Sensitive and quantitative method to evaluate DNA methylation of the positive regulatory domains (PRDI, PRDII) and cAMP response element (CRE) in human endothelial nitric oxide synthase promote

Nitric oxide plays a prominent role in the cardiovascular system and much attention has been devoted in the last years on deciphering the regulation of human endothelial nitric oxide synthase (eNOS) expression. Epigenetic-based mechanisms have a key role in the eNOS expression and their pathologic perturbations may have profound effects on the steady state RNA levels in the endothelium. The human eNOS promoter lacks a canonical TATA box and it does not contain a proximal CpG island. A differentially DNA methylated region (DMR) in the native eNOS proximal promoter is involved in gene expression regulation. Here we describe a quantitative, sensitive and cost-effective method that, relying on a novel normalization strategy, allows the quantification of DNA methylation status of the positive regulatory domains (PRDI, PRDII) and cAMP response element (CRE) in human eNOS promoter. This technique will enable to explore the functional relevance of DNA methylation perturbations of eNOS promoter both under pathological and physiological conditions

Adventitial vessel growth and progenitor cells activation in an ex vivo culture system mimicking human saphenous vein wall strain after coronary artery bypass grafting

Abstract Saphenous vein graft disease is a timely problem in coronary artery bypass grafting. Indeed, after exposure of the vein to arterial blood flow, a progressive modification in the wall begins, due to proliferation of smooth muscle cells in the intima. As a consequence, the graft progressively occludes and this leads to recurrent ischemia. In the present study we employed a novel ex vivo culture system to assess the biological effects of arterial-like pressure on the human saphenous vein structure and physiology, and to compare the results to those achieved in the presence of a constant low pressure and flow mimicking the physiologic vein perfusion. While under both conditions we found an activation of Matrix Metallo-Proteases 2/9 and of microRNAs-21/146a/221, a specific effect of the arterial-like pressure was observed. This consisted in a marked geometrical remodeling, in the suppression of Tissue Inhibitor of Metallo-Protease-1, in the enhanced expression of TGF-\u3b21 and BMP-2 mRNAs and, finally, in the upregulation of microRNAs-138/200b/200c. In addition, the veins exposed to arterial-like pressure showed an increase in the density of the adventitial vasa vasorum and of cells co-expressing NG2, CD44 and SM22\u3b1 markers in the adventitia. Cells with nuclear expression of Sox-10, a transcription factor characterizing multipotent vascular stem cells, were finally found in adventitial vessels. Our findings suggest, for the first time, a role of arterial-like wall strain in the activation of pro-pathologic pathways resulting in adventitial vessels growth, activation of vasa vasorum cells, and upregulation of specific gene products associated to vascular remodeling and inflammation

Reducing the environmental impact of surgery on a global scale: systematic review and co-prioritization with healthcare workers in 132 countries

Background Healthcare cannot achieve net-zero carbon without addressing operating theatres. The aim of this study was to prioritize feasible interventions to reduce the environmental impact of operating theatres. Methods This study adopted a four-phase Delphi consensus co-prioritization methodology. In phase 1, a systematic review of published interventions and global consultation of perioperative healthcare professionals were used to longlist interventions. In phase 2, iterative thematic analysis consolidated comparable interventions into a shortlist. In phase 3, the shortlist was co-prioritized based on patient and clinician views on acceptability, feasibility, and safety. In phase 4, ranked lists of interventions were presented by their relevance to high-income countries and low–middle-income countries. Results In phase 1, 43 interventions were identified, which had low uptake in practice according to 3042 professionals globally. In phase 2, a shortlist of 15 intervention domains was generated. In phase 3, interventions were deemed acceptable for more than 90 per cent of patients except for reducing general anaesthesia (84 per cent) and re-sterilization of ‘single-use’ consumables (86 per cent). In phase 4, the top three shortlisted interventions for high-income countries were: introducing recycling; reducing use of anaesthetic gases; and appropriate clinical waste processing. In phase 4, the top three shortlisted interventions for low–middle-income countries were: introducing reusable surgical devices; reducing use of consumables; and reducing the use of general anaesthesia. Conclusion This is a step toward environmentally sustainable operating environments with actionable interventions applicable to both high– and low–middle–income countries

Epigenetic Programming and Risk : the Birthplace of Cardiovascular Disease?

Epigenetics, through control of gene expression circuitries, plays important roles in various physiological processes such as stem cell differentiation and self renewal. This occurs during embryonic development, in different tissues, and in response to environmental stimuli. The language of epigenetic program is based on specific covalent modifications of DNA and chromatin. Thus, in addition to the individual identity, encoded by sequence of the four bases of the DNA, there is a cell type identity characterized by its positioning in the epigenetic "landscape". Aberrant changes in epigenetic marks induced by environmental cues may contribute to the development of abnormal phenotypes associated with different human diseases such as cancer, neurological disorders and inflammation. Most of the epigenetic studies have focused on embryonic development and cancer biology, while little has been done to explore the role of epigenetic mechanisms in the pathogenesis of cardiovascular disease. This review highlights our current knowledge of epigenetic gene regulation and the evidence that chromatin remodeling and histone modifications play key roles in the pathogenesis of cardiovascular disease through (re)programming of cardiovascular (stem) cells commitment, identity and function

Methylation profiling by bisulfite sequencing analysis of the mtDNA Non-Coding Region in replicative and senescent Endothelial Cells

The regulation and function of Mitochondrial DNA (mtDNA) cytosine methylation (5mC) are largely unexplored. Mitochondria, Endothelial Cell (EC) senescence, and cardiovascular dysfunction are closely related. We extensively investigated the mtDNA Non-Coding Region (NCR) methylation pattern and its variations in EC replicative senescence. We observed previously undescribed 5mC clusters and a biased distribution of 5mC among DNA sites and throughout the NCR. The methylation pattern in senescent EC showed non-random variations, including the hypo-methylation of mtDNA replication regulatory sites. Additional experiments opened to a possible role for 5mC in D-loop formation, rather than in mitochondrial gene expression

The Janus face of Bartonella quintana recognition by Toll-like receptors (TLRs): a review.

Contains fulltext : 70849.pdf (publisher's version ) (Closed access)Bartonella quintana (B. quintana) is a facultative, intracellular bacterium, which causes trench fever, chronic bacteraemia and bacillary angiomatosis. Little is known about the recognition of B. quintana by the innate immune system. In this review, we address the impact of Toll-like receptors (TLRs) on the recognition of B. quintana and the activation of the host defense. When experimental models using human mononuclear cells, transfected CHO cells, or TLR2-/- and TLR4-/- mice were used, differential effects of TLR2 and TLR4 have been observed. B. quintana micro-organisms stimulated cytokine production through TLR2-mediated signals, whereas no role for TLR4 in the recognition of this pathogen was observed. When single, water-phenol extraction was performed, B. quintana LPS, stimulated cytokine production in a TLR2-dependent manner. However, when double extraction was performed in order to generate highly purified LPS, B. quintana LPS entirely lost its capacity to stimulate cytokines, demonstrating that non-LPS components of B. quintana are responsible for the recognition through TLR2. Moreover, B. quintana LPS was shown to be a potent antagonist of Toll-like receptor 4 (TLR4). In conclusion, B. quintana is an inducer of cytokines through TLR2-, but not TLR4-, dependent mechanisms. This stimulation is induced by bacterial components other than lipopolysaccharide. B. quintana LPS is a naturally occurring antagonist of Toll-like receptor 4 (TLR4). In view of the role played by TLR4 in inflammation, B. quintana LPS may be useful as an anti-TLR4 agent with therapeutic potential in both infections and autoimmune inflammation