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The major issue in coronary heart disease (CHD) diagnosis and management is that symptoms onset in an advanced state of disease. Despite the availability of several clinical risk scores, the prediction of cardiovascular events is lacking, and many patients at risk are not well stratified according to the canonical risk factors alone. Therefore, adequate risk assessment remains the most challenging issue. Recently, the integration of imaging data with biochemical markers in a radiogenomic framework has been proposed in many fields of medicine as well as in cardiology. Multimodal imaging and advanced processing techniques can provide both direct (e.g., remodeling index, calcium score, total plaque volume, plaque burden) and indirect (e.g., myocardial perfusion index, coronary flow reserve) imaging features of CHD. Furthermore, the identification of novel non-invasive biochemical markers, mainly focused on plasma and/or serum samples, has increased the specificity of findings, reflecting several pathophysiological pathways of atherosclerosis, the principal actor in CHD. In this context, a multifaced approach, derived from the strengths of all these modalities, appears promising for finer risk stratification and treatment strategies, facilitating the decision-making and clinical management of patients. This review underlines the role of different imaging modalities in the quantification of coronary atherosclerosis and describes novel blood-based markers that could improve diagnosis and have a better predictive value in CHD.

Male breast cancer (MBC) is a rare disease, accounting for ~1% of all breast cancer cases worldwide. Although other genes are also involved, predisposing genetic factors to MBC include germline mutations in the BRCA genes (BRCA2). Among the other genes, partner and localizer of BRCA2 (PALB2) is considered a moderate-penetrance breast cancer susceptibility gene that may also play a role in MBC predisposition. Thus, the aim of the present study was to determine the PALB2 gene status in 8 MBC cases selected from a cohort of 181 hereditary breast and/or ovarian cancer probands. We performed PALB2 mutational analysis by direct sequencing of 13 exons and adjacent intronic regions. This study showed the presence of a PALB2 truncating mutation in 1/8 (12.5%) cases. This novel mutation was named c.1285_1286delAinsTC (p.I429SfsX12) and is localized in exon 4 of PALB2, in the region encoding for the ChAM motif which is important for the efficient association of PALB2 to chromatin and for recruitment of the BRCA complex to accumulate RAD51 at double-strand break sites. Our findings indicate that PALB2 could be added to the list of breast cancer susceptibility genes also in families with MBC cases.

From the 1980s, extracorporeal photochemotherapy (ECP) has been shown to be effective in a variety of pathological conditions such as erythrodermic cutaneous T-cell lymphoma, autoimmune diseases, solid organ allograft rejection and graft versus host disease. To date, ECP represents a non-aggressive immune modulatory therapy with a low spectrum of toxicity. ECP reduces the alloreactivity promoting the immune tolerance to self. At the same time, it allows the maintenance of immune response integrity of both naive and memory T-cells. However, the molecular mechanisms of action by which ECP exerts its therapeutic activity are still under investigation. Here, we review molecular mechanisms and clinical applications involved in ECP. The outcome of ECP is difficult due to the lack of reliable predictor factors for the selection of patients and their adequate follow-up. Since the study of such predictors is important, we also describe some biological markers that enable us to investigate the clinical management of the patients considered for the use of ECP.

Background: The main challenge in type 2 diabetes (T2D) is to detect the regulators of pathogenic events during early stages of disease, as well as prevention and progression follow-up of cardiovascular (CV) complications. DNA methylation and micro-RNAs (miRNAs) are major components of the epigenome, which are involved in the diabetic interactome. This study protocol may contribute to advance our knowledge on molecular basis underlying T2D and its CV complications, as well as provide putative useful prognostic biomarkers.Methods: The perturbation of interactome through micro-RNA and methylome analysis in diabetes endophenotypes: the PIRAMIDE pathogenic clinical study protocol is a cross-sectional research program planned to combine big data and network-based analysis aimed to investigate whether DNA methylation and miRNAs may act as simultaneous regulators of the interactome in T2D patients. Clinical datasets will be aggregate to large-scale DNA methylation, mRNA-Seq, and miRNA-Seq analysis performed both on purified CD4+ and CD8+ T cells isolated from 35 T2D patients and 35 sex and age-matched controls. DNA methylome data will be used as input for the weighted human DNA methylation PPI network (WMPN) algorithm. RNA sequencing data will be used as input data for the TargetScan algorithm. The primary endpoint will be to integrate both DNA methylation and miRNA networks to potentially capture which genes are simultaneously modulate by interactions between epigenetic changes. Then, statistical analysis will be performed to correlate these molecular modifications with development of T2D-related CV complications.Conclusions: PIRAMIDE pathogenic clinical study protocol will test the hypothesis that simultaneous interactions between DNA methylation and miRNAs may hit T2D-associated candidate genes and predict the development of T2D-related CV complications.Trial Registration: The ongoing PIRAMIDE pathogenic clinical study protocol has been registered on NIH website (NCT03792607).

Objective: To report on the clinical benefits of platelet gel application in a non-regenerating skin wound. Clinical Presentation and Intervention: An 84-year-old man presented with a severe wound with a regular circumference in the frontal region which resulted in a complete loss of epidermis and dermis. The skin lesion, induced by cryosurgery used to remove a basal-cell carcinoma, had previously been treated with a dermal substitute application (Integra®). After the failure of the skin graft, the patient was treated using a platelet gel therapeutic protocol which achieved the complete healing of the injured area. Conclusion: This case showed the clinical efficacy of using platelet gel in this elderly patient in whom the dermal substitute graft had been ineffective.

In the last few years, therapeutic apheresis (TA) has emerged as a valuable treatment option for certain life-threatening cardiovascular diseases (CVDs) and for all the cardiac dysfunctions caused by autoimmune or metabolic disorders. Besides the well-established indications for apheresis treatment, such as familial hypercholesterolaemia, hyperviscosity syndrome and thrombotic thrombocytopenic purpura (TTP), we discuss the novel approaches in the therapy of dilated cardiomyopathy, cardiac failure and some specific syndromes of severe dysfunction occurring after heart transplantation. The rationale for using apheresis in such patients is the contribution in immune modulation that this procedure can undoubtedly provide. The clinical course of TTP has dramatically changed, thanks to the introduction of therapeutic plasma exchange. Low-density lipoprotein apheresis has been extremely efficacious, safe and suitable for lowering cholesterol levels and can be used even for long-term treatment. In Waldenstrom macroglobulinaemia and other hyperviscosity syndromes, plasma exchange has demonstrated to be an efficient tool for reducing blood viscosity and the risk of a consequent cardiac dysfunction. However, TA may induce oxidative injury to erythrocytes, making these cells more prone to haemolysis and causing a significant reduction in their half-life. Ascorbate administration can be useful to lower the levels of hydrogen peroxide and proinflammatory mediators in patients undergoing apheresis. Further data are needed to support this benefit and to test other potential antioxidant therapies. Many of these therapeutic indications need further studies to be definitively approved, but preliminary data are encouraging.

Intravenous immunoglobulin (IVIG) products are known to have beneficial immunomodulatory effects on several inflammatory and autoimmune disorders. These effects could be attributed to a different inhibitory action on complement factors, but other mechanisms could be implicated, e.g., immunocomplexes development and/or anti-idiotypic antibodies. Positive results on the reduction of anti-Human Leukocyte Antigens (HLA) antibodies in highly sensitized patients have also been found. The present study focuses on the effect of IVIG on the reduction of Panel Reactive Antibody level and crossmatch positivity in sensitized patients awaiting kidney transplantation.The study was performed adapting an in vitro assay on sensitized patients' sera in waiting list for kidney transplantation. Sera of twelve highly sensitized patients were evaluated for the cytotoxicity inhibition after 10% IVIG treatment.A reduction of anti- HLA antibody levels was observed in 75% (9/12) of treated patients in vitro, while 25% (3/12) resulted unresponsiveness. Particularly, our data showed a significantly higher Panel Reactive Antibody reduction for T lymphocytes (p0.010) than B lymphocytes (p0.032).In this study, we have used an in vitro assay to investigate susceptibility to desensitization with IVIG treatment of sensitized patient sera. These findings reveal that the variable effect of IVIG on reducing Panel Reactive Antibody in our immunized patients could be attributed to a different inhibitory action on complement, likely due to the type and the titre of anti-HLA antibodies.

Human leukocyte antigen (HLA) antibodies represent a significant risk factor for transplant failure. It is very important to characterize anti-HLA antibodies as epitopes rather than antigens so that this knowledge can be applied clinically. The aim of the study was to investigate the extra reactivity patterns in sensitized multipare. Here, we have used the HLAMatchmaker program, a theoretical algorithm, to explain these unexpected antibody reactivity patterns in multipare awaiting for heart transplant. The patient was sensitized during pregnancy by alleles HLA-A(*)24:02, HLA-DRB1(*)07:01, HLA-DRB4(*)01:01, DQB1(*)02:02 and DQA1(*)02:01 mismatches with development of respective antibodies. However, the patient' sera were shown an unexpected reactivity not directed toward HLA mismatches of daughters: A(∗)23:01, A(*)24:03 and B(*)15:12 for class I and DRB4(*)01:03, DRB1(*)09:02, DRB1(*)09:01, DQB1(*)03:01, DQB1(*)03:03, DQB1(*)03:02, DQB1(*)04:02, DQB1(*)04:01 and DQB1(*)02:01 for class II. By HLAMatchmaker analysis we found that these antibodies reacted with eplet shared by antigens in single allele Luminex panels. These eplets were: 62EE, 66GKH, 70KAH, 71HS, 127K, 113YH, 144KR, 150AAH, 151AHV, 163TG and 167DG for class I and 4Q, 74RRAE, 71RRA, 98KN, 120N, and 135G, 25FT, 34HE, 41ER, 47EK2, 48LF for class II. Thus, HLAMatchmaker software together with to solid phase techniques could open new horizons for a more precise characterization of the HLA-antibodies.

Nitric oxide (NO) has been suggested to be a pathophysiological modulator of cell proliferation, cell cycle arrest, and apoptosis. In this context, NO can exert opposite effects under diverse conditions. Indeed, several studies have indicated that low relative concentrations of NO seem to favor cell proliferation and antiapoptotic responses and higher levels of NO favor pathways inducing cell cycle arrest, mitochondria respiration, senescence, or apoptosis. Here we report the effects of NO on both promotion and inhibition of cell proliferation, in particular in regard to cardiovascular disease, diabetes, and stem cells. Moreover, we focus on molecular mechanisms of action involved in the control of cell cycle progression, which include both cyclic guanosine monophosphate–dependent and –independent pathways. This growing field may lead to broad and novel targeted therapies against cardiovascular diseases, especially concomitant type 2 diabetes, as well as novel bioimaging NO-based diagnostic tools.

Different desensitization strategies are available for treating patients with preformed human leukocyte antigen (HLA) antibodies. A highly presensitized heart recipient received immunoadsorption and rituximab therapy. The patient, with end-stage heart failure, was positive only for antibodies of HLA class I (anti-A2, A10, B17), and Luminex platform (One Lambda kit) showed a panel-reactive antibody score of 64%. The patient's serum was tested repeatedly in both complement-dependent cytotoxicity and flow-cytometry crossmatches against cells from different potential organ donors. The results of these crossmatches were positive on flow cytometry when tested with HLA-A2, A10, and B17 but were still negative on cytotoxicity. The patient was treated with a desensitization regimen; this treatment immediately decreased antibody levels of 70% and the patient subsequently received a transplant with donor-specific HLA antibody (HLA-A2). After more than 2 years, graft function remains normal and the clinical status of the patient is stable.

Endothelial progenitor cells are produced in the bone marrow and have the hemangioblast as common precursor to hematopoietic stem cells. They have been identified in 1997 in the peripheral blood stream. The EPC are able to migrate, proliferate and differentiate into mature endothelial cells and to determine the formation of new blood vessels even in the postnatal period. These cells can be infected by Bartonella henselae, a Gram-negative pathogen, aetiological agent of a series of human diseases known as bartonellosis. Polysaccharide A pro-duced by Bacteroides fragilis, a human intestinal bacterium, has immunoregulatory properties, among which is proven in mice to provide protection from inflammation caused by B. henselae. We have infected the EPC with B. henselae, B. fragilis WT or B. fragilis PSA (not able to produce the polysaccharide A), or coinfected with B. henselae and B. fragilis, WT or PSA respectively, to assess whether the polysaccharide A had a role in the response of these cells to the infection. After the ultrastructural characterization of bacterial strains used in our experiments, Bacteroides ability to infect EPC was assessed by CLSM. Both bacterial strains are internalized already at 24h after infection and at a multiplicity of infection of 100. By TEM it was observed that Bacteroides in contrast to Bartonella, are internalized activating cytoplasmic lysosomes that digest them. B. henselae instead infects those cells forming invasomes in which they continues to proliferate. So polysaccharide A accentuates the macrophagic features that these cells have in their early differentiation stage. Analysis of genic expression using real time PCR from total RNA extracted by the cell cultures have shown the up-regulation of the gene coding for the IL-10 anti-inflammatory cytokine in cells co-infected with B. fragilis WT and B. henselae compared to the cells infected only with B. henselae. The main inflammatory cytokines secretion evaluated by ELISA has also evidenced an increase of IL-10 produced by cells coinfected with B. fragilis WT and B. henselae. These cells react to the co-infection in the same manner of macrophages. These data assert that the EPC does not play only a key role in vasculogenesis, but in an adult organism they could also play an essential role in the immune response. The EPC may collaborate with macrophages and T cells CD4+, to the inflammatory process triggered by the polysaccharide A after infection.

In this chapter we will describe the use of the tubular network assay as a useful tool in the evaluation of cancer neoangiogenesis (as reported by Arnaoutova et al. [Angiogenesis 12:267–274, 2009]). Angiogenesis is involved not only in pathological conditions including cancer biology and non-neoplastic diseases, but also many biological processes including reproduction, development and repair. A detailed working protocol applicable to the study of any type of endothelial cells is provided below and in addition, details on data interpretation analysis and troubleshooting.

none 8 si Combinations between an angiotensin converting enzyme (ACE) inhibitor or an angiotensin II receptor blocker (ARB) and hydrochlorothiazide (HCTZ) are among the recommended treatments for hypertensive patients uncontrolled by monotherapy. Four randomized, double-blind, parallel group studies with a similar design, including 1469 hypertensive patients uncontrolled by a previous monotherapy and with ≥1 cardiovascular risk factor, compared the efficacy of a combination of a sulfhydryl ACE inhibitor (zofenopril at 30 or 60 mg) or an ARB (irbesartan at 150 or 300 mg) plus HCTZ 12.5 mg. The extent of blood pressure (BP)-lowering was assessed in the office and over 24 h. Pleiotropic features of the treatments were evaluated by studying their effect on systemic inflammation, organ damage, arterial stiffness, and metabolic biochemical parameters. Both treatments similarly reduced office and ambulatory BPs after 18–24 weeks. In the ZODIAC study a larger reduction in high sensitivity C reactive protein (hs-CRP) was observed under zofenopril (−0.52 vs. +0.97 mg/dL under irbesartan, p = 0.001), suggesting a potential protective effect against the development of atherosclerosis. In the ZENITH study the rate of carotid plaque regression was significantly larger under zofenopril (32% vs. 16%; p = 0.047). In the diabetic patients of the ZAMES study, no adverse effects of treatments on blood glucose and lipids as well as an improvement of renal function were observed. In patients with isolated systolic hypertension of the ZEUS study, a slight and similar improvement in renal function and small reductions in pulse wave velocity (PWV), augmentation index (AI), and central systolic BP were documented with both treatments. Thus, the fixed combination of zofenopril and HCTZ may have a relevant place in the treatment of high-risk or monotherapy-treated uncontrolled hypertensive patients requiring a more prompt, intensive, and sustained BP reduction, in line with the recommendations of current guidelines. none Omboni, Stefano; Malacco, Ettore; Napoli, Claudio; Modesti, Pietro Amedeo; Manolis, Athanasios; Parati, Gianfranco; Agabiti-Rosei, Enrico; Borghi, Claudio Omboni, Stefano; Malacco, Ettore; Napoli, Claudio; Modesti, Pietro Amedeo; Manolis, Athanasios; Parati, Gianfranco; Agabiti-Rosei, Enrico; Borghi, Claudio

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.

HBV still represents a global risk factor in transfusion medicine. The residual risk of HBV is not limited to pre-seroconversion window period but it extends to donors with occult HBV infection (OBI) characterized by the presence of HBV DNA in liver and by the absence of the virus surface antigen. Each country developed an appropriate blood screening policy according to local HBV prevalence, yields of infectious units per different screening methods and cost-effectiveness. We underline the need of maintaining a high level of attention for OBI carrier identification in all blood banks worldwide where the screening procedures are generally based on a combination of both serological markers and nucleic acid amplification test. In this context, markers such as hepatitis B surface antibodies and hepatitis B core antibodies (anti-HBc) might be useful, although the use of this latter is highly debated and still controversial. Our aim is to give an overview on the relevant diagnostic approaches for the routine screening for HBV focusing on the feasibility of anti-HBc testing as precautionary measure in preventing OBI transmission worldwide. In our tailored algorithm, the loss of about 1% of ‘anti-HBc only' donors, does not significantly affect the blood supply while improving recipient safety.

Red cell alloimmunization is a serious problem in chronically transfused patients. A number of high-throughput DNA assays have been developed to extend or replace traditional serologic antigen typing. DNA-based typing methods may be easily automated and multiplexed, and provide reliable information on a patient. Molecular genotyping promises to become cheaper, being not dependent on serologic immunoglobulin reagents. Patients with hemoglobinopathies could benefit from receiving extended genomic typing. This could limit post transfusional complications depending on subtle antigenic differences between donors and patients. Patient/donor compatibility extended beyond the phenotype Rh/Kell may allows improved survival of transfused units of red blood cells (RBC) and lead to reduced need for blood transfusion and leading to less iron overload and reduced risk of alloimmunization. Here we discuss the advantages and limitations of current techniques, that detect only predefined genetic variants. In contrast, target enrichment next-generation sequencing (NGS) has been used to detect both known and de novo genetic polymorphisms, including single-nucleotide polymorphisms, indels (insertions/deletions), and structural variations. NGS approaches can be used to develop an extended blood group genotyping assay system.

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.

OBJECTIVE Whether all antihypertensive drugs are equally effective in patients with metabolic syndrome is still unclear. The goal of the Zofenopril in Advanced MEtabolic Syndrome (ZAMES) study was to investigate whether treatment with the fixed-dose combination of sulphydril-containing angiotensin-converting enzyme inhibitor zofenopril plus hydrochlorothiazide is at least as effective as that with the angiotensin receptor blocker irbesartan plus hydrochlorothiazide in patients with metabolic syndrome and essential hypertension, uncontrolled by a previous monotherapy. METHODS We enrolled 721 patients in a multicenter, international (Italy and Romania), randomized, double-blind, parallel group, phase III study. Following a 1-week screening withdrawal period, 482 patients (mean age 59 ± 10 years, 53% men) bearing a SBP at least 140 mmHg and/or DBP at least 90 mmHg plus metabolic syndrome (ATP-III criteria) were randomly allocated to a fixed-dose combination of zofenopril 30 mg plus hydrochlorothiazide 12.5 mg or irbesartan 150 mg plus hydrochlorothiazide 12.5 mg once daily for a cumulative period of 24 weeks. After 8 and 16 weeks, zofenopril and irbesartan doses were doubled in nonnormalized study participants. The study endpoint was the office DBP reduction at study end. In 20% of patients, an ambulatory blood pressure monitoring was performed. RESULTS The prevalence of diabetes at baseline was significantly (P

Recent efforts in transfusion medicine are focused on improving blood safety as well as establishing effective and efficient diagnostic algorithms for donor screening. To date, syphilis is a transfusion-transmitted infection re-emerged in many countries as a public health threat especially among populations at specific risk. This task requires new diagnostic tools and hemovigilance programs. The current diagnostic methodologies are debated, since presenting limitations and unresolved issues with special regard to the clinical interpretation of serological patterns, especially in asymptomatic patients and in blood donors. Furthermore, the switch from the traditional to alternative diagnostic algorithms underlines the lack of a gold standard, which has not been supported by shared guidelines. Besides, a lot of ongoing clinical trials on the performance of diagnostic assays, on the serological response associated with different pharmacological treatments, as well as on the prevention programs are currently under investigation. Here, we review the recent literature about the diagnosis of syphilis especially for low-risk populations proposing the adoption of an algorithm for blood donor screening that should satisfy the need of increasing safety for transfusion-transmitted infections in the modern blood transfusion centers.

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

Aim. Sulfhydryl angiotensin-converting enzyme (ACE) inhibitors exert antiatherosclerotic effects in preclinical models and antioxidant effects in patients. However, whether ACE inhibitors have any clinically significant antiatherogenic effects remains still debated. Objectives. In mildly hypertensive patients, we evaluated the effect of the sulfhydryl ACE inhibitor zofenopril in comparison with the carboxylic ACE inhibitor enalapril on carotid atherosclerosis (intima-media thickness [IMT] and vascular lumen diameter) and systemic oxidative stress (nitrite/nitrate, asymmetrical dimethyl-L-arginine, and isoprostanes). Material and methods. In 2001, we started a small prospective randomized clinical trial on 48 newly diagnosed mildly hypertensive patients with no additional risk factors for atherosclerosis (eg, hyperlipidemia, smoke habit, familiar history of atherosclerosisrelated diseases or diabetes). Patients were randomly assigned either to the enalapril (20 mg/d, n = 24) or the zofenopril group (30 mg/d, n = 24); the planned duration of the trial was 5 years. Carotid IMT and vascular lumen diameter were determined by ultrasonography for all patients at baseline and at 1, 3, and 5 years. Furthermore, nitrite/nitrate, asymmetrical dimethyl-L-arginine, and isoprostane levels were measured. Results. In our conditions, IMT of the right and left common carotid arteries was similar at baseline in both groups (P = NS). Intima-media thickness measurements until 5 years revealed a significant reduction in the zofenopril group but not in the enalapril group (P b .05 vs enalapril-treated group). This effect was coupled with a favorable nitric oxide/oxidative stress profile in the zofenopril group. Conclusion. Long-term treatment with the sulfhydryl ACE inhibitor zofenopril besides its blood pressure–lowering effects may slow the progression of IMT of the carotid artery in newly diagnosed mildly hypertensive patients. (Am Heart J 2008;156:1154.e1-1154.e8.)

Sensitized candidates for heart transplant usually end up on a long waiting list and have an increased risk of rejection, graft loss, and incidence of cardiac allograft vasculopathy. An increasing number of studies have demonstrated the negative effect of preformed and posttransplant antibodies on graft survival. Thus, in sensitized patients, the combination of new, appropriate, desensitization protocols, and monitoring of posttransplant development of donor-specific antibodies may improve short-term and long-term outcomes. Introduction of more-sensitive and more-specific techniques for antibody detection provides a valid tool for assessing the degree of pretransplant HLA histocompatibility, and, therefore, predicting the results of crossmatch in sensitized patients, which are difficult to transplant. Currently, there are no accurate and standard methods to determine the functional characteristics of antibodies detected by solid-phase assay and, therefore, to predict their clinical relevance. Therefore, the future of heart transplantation requires a better understanding of tissue typing techniques and the effect of anti-HLA antibodies on clinical outcome to prevent discrimination against sensitized patients at the time of organ allocation.

The endothelium represents the largest functional organ in the human body playing an active role in vasoregulation, coagulation, inflammation, and microvascular permeability. Endothelium contributes to maintain vascular integrity, intravascular volume, and tissue oxygenation promoting inflammatory network response for local defense and repair. Acid-basis homeostasis is an important physiologic parameter that controls cell function, and changes in pH can influence vascular tone by regulating endothelium and vascular smooth muscle cells. This review presents a current perspective of the effects of intracellular acidosis on the function and the basic regulatory mechanisms of endothelial cells.

Mediator (MED) is a fundamental component of the RNA polymerase II-mediated transcription machinery. This multiprotein complex plays a pivotal role in the regulation of eukaryotic mRNA synthesis. The yeast Mediator complex consists of 26 different subunits. Recent studies indicate additional pathogenic roles for Mediator, for example during transcription elongation and non-coding RNA production. Mediator subunits have been emerging also to have pathophysiological roles suggesting MED-dependent therapeutic targets involving in several diseases, such as cancer, cardiovascular disease (CVD), metabolic and neurological disorders.

The CD146 cell membrane adhesion molecule is highly expressed on the cell surface of several tumours. The level of its expression has been found to correlate directly with tumour progression and metastatic potential, thus establishing CD146 as an important candidate of tumour growth and metastasis. In order to characterize its expression in human osteosarcoma (OS) cell lines, we have examined the CD146 expression at protein and RNA levels in both normal and tumour osteoblast-like cell lines by several methods. Our results indicate that CD146 protein is expressed at low levels in normal osteoblast cells whereas it is highly expressed in all OS cell lines analysed, (SaOS, MG-63, U-2OS). Moreover, CD146 overexpression was partially reduced in shYY1 cells, where the Yin Yang 1 transcription factor, also found over-expressed in human OS cells, has been silenced.

In the 1970s, pharmacological therapy interrupting the renin-angiotensin system was considered beneficial for patients with high-renin hypertension. Angiotensin-converting enzyme (ACE) inhibitors proved to be effective not only in patients with high renin and elevated blood pressure, but also in many hypertensive patients with normal levels of plasma renin activity. ACE inhibitors are used in a wide range of chronic illnesses such as atherosclerosis, hypertension, myocardial infarction, heart failure, diabetic complications, and stroke. To date, more than ninety controlled clinical trials evaluating the beneficial effects of 14 different ACE inhibitors were conducted. Moreover, data from experimental studies showed that ACE inhibitors can attenuate the development of atherosclerosis, oxidative stress, and vascular inflammation in a wide range of species indicating that ACE inhibition also favourably affects the vasculature. More than fifteen years ago, the bi-sulfydryl ACE-inhibitor zofenopril has shown an excellent clinical safety and efficacy in patients with hypertension and in those with myocardial infarction. More recently, this compound exhibited a potent antioxidant and antiatherosclerotic effect indicating a clinical useful vasoprotective action.

Background: Age-related effects on the ability of 6-min walking test (6MWT) and ejection fraction (EF) to predict mortality in coronary artery bypass grafting (CABG) patients undergoing cardiac rehabilitation (CR) is still debated. Design and methods: In order to verify the role of 6MWT and EF on all-cause mortality in patients undergoing CR following CABG, 882 CABG patients undergoing CR stratified in adults (

Several studies indicate that impaired foetal growth, and in utero exposure to risk factors, especially maternal hypercholesterolaemia, may be relevant for the early onset of cardiovascular damage. The exact molecular mechanisms of such foetal programming are still unclear. Epigenetics may represent one of the possible scientific explanations of the impact of such intrauterine risk factors for the subsequent development of cardiovascular disease (CVD) during adulthood. Translational studies support this hypothesis; however, a direct causality in humans has not been ascertained. This hypothesis could be investigated in primates and in human post-mortem foetal arteries. Importantly, some studies also suggest the transgenerational transmission of epigenetic risk. The recently launched International Human Epigenome Consortium and the NIH Roadmap Epigenomics Mapping Consortium will provide the rationale for a useful clinical scenario for primary prevention and therapy of CVD. Despite the heritable nature of epigenetic modification, the clinically relevant information shows that it could be reversible through therapeutic approaches, including histone deacetylase inhibitors, histone acetyltransferase inhibitors, and commonly used drugs such as statins.

PURPOSE: The pathogenic role of angiotensin-converting enzyme (ACE) inhibition in hypertensive patients regarding endothelial progenitor-cell (EPC) function is still poorly understood. The aim of the study was to evaluate EPC number, function, and relationship to carotid intima media thickness (IMT) progression. METHODS: We studied 36 newly diagnosed mildly hypertensive patients free of cardiovascular disease and related risk factors without prior or concurrent therapy with ACE inhibitors. Patients were randomized to receive enalapril 20 mg/day (n = 18) or zofenopril 30 mg/day (n = 18). EPC number and migrating capacity, plasma nitrite and nitrate (NOx), and isoprostane concentrations were evaluated. Carotid IMT was determined by ultrasonography at baseline and after 1 and 5 years of follow-up. RESULTS: EPC number increased during the follow-up, with no statistical differences between treatment groups. There was an inverse correlation between circulating EPCs and IMT increase over time. Plasma NOx decreased during the study without evident differences between treatment groups. Isoprostanes decreased more markedly in zofenopril-treated patients. Multiple linear regression model demonstrated that carotid IMT was significantly inversely correlated with EPC but not with migratory cells after adjusting for confounders. CONCLUSIONS: The study demonstrated that EPC levels increased during the follow-up in both groups of newly diagnosed hypertensive patients treated with ACE inhibitors. These drugs prevented progression of vascular damage, with an inverse correlation between circulating EPC levels and IMT values.

The endothelial progenitor cells (EPCs) are angiogenic cells having properties similar to those of embryonal angioblasts. The number and function of EPCs are affected by a variety of conditions, including cytokines and chemokines, which are pivotal inflammatory signaling molecules. The purpose of this paper is to review current knowledge about the role of these progenitor in different vascular diseases, emphasizing the important biological role played from the CXCR4-CXCL12 axis in the cellular trafficking. Indeed, as described in detail in this review, the CXCR4/CXCL12 interaction produces pleiotropic effects in stem cells and plays a pivotal role in several processes related to development, tissue regeneration and development/progression of malignancies.

Mediator of RNA polymerase II transcription subunits (MEDs) serve to promote the assembly, activation, and regeneration of transcription complexes on core promoters during the initiation and re-initiation phases of transcription. There are no studies on the Mediator complex during development of endothelial progenitors (EPCs). Here, we have analysed all known MEDs during the differentiation of EPCs, by expression profile studies at RNA level and, for a limited subset of MED subunits, also at protein level. Since beneficial effects of l-arginine on EPCs have been described, we have also examined its effect on the expression of Mediator subunit coding genes. Through RT-PCR we have found increased expression for MED12 and decreased levels for MED30 after l-arginine treatment; Western blot analysis do not agree entirely with the RNA data in the identification of a putative protein product. Furthermore, we have analysed the three-dimensional nuclear positions of MED12 and MED30 genes in the presence of l-arginine treatment. Our major finding is the identification of a novel transcript of MED30, termed MED30 short (MED30s) generating by alternative splicing. Our results showed that the mRNA of this novel isoform is present only in circulating cells, but it is not expressed in cultured adherent cells. These findings are broadly relevant and will contribute to our understanding of the role of Mediator in eukaryotic gene expression. Despite the need to confirm the in vivo presence of the protein of this novel isoform, the presence of this novel RNA raises the possibility of regulating pathophysiological mechanism in progenitors. © 2010 Elsevier B.V. All rights reserved.

The prognostic role of human leukocyte antigen (HLA) matching in heart transplantation (HTx) is still unclear. We previously demonstrated an association of DR-mismatch with early mortality after HTx. Aim of the study was to evaluate the impact of HLA mismatch on 1 year acute cellular rejection (ACR

Angiotensin-converting enzyme (ACE) 2 is a recently identified homologue of ACE. There is great interest in the therapeutic benefit for ACE2 overexpression in the heart. However, the role of ACE2 in the regulation of cardiac structure and function, as well as maintenance of systemic blood pressure, remains poorly understood. In cell culture, ACE2 overexpression led to markedly increased myocyte volume, assessed in primary rabbit myocytes. To assess ACE2 function in vivo, we used a recombinant adeno-associated virus 6 delivery system to provide 11-week overexpression of ACE2 in the myocardium of stroke-prone spontaneously hypertensive rats. ACE2, as well as the ACE inhibitor enalapril, significantly reduced systolic blood pressure. However, in the heart, ACE2 overexpression resulted in cardiac fibrosis, as assessed by histological analysis with concomitant deficits in ejection fraction and fractional shortening measured by echocardiography. Furthermore, global gene expression profiling demonstrated the activation of profibrotic pathways in the heart mediated by ACE2 gene delivery. This study demonstrates that sustained overexpression of ACE2 in the heart in vivo leads to the onset of severe fibrosis.

Studies have shown that some of statin's pleiotropic effects were achieved by either promotion or inhibition of angiogenesis, depending on the underlying disease. This study tested the hypothesis that the angiogenic potential of simvastatin is related to the microenvironmental conditions.Human umbilical vein endothelial cells (HUVEC) were studied after exposure to hypoxia or the inflammatory factors tumor necrosis factor (TNF)-alpha, with or without co-incubation with simvastatin (1 micromol/L) and mevalonate. HUVEC angiogenesis was evaluated by tube formation, migration, and proliferation assays. Hypoxia inducible factor (HIF)-1alpha, vascular endothelial growth factor (VEGF), Akt, endothelium nitric oxide synthase (e-NOS), and oxidative stress were evaluated.HUVEC angiogenesis increased during hypoxia (tube length 14.7+/-0.5 vs. 7.8+/-0.6 mm, p0.05) and further enhanced by simvastatin (19.3+/-1.1 mm, p0.05 vs. hypoxia alone), which downregulated the expression of the HIF-1 inhibitor PHD2 and upregulated HIF-1alpha, VEGF, and Akt, without changing oxidative stress or eNOS. Incubation with TNF-alpha promoted HUVEC angiogenesis (7.4+/-0.2 vs. 6.5+/-0.2 mm, p0.05) with increased oxidative stress. However, simvastatin inhibited this promotion (2.5+/-0.3 mm, p0.001 vs. TNF-alpha alone) by decreasing oxidative stress, VEGF, Akt, and eNOS.We conclude that at the same dosage, simvastatin can either promote or inhibit angiogenesis, possibly by activating upstream regulators of HIF-1alpha in hypoxia, but conversely interfering with angiogenic signaling downstream to inflammation. These opposing angiogenic effects should be considered in the therapeutic strategies with statins.

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, p0.01). 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 (p0.05) and those receiving carvedilol (p0.05). 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.

Increasing evidence indicates that mammalian SIRT1 mediates calorie restriction and influences lifespan regulating a number of biological molecules such as FoxO1. SIRT1 controls the angiogenic activity of endothelial cells via deacetylation of FoxO1. Endothelial dysfunction and reduced new blood vessel growth in diabetes involve a decreased bioactivity of endothelial progenitor cells (EPCs) via repression of FoxO1 transcriptional activity. The relative contribution of SIRT1 with respect to the direct effects of high glucose on EPC number is poorly understood. We report that treatment of EPCs with high glucose for 3 days determined a consistent downregulation of EPC positive to DiLDL/lectin staining and, interestingly, this was associated with reduced SIRT1 expression levels and enzyme activity, and increased acetyl-FoxO1 expression levels. Moreover, EPCs responded to high glucose with major changes in the expression levels of cell metabolism-, cell cycle-, and oxidative stress-related genes or proteins. Proteomic analysis shows increased expression of nicotinamide phosphorybosyl transferase and mitochondrial superoxide dismutase whereas a glucose-related heat shock protein is reduced. These findings show that SIRT1 is a critical modulator of EPCs dysfunction during alteration of glucose metabolism. © 2008 Elsevier B.V. All rights reserved.

Background The custom microenvironment ‘vascular niche’ is a potential therapeutic target for several pathophysiological conditions. Osteoblasts regulate the hematopoietic stem cell niche, and activation of the parathyroid hormone (PTH) receptor can increase the number of cells mobilized into the bloodstream. Methods C57Bl/6 mice were randomly assigned treatment with granulocyte-colony stimulating factor (G-CSF), PTH, G-CSF plus PTH or saline. All mice underwent hindlimb ischemia. Blood flow was measured by laser Doppler imaging. Indices of capillary activity were determined by electron microscopy in muscle tissue. CD34+ and Ki67+ cells were detected and evaluated by immunofluorescence, apoptosis by TUNEL, surface antigen and endothelial progenitor cells by fluorescence-activated cell sorting analysis, and vascular endothelial growth factor-164 and angiopoietin-1 expression by reverse-transcriptase polymerase chain reaction. Frozen bone marrow sections were stained for antigen-specific B cells and fibronectin and analyzed by confocal laser scanning microscopy. Results Following mobilization induced by G-CSF treatment, mice also treated with PTH showed increases in blood flow, capillary density, nitrite/nitrate release, angiogenic factors and circulating progenitor cells, as well as reduced apoptosis, fibrosis, oxidative stress and inflammation in ischemic muscles. Furthermore, hematopoietic antigen-specific B cells in the bone marrow were also increased by G-CSF alone and in combination with PTH. Conclusions PTH might increase the efficiency of hematopoietic stem-cell-based therapy in a recognized model of peripheral ischemia. Our translational experimental therapeutic targeting of the vascular niche points to novel clinical targets for the hematopoietic stem-cell treatment of ischemic vascular diseases.

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.

The circulating form of endothelial progenitors cells (EPCs) are derived from bone marrow (BM)-derived hematopoietic stem cells (HSCs). Enhanced mobilization of EPCs was shown to be linked to cardiac diseases. This study investigated whether reduced EPC levels in advanced coronary heart disease (CHD) are secondary to a functional exhaustion of HSCs in the BM or to reduced mobilization. Number and functional properties of EPCs were assessed in 15 healthy controls, and 40 patients with CHD. The colony-forming unit (CFU) capacity of BM-derived mononuclear cells and the CD34+ HSC number were examined in four healthy volunteers, and 15 CHD patients. EPC number was reduced in CHD patients (P < 0.01 vs. controls). Moreover, the migratory capacity was significantly impaired in EPCs of CHD patients (P < 0.05 vs. controls). On multivariate analysis, CHD was an independent predictor of functional EPC impairment. CFUs were reduced in CHD patients (59.6 +/- 21.2 vs. 75.4 +/- 25.8 in controls, P < 0.05). CHD was also predictor of impaired CFU capacity. In this small clinical study, CHD is associated with selective impairment of HSC function in the BM and in the peripheral blood, which may contribute to impairment of cardiac function.

SummaryAdenoviruses are used extensively as gene transfer agents, both experimentally and clinically. However, targeting of liver cells by adenoviruses compromises their potential efficacy. In cell culture, the adenovirus serotype 5 fiber protein engages the coxsackievirus and adenovirus receptor (CAR) to bind cells. Paradoxically, following intravascular delivery, CAR is not used for liver transduction, implicating alternate pathways. Recently, we demonstrated that coagulation factor (F)X directly binds adenovirus leading to liver infection. Here, we show that FX binds to the Ad5 hexon, not fiber, via an interaction between the FX Gla domain and hypervariable regions of the hexon surface. Binding occurs in multiple human adenovirus serotypes. Liver infection by the FX-Ad5 complex is mediated through a heparin-binding exosite in the FX serine protease domain. This study reveals an unanticipated function for hexon in mediating liver gene transfer in vivo.

To date, there is no report on the effect of antioxidants on endothelial progenitor cells (EPCs). This study shows that in vitro incubation of EPCs with vitamin C and E reverted the already well documented lowering effect of TNF-alpha on EPC number and increased p-p38 expression levels. In order to document major changes of gene expression levels and gain insight into signalling pathways, microarray analysis was performed and a significant variation of the expression of 5389 genes in EPCs following antioxidant treatment was detected. Also in vivo evidence is provided about the positive effect of antioxidant vitamins on EPCs, since vitamin C and E supplementation potentiated the physical training-induced increase of EPC number and VEGF levels. Together, these data indicate that antioxidant treatment ameliorates EPC number and causes major changes of gene expression within these cells in vitro. Furthermore, concomitant antioxidant supplementation and physical training in vivo raised the levels of circulating EPCs and serum VEGF more than physical training alone.

Despite the use of systems biology is still at the beginning in the study of complex diseases, the '-omics'strategies are already contributing to novel insights into atherosclerosis and CVDs. The promise of this approach includes disease risk prediction and prevention as well as the possibility of personalized therapy. In this concern, research interest has been recently focused on evaluating the role of endothelial progenitor cells in tissue vascularization and regeneration. This circulating cell population has also been proposed as potential clinical marker for several diseases including cardiovascular ones, where they have begun to be used as a new therapeutic option [23]. Recent in vivo proteomic data suggest a potential effect of acute coronary syndrome in the protein profiles of these cells [24]. These findings suggest that integrated 'omics technologies, including metabolomics, may help to better characterize these cells and understand their biology as well as their pathophysiological role in CVD and tissue regeneration.

The growing improvements of computed tomography have made this technique more and more available for cardiac evaluation. Coronary artery anomalies (CAAs) are often incidental findings in subjects with suspected coronary artery disease (CAD) undergoing coronary angiography or computed tomography coronary angiography (CTCA). In some cases, CAAs can be clinically relevant so their identification could change radically patient management and treatment. We report the case of a 68-year-old male patient with known CAD and associated anomalous origination of the left coronary artery from the opposite sinus.

The complex polymorphism of the HLA genes and the need of a proper identification of anti-HLA antibodies have led to continuously develop novel practical and feasible technologies in the field of organ and tissue transplantation. Technologies to identify HLA molecules have evolved from the serological to the molecular methods and a true innovation in the DNA sequencing has taken place with the development of next generation sequencing. An interesting field to explore is how the information resulting from the HLA-DNA sequencing can be applied in the clinical setting by including the alloimmunization assessment. Indeed, a good characterization of anti-HLA antibody at epitope level can reduce the risk of immunization. Many anti-HLA antibodies are specific for epitopes rather than for HLA antigens and the knowledge of unacceptable epitopes allows to reduce the number of mismatched antigens. Furthermore, high resolution HLA allele typing could help to understand the epitopes against which antibodies are developed. However, the improvements should not only concern the diagnostic tools in the pre-transplantation phase, but also the monitoring of post transplantation outcome. There is a growing interest in developing new non-invasive biomarkers to monitor the rejection. Currently, increasing evidence has focused on miRNAs, epigenetic markers emerged as regulators of molecular events that are differently expressed in biopsies and blood as well as in urinary samples of transplanted recipients. The implementation of next generation sequencing and genome-wide expression analysis together with functional assays may provide useful tools to evaluate the epigenetic modulation in transplantation biology but many efforts are requested for translating in the clinical arena the results obtained in experimental models.

Background Circulating endothelial progenitors cells (EPCs) play a critical role in neovascularization and endothelial repair. There is a growing evidence that hyperglycemia related to Diabetes Mellitus (DM) decreases EPC number and function so promoting vascular complications. Aim of the Study This study investigated whether an intensive glycemic control regimen in Type 2 DM can increase the number of EPCs and restores their function. Methods Sixty-two patients with Type 2 DM were studied. Patients were tested at baseline and after 3 months of an intensive regimen of glycemic control. The Type 2 DM group was compared to control group of subjects without diabetes. Patients with Type 2 DM (mean age 58.2±5.4 years, 25.6% women, disease duration of 15.4±6.3 years) had a baseline HgA1c of 8.7±0.5% and lower EPC levels (CD34+/KDR+) in comparison to healthy controls (p

Epigenetic modifications include DNA methylation, histone modifications, and microRNA. Gene alterations have been found to be associated with cardiovascular diseases, and epigenetic mechanisms are continuously being studied to find new useful strategies for the clinical management of afflicted patients. Numerous cardiovascular disorders are characterized by the abnormal methylation of CpG islands and so specific drugs that could inhibit DNA methyltransferase directly or by reducing its gene expression (e.g., hydralazine and procainamide) are currently under investigation. The anti-proliferative and anti-inflammatory properties of histone deacetylase inhibitors and their cardio-protective effects have been confirmed in preclinical studies. Furthermore, the regulation of the expression of microRNA targets through pharmacological tools is still under development. Indeed, large controlled trials are required to establish whether current possible candidate antisense microRNAs could offer better therapeutic benefits in clinical practice. Here, we updated therapeutic properties, side effects, and feasibility of emerging epigenetic-based strategies in cardiovascular diseases by highlighting specific problematic issues that still affect the development of large scale novel therapeutic protocols.