinical trials, to define the normal protein maps and the global patterns of normal protein levels, as well as those specific for the different expressions of CVD.

Endothelial Dysfunction in COVID-19: A Unifying Mechanism and a Potential Therapeutic Target.

The novel severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) generated a worldwide emergency, until the declaration of the pandemic in March 2020. SARS-CoV-2 could be responsible for coronavirus disease 2019 (COVID-19), which goes from a flu-like illness to a potentially fatal condition that needs intensive care. Furthermore, the persistence of functional disability and long-term cardiovascular sequelae in COVID-19 survivors suggests that convalescent patients may suffer from post-acute COVID-19 syndrome, requiring long-term care and personalized rehabilitation. However, the pathophysiology of acute and post-acute manifestations of COVID-19 is still under study, as a better comprehension of these mechanisms would ensure more effective personalized therapies. To date, mounting evidence suggests a crucial endothelial contribution to the clinical manifestations of COVID-19, as endothelial cells appear to be a direct or indirect preferential target of the virus. Thus, the dysregulation of many of the homeostatic pathways of the endothelium has emerged as a hallmark of severity in COVID-19. The aim of this review is to summarize the pathophysiology of endothelial dysfunction in COVID-19, with a focus on personalized pharmacological and rehabilitation strategies targeting endothelial dysfunction as an attractive therapeutic option in this clinical setting.

Resveratrol regulates p66Shc activation in HaCaT cells.

Skin is exposed to both endogenous and environmental oxidant agents, leading to the harmful generation of reactive oxygen species. Particular interest has been pointed on plant antioxidants, such as resveratrol, because of their wide-ranging biological activity and clinical potential. Resveratrol exerts antioxidant, metabolism-regulating and pro-apoptotic/anti-cancer effects on a variety of experimental models and has been suggested to protect skin from ultraviolet-induced photodamaging and photoaging. In parallel, also the biological significance of p66Shc, a member of the Src Homologue and Collagene homologue family with redox activity, is getting further attention. Because of the striking intersection among the activities of resveratrol with those of p66Shc, we investigated whether resveratrol would activate p66Shc in human immortalised keratinocytes (HaCaT cells), a well known and largely used model for skin keratinocytes. HaCaT cells were treated with resveratrol (10-150 µm) for different times. The effect of resveratrol on the proliferation of HaCaT cells and the activation of ERK1/2, AKT, and p66Shc was investigated by cell counting, fluorescence-activated cell sorting, and western blot analysis of total or immunoprecipitated cell extracts. In HaCaT cells, resveratrol induces dose- and time-dependent growth arrest, p66Shc-Ser36 phosphorylation, ERK1/2 phosphorylation and AKT dephosphorylation. Finally, we showed that resveratrol-induced p66Shc-Ser36 phosphorylation is dependent on ERK1/2 activation. Interestingly, these resveratrol-induced molecular effects were associated with reduced adhesion and reversible growth arrest rather than cell death pathways. This is the first evidence linking resveratrol with p66Shc and suggests that p66Shc may contribute to the effect of resveratrol on cell proliferation and function in the outermost layer of the skin.

The Diversity of Muscles and Their Regenerative Potential across Animals.

Cells with contractile functions are present in almost all metazoans, and so are the related processes of muscle homeostasis and regeneration. Regeneration itself is a complex process unevenly spread across metazoans that ranges from full-body regeneration to partial reconstruction of damaged organs or body tissues, including muscles. The cellular and molecular mechanisms involved in regenerative processes can be homologous, co-opted, and/or evolved independently. By comparing the mechanisms of muscle homeostasis and regeneration throughout the diversity of animal body-plans and life cycles, it is possible to identify conserved and divergent cellular and molecular mechanisms underlying muscle plasticity. In this review we aim at providing an overview of muscle regeneration studies in metazoans, highlighting the major regenerative strategies and molecular pathways involved. By gathering these findings, we wish to advocate a comparative and evolutionary approach to prompt a wider use of "non-canonical" animal models for molecular and even pharmacological studies in the field of muscle regeneration.

Overexpression of chromatin assembly factor-1 (CAF-1) p60 is predictive of adverse behaviour of prostatic cancer.

AIMS: Prostatic cancer may remain organ-confined indefinitely; in a number of patients, however it gives rise to clinical symptoms and death. The biological behaviour of this tumour mostly remains difficult to predict. A promising tool for diagnosis and prognosis of some human tumours is the chromatin assembly factor-1 (CAF-1), involved in the control of higher order chromatin organization. The aim was to explore the role of CAF-1/p60 protein as a new prognostic marker for prostatic cancer. METHODS AND RESULTS: The expression of CAF-1/p60 was evaluated by immunohistochemistry and Western blotting in a selected series of prostatic cancers and in prostatic cancer cell lines. Results were compared with clinicopathological data and outcome of patients. CAF-1/p60 was expressed in all cases, with a linear increase from low-grade tumours (Gleason score <7) to high-grade prostatic cancers (Gleason score >7). By comparing results with follow-up data, a significant association between overexpression of CAF-1/p60 and unfavourable behaviour of prostatic cancer emerged, and its predictive value was independent of classical prognostic factors. CONCLUSIONS: In our series of cases, overexpression of CAF-1/p60 characterized prostatic cancers with a worse prognosis. CAF-1/p60 has a potential role as a new reliable prognostic biomarker for prostatic cancer.

Understanding the immunoangiostatic CXC chemokine network.

Chemokines, originally discovered as mediators of directional migration of immune cells to sites of inflammation and injury, have a function beyond their role in leukocyte chemotaxis. Indeed, they participate in organ development, angiogenesis, tumourigenesis and, more importantly, in the immune response. The chemokine family characterized by four highly conserved cysteine amino acid residues, with two cysteine residues (C) and a non-cysteine amino acid (X) between them (CXC), is known for its ability to promote trafficking of various leukocytes and to regulate angiogenesis and vascular remodelling. Intriguingly, the presence or absence of a structural-functional domain constituted by glutamic acid-leucine-arginine motif that precedes the first cysteine amino acid residue accounts for their unique property to induce or inhibit angiogenesis (angiogenic or angiostatic activity). The ability of CXC chemokine receptor 3 to promote Th1-dependent immunity and, at the same time, inhibit angiogenesis (immunoangiostasis) is of critical importance for inducing tumour regression. Agents that are able to inhibit angiogenic activities or promote angiostatic activities of CXC chemokines are future targets for research on cancer treatment. Here, we review insights on CXC chemokines in the context of immunoangiostasis and vascular damage.

Proteomics and cardiovascular disease: an update.

Proteomics has unraveled important questions in the biology of cardiovascular disease and holds even greater promise for the development of novel diagnostic and prognostic biomarkers. This approach may establish early detection strategies, and monitor responses to therapies. Technological advances (most notably blue native polyacrylamide gel electrophoresis, electrospray ionization, matrix-assisted laser desorption/ionization (MALDI), analysis of MALDI-derived peptides in Time-of-Flight (TOF) analyzers, and multidimensional protein identification technology (MudPIT) and bioinformatics for data handling and interpretation allow a large-scale identification of peptide sequence and post-translational modifications. Moreover, combination of proteomic biomarkers with clinical phenotype, metabolite changes, and genetic haplotype information is promising for the physician assessment of individual cardiovascular risk profile.

Therapeutic dose of nebivolol, a nitric oxide-releasing beta-blocker, reduces atherosclerosis in cholesterol-fed rabbits.

Nitric oxide (NO) exerts a plethora of vascular beneficial effects. The NO-releasing beta-blocker nebivolol is a racemic mixture of D/L-enantiomers that displays negative inotropic as well as direct vasodilating activity. The in vivo antiatherogenic activity of therapeutic doses of the beta-blocker with antioxidant properties carvedilol (12.5mg/day) or nebivolol (5mg/day) was tested in cholesterol-fed rabbits. Animals received a 1% cholesterol-rich diet alone (controls) or mixed with drugs (treated animals) for 8 weeks. While it did not affect hyperlipidemia, nebivolol inhibited the development of atherosclerosis, expressed as computer-assisted imaging analysis of aortic area covered by lesions (23.3+/-4.1% in treated vs 38.2+/-6.4% in control animals, p<0.01). Differently, in our experimental condition of therapeutic drug doses, this antiatherogenic effect did not reach statistical significance in rabbits treated with carvedilol (32.5+/-5.1% aortic area covered by lesions, p=NS vs controls). Plasma nitrates increased in rabbits treated with nebivolol while both beta-blockers reduced LDL oxidation. Moreover, nebivolol induced a consistent increase of endothelial reactivity and aortic eNOS expression compared with control animals (p<0.05) and those receiving carvedilol (p<0.05). Since NO may exert beneficial effects in atherosclerosis, a NO-dependent mechanism could explain this data. These observations suggest that the NO-releasing beta-blocker, nebivolol, might represent an effective pharmacological approach for preventing atherosclerotic lesion progression.

Potential clinical benefits of cell therapy in coronary heart disease: an update.

Cell therapy is a central issue of regenerative medicine and is raising a growing interest in the scientific community, but its full therapeutic potential in coronary heart disease (CHD) has not been reached yet. Several different methods, cell types, delivery routes, and supporting techniques have been attempted and improved to elicit cardiac regeneration in CHD, but only some of them showed a really convincing potential for the use in clinical practice. Here we provide an update on approaches and clinical trials of cell therapy applied to CHD, which are ongoing or that have been realized in the last 5 years. Moreover, we discuss the evidence collected so far in favor or against the validity of stem cell therapy for CHD. In particular, we review and comment the recent advances in cell therapy applied to CHD, the most promising cell types, delivery strategies, biochemical and engineering techniques that have been adopted in this context.

p27Kip1 and human cancers: A reappraisal of a still enigmatic protein.

p27Kip1 is a cell cycle regulator firstly identified as a cyclin-dependent kinase inhibitor. For a long time, its function has been associated to cell cycle progression inhibition at G1/S boundary in response to antiproliferative stimuli. The picture resulted complicated by the discovery that p27Kip1 is an intrinsically unstructured protein, with numerous CDK-dependent and -independent functions and involvement in many cellular processes, such as cytoskeleton dynamics and cell motility control, apoptosis and autophagy activation. Depending on the cell context, these activities might turn to be oncogenic and stimulate cancer progression and metastatization. Nevertheless, p27Kip1 role in cancer biology suppression was underscored by myriad data reporting its down-regulation and/or cytoplasmic relocalization in different tumors, while usually no genetic alterations were found in human cancers, making the protein a non-canonical oncosuppressor. Recently, mostly due to advances in genomic analyses, CDKN1B, p27Kip1 encoding gene, has been found mutated in several cancers, thus leading to a profound reappraisal of CDKN1B role in tumorigenesis. This review summarizes the main p27Kip1 features, with major emphasis to its role in cancer biology and to the importance of CDKN1B mutations in tumor development.

Clinical relevance of epigenetics in the onset and management of type 2 diabetes mellitus.

Epigenetics is involved in the altered expression of gene networks that underlie insulin resistance and insufficiency. Major genes controlling ß-cell differentiation and function, such as PAX4, PDX1, and GLP1 receptor, are epigenetically controlled. Epigenetics can cause insulin resistance through immunomediated pro-inflammatory actions related to several factors, such as NF-kB, osteopontin, and Toll-like receptors. Hereafter, we provide a critical and comprehensive summary on this topic with a particular emphasis on translational and clinical aspects. We discuss the effect of epigenetics on ß-cell regeneration for cell replacement therapy, the emerging bioinformatics approaches for analyzing the epigenetic contribution to type 2 diabetes mellitus (T2DM), the epigenetic core of the transgenerational inheritance hypothesis in T2DM, and the epigenetic clinical trials on T2DM. Therefore, prevention or reversion of the epigenetic changes occurring during T2DM development may reduce the individual and societal burden of the disease.

Evidence of epigenetic tags in cardiac fibrosis.

In cardiac fibrosis, following an injury or a stress, non-functional fibrotic tissue substitutes normal myocardium, thus leading to progressive heart failure. Activated fibroblasts are principal determinants of cardiac fibrosis by producing excessive fibrotic extracellular matrix and causing hypertrophy of cardiomyocytes. Epigenetic changes, such as DNA methylation, histone modifications, and miRNAs have been involved in these mechanisms. Therefore, there is a strong interest in reverting such epigenetic transformations in order to arrest myocardial fibrotic degeneration. Demethylating agents, such as 5-aza-2'-deoxycytidine, 5-azacytidine, some selective histone deacetylase inhibitors, including mocetinostat, trichostatin A, and MPT0E014, have a direct action on important inducers of cardiac fibrosis. Also dietary compounds, such as resveratrol, can suppress the differentiation of fibroblasts to myofibroblasts. Although in vivo and in vitro studies suggest specific epigenetic therapies to treat cardiac fibrosis, the related clinical trials are still lacking. A better understanding of the epigenetic effects of dietary compounds (e.g. curcumin and green tea catechins) on the onset and progression of cardiac fibrosis, will allow the identification of protective dietary patterns and/or the generation of novel potential epidrugs.

Multiple pathways of SIRT6 at the crossroads in the control of longevity, cancer, and cardiovascular diseases.

Sirtuin 6 (SIRT6) is a member of the sirtuin family NAD+-dependent deacetylases with multiple roles in controlling organism homeostasis, lifespan, and diseases. Due to its complex and opposite functional roles, this sirtuin is considered a two-edged sword in health and disease. Indeed, SIRT6 improves longevity, similarly to the founding yeast member, silent information regulator-2 (Sir2), and modulates genome stability, telomere integrity, transcription, and DNA repair. Its deficiency is associated with chronic inflammation, diabetes, cardiac hypertrophy, obesity, liver dysfunction, muscle/adipocyte disorders, and cancer. Besides, pieces of evidence showed that SIRT6 is a promoter of specific oncogenic pathways, thus disclosing its dual role regarding cancer development. Collectively, these findings suggest that multiple mechanisms, to date not entirely known, underlie the intriguing roles of SIRT6. Here we provide an overview of the current molecular mechanisms through which SIRT6 controls cancer and heart diseases, and describe its recent implications in the atherosclerotic plaque development.

Imaging techniques to evaluate cell therapy in peripheral artery disease: state of the art and clinical trials.

Cell-based therapies, as potential approach to cure peripheral artery disease (PAD), have been clinically investigated after promising results in preclinical models. The so far published studies are very heterogeneous, as different cell sources, cell types, amounts of administered cells and delivering strategies have been used. Overall, cell therapies for PAD bring about a general improvement of patient's clinical condition, even though conclusions cannot be established due to the small size and non-randomized design of these trials. In this context, non-invasive imaging techniques, aimed to monitor angiogenesis and neovascularization after cell therapy, will help the follow-up of clinical studies. However, still much work is needed to establish advanced imaging procedure to overcome the limitation of the current techniques and to accumulate more data in large populations of patients. Here, we report the main imaging techniques employed to evaluate the outcome of the different cell-based therapies in PAD. Moreover, we focus on both published and ongoing clinical trials utilizing cell therapy in PAD.

Infections and cardiovascular disease: is Bartonella henselae contributing to this matter?

Cardiovascular disease is still the major cause of death worldwide despite the remarkable progress in its prevention and treatment. Endothelial progenitor cells (EPCs) have recently emerged as key players of vascular repair and regenerative medicine applied to cardiovascular disease. A large amount of effort has been put into discovering the factors that could aid or impair the number and function of EPCs, and also into characterizing these cells at the molecular level in order to facilitate their therapeutic applications in vascular disease. Interestingly, the major cardiovascular risk factors have been associated with reduced number and function of EPCs. The bacterial contribution to cardiovascular disease represents a long-standing controversy. The discovery that Bartonella henselae can infect and damage EPCs revitalizes the enduring debate about the microbiological contribution to atherosclerosis, thus allowing the hypothesis that this infection could impair the cardiovascular regenerative potential and increase the risk for cardiovascular disease. In this review, we summarize the rationale suggesting that Bartonella henselae could favour atherogenesis by infecting and damaging EPCs, thus reducing their vascular repair potential. These mechanisms suggest a novel link between communicable and non-communicable human diseases, and put forward the possibility that Bartonella henselae could enhance the susceptibility and worsen the prognosis in cardiovascular disease.