Alberto Zullo

Functional Characterization of Ryanodine Receptor (RYR1) Sequence Variants Using a Metabolic Assay in Immortalized B-Lymphocytes

Mutations in the RYR1 gene are linked to malignant hyperthermia (MH), central core disease and multi‐minicore disease. We screened by DHPLC the RYR1 gene in 24 subjects for mutations, and characterized functional alterations caused by some RYR1 variants. Three novel sequence variants and twenty novel polymorphisms were identified. Immortalized lymphoblastoid cell lines from patients with RYR1 variants and from controls were stimulated with 4‐chloro‐m‐cresol (4‐CmC) and the rate of extracellular acidification was recorded. We demonstrate that the increased acidification rate of lymphoblastoid cells in response to 4‐CmC is mainly due to RYR1 activation. Cells expressing RYR1 variants in the N‐terminal and in the central region of the protein (p.Arg530His, p.Arg2163Pro, p.Asn2342Ser, p.Glu2371Gly and p.Arg2454His) displayed higher activity compared with controls; this could account for the MH‐susceptible phenotype. Cell lines harboring RYR1Cys4664Arg were significantly less activated by 4‐CmC. This result indicates that the p.Cys4664Arg variant causes a leaky channel and depletion of intracellular stores. The functional changes detected corroborate the variants analyzed as disease‐causing alterations and the acidification rate measurements as a means to monitor Ca2+‐induced metabolic changes in cells harboring mutant RYR1 channels.

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.

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. J. Cell. Biochem. 114: 7–20, 2012.

Possible Muscle Repair in the Human Cardiovascular System

The regenerative potential of tissues and organs could promote survival, extended lifespan and healthy life in multicellular organisms. Niches of adult stemness are widely distributed and lead to the anatomical and functional regeneration of the damaged organ. Conversely, muscular regeneration in mammals, and humans in particular, is very limited and not a single piece of muscle can fully regrow after a severe injury. Therefore, muscle repair after myocardial infarction is still a chimera. Recently, it has been recognized that epigenetics could play a role in tissue regrowth since it guarantees the maintenance of cellular identity in differentiated cells and, therefore, the stability of organs and tissues. The removal of these locks can shift a specific cell identity back to the stem-like one. Given the gradual loss of tissue renewal potential in the course of evolution, in the last few years many different attempts to retrieve such potential by means of cell therapy approaches have been performed in experimental models. Here we review pathways and mechanisms involved in the in vivo repair of cardiovascular muscle tissues in humans. Moreover, we address the ongoing research on mammalian cardiac muscle repair based on adult stem cell transplantation and pro-regenerative factor delivery. This latter issue, involving genetic manipulations of adult cells, paves the way for developing possible therapeutic strategies in the field of cardiovascular muscle repair.

Potential benefits of cell therapy in coronary heart disease

Cardiovascular disease is the leading cause of morbidity and mortality in the world. In recent years, there has been an increasing interest both in basic and clinical research regarding the field of cell therapy for coronary heart disease (CHD). Several preclinical models of CHD have suggested that regenerative properties of stem and progenitor cells might help restoring myocardial functions in the event of cardiac diseases. Here, we summarize different types of stem/progenitor cells that have been tested in experimental and clinical settings of cardiac regeneration, from embryonic stem cells to induced pluripotent stem cells. Then, we provide a comprehensive description of the most common cell delivery strategies with their major pros and cons and underline the potential of tissue engineering and injectable matrices to address the crucial issue of restoring the three-dimensional structure of the injured myocardial region. Due to the encouraging results from preclinical models, the number of clinical trials with cell therapy is continuously increasing and includes patients with CHD and congestive heart failure. Most of the already published trials have demonstrated safety and feasibility of cell therapies in these clinical conditions. Several studies have also suggested that cell therapy results in improved clinical outcomes. Numerous ongoing clinical trials utilizing this therapy for CHD will address fundamental issues concerning cell source and population utilized, as well as the use of imaging techniques to assess cell homing and survival, all factors that affect the efficacy of different cell therapy strategies.

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 patients 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.

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. J. Cell. Physiol. 228: 846–852, 2013.

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

ABSTRACT Epigenetics is involved in the altered expression of gene networks that underlie insulin resistance and insufficiency. Major genes controlling β-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 β-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.

Epigenetics and type 1 diabetes: mechanisms and translational applications

Type 1 diabetes (T1D) is an irreversible degenerative disease with severe complications such as heart disease, nephropathy, neuropathy, and retinopathy. Although exogenous insulin administration is a life-saving therapy, it does not cure the disease. This review addresses the epigenetic mechanisms responsible for the development of T1D and discusses epigenetic-based strategies for prevention and treatment of the disease. We describe novel epigenetic biomarkers for the identification of susceptible individuals and the establishment of innovative therapies with epidrugs and cell therapy to regenerate the lost β-cells. Despite the wealth of promising data regarding the potential benefits of epigenetic tools to reduce the burden of T1D, clinical trials are still very few, and this issue needs to be resolved in the near future.

Novel deletion mutation in the cardiac sodium channel inactivation gate causes long QT syndrome.

Disturbances in cardiac sodium channel function are associated with inherited arrhythmia susceptibility. Mutations in SCN5A, which encodes the cardiac sodium channel (NaV1.5), cause congenital long QT syndrome type 3 (LQT3), Brugada syndrome (BrS) and a variety of cardiac conduction disorders (CCD) [1,2]. These disorders have can have complex genotype-phenotype relationships [3,4]. Here we report the clinical features of an LQTS family segregating a novel amino acid deletion mutation (N1472del) in SCN5A that produces a unique pattern of biophysical disturbances consistent with the clinical phenotype.