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We report the case of a 60-year-old man with patent coronary by-pass grafts on the left anterior descending and circumflex coronary arteries, who experienced recurrent Canadian class IV angina refractory to medical and interventional treatments for a dominant right coronary artery occlusion. He underwent autologous PBSC transplant into the inferior ventricular wall through a minimally invasive approach as a stand-alone therapy, in an attempt to induce therapeutic angiogenesis. Six months after the operation, angina significantly ameliorated; scintigraphy and coronary angiography showed a marked improvement in perfusion of the target injection area. These benefits had persisted at 25 months after stem cell transplant. [ABSTRACT FROM AUTHOR]

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. • eNOS, as principal NO producer, play a fundamental role in pathological and physiological cardiovascular conditions. • Epigenetic-based mechanisms have a key role in the eNOS expression. • Differentially DNA methylated region in eNOS proximal promoter determines gene expression regulation. • Sensitive and cost-effective technique to quantify the DNA methylation of PRDI, PRDII and CRE regions of eNOS promoter. [ABSTRACT FROM AUTHOR]

Funding Acknowledgements Type of funding sources: Public grant(s) – National budget only. Main funding source(s): This research was funded by the Department of Innovation, Research and University of the Autonomous Province of Bolzano-South Tyrol (Italy), and by the Joint Project Südtirol- FWF (Italy-Austria) for A.R. J.T. A.M. R.P.. Background Arrhythmogenic cardiomyopathy (ACM) is a genetic disease associated with sudden cardiac death and fibro-fatty replacement of myocardium. Recently, it has been demonstrated that oxidized lipid contributes to cardiac adipogenesis and that ACM hearts are characterized by an increase in oxidative stress. Purpose As mitochondria are an important source of reactive oxygen species (ROS) within mammalian cells, the present work aims to evaluate if increased oxidative stress observed in ACM hearts is associated with altered mitochondrial function. Methods The oxidative stress marker 4HNE was investigated together with the cardiomyocyte marker cardiac Troponin T on paraffin embedded human ventricular samples and analyzed by confocal microscopy. Human primary cardiac stromal cells (CStCs), obtained from either right ventricle biopsies of ACM patients or healthy cadaveric tissue donor (CTR), were used as cellular model as they are known for their contribution to adipogenesis in the ACM pathology. CStCs were cultured either in basal medium or adipogenic medium (ADIPO) in presence or absence of 500 nM of the ROS scavenger MitoTEMPO. After 7 days of adipogenic differentiation, intracellular lipid droplets accumulation and mitochondrial superoxide levels were measured in CStCs by confocal microscopy using BODIPY 493/503 (0.5 µM) and MitoSOX Red (5 µM) dyes, respectively. The oxidative phosphorylation (OXPHOS) and fatty acid oxidation (FAO) were also evaluated in CStCs by high resolution respirometry. Results 4HNE staining was increased in heart tissues from ACM patients compared to CTRs and particularly evident in non-cardiomyocyte cells. In agreement, a higher MitoSOX fluorescence intensity was found in ACM-CStCs compared to CTR-CStCs in ADIPO medium, confirming higher ROS presence in patient cells. The treatment with MitoTEMPO was efficient in reducing ROS abundance, strongly suggesting mitochondrial origin. Lipid accumulation in ACM-CStCs was also prevented by MitoTEMPO treatment. Preliminary evidence further indicates that mitochondrial respiratory capacity is increased in ACM-CStCs versus CTR-CStCs exposed to adipogenic medium for 7 days. However, transmission electron microscopy analysis and Western blot analysis of Mfn1/2, Opa1, Fis1 and Drp1 did not show an evident alteration neither in mitochondria ultrastructure nor in the expression of proteins regulating mitochondrial dynamics. Conclusions Our data support an alteration of mitochondrial activity in CStCs from ACM patients, apparently not linked to a modified network or morphology of mitochondria but associated with a higher ROS production. [ABSTRACT FROM AUTHOR]

Funding Acknowledgements Type of funding sources: Public grant(s) – National budget only. Main funding source(s): Italian Ministry of Health Background Heart failure with preserved ejection fraction (HFpEF) is a heterogeneous syndrome characterized by impaired left ventricular (LV) diastolic function, with normal LV ejection fraction. The most common cause is aortic valve stenosis, which provokes sustained pressure overload (PO) (1). HFpEF cardiac remodeling comprises cardiomyocyte (CM) hypertrophy and fibrotic matrix deposition (2). A link between HFpEF pathogenesis and DNA damage response (DDR) activation emerged from in vivo studies (3). DNA damage activates DDR kinases, producing the phosphorylation of H2AX histone and checkpoint proteins, CHK1 and CHK2, to orchestrate cell recovery. DDR activation contributes to mouse CM hypertrophy and inflammation, promoting cardiac remodeling and HF (4)(5). No studies of this molecular mechanism have been performed on HFpEF patient samples yet. Moreover, known the pivotal role of the stromal compartment in the myocardial response to PO (6)(7), the effects of DDR activation on cardiac mesenchymal stromal cells (C-MSC) are still to be investigated. Purpose Our aim is to address the aforementioned gaps, unraveling the effects of C-MSC DDR persistent activation on C-MSC phenotypes. Methods We collected LV septum samples from patients with HFpEF undergoing aortic valve surgery (n=7) and healthy controls (HC; n=7), both for tissue analyses and C-MSC isolation. PO-induced mechanical stimuli has been simulated in vitro by cyclic unidirectional stretch. Results Histological analyses of HFpEF tissues showed enlarged nuclei and hypertrophic cardiac fibers, increased collagen deposition, higher apoptosis and oxidative stress than HC tissues. HFpEF samples revealed DNA damage (% γH2AX positive cells p=0.047), in both CM and C-MSC. γH2AX, pCHK1, pCHK2 protein expression was higher in HFpEF total tissue (γH2AX/GAPDH p= 0.033; pCHK1/GAPDH p= 0.018; pCHK2/GAPDH p= 0.049). Primary human C-MSC isolated from HFpEF and HC cardiac tissues confirmed the increased γH2AX (γH2AX/GAPDH p=0.0030) and phosphorylated checkpoint protein expression (pCHK1/GAPDH p= 0.045; pCHK2/GAPDH p= 0.039), suggesting C-MSC involvement in DDR-driven remodeling. HFpEF C-MSC express also more pro-fibrotic and pro-inflammatory factors than HC cells. In response to in vitro mechanical stimulation, HC C-MSC increased DNA damage (% γH2AX positive cells non-stretched vs. stretched cells p=0.022) and phosphorylated checkpoint proteins, suggesting PO-guided activation of DDR. Stretched C-MSC secreted more pro-inflammatory and pro-fibrotic molecules compared to static control. Conclusion HFpEF sustained PO induces DDR persistent activation not only in CM but also in C-MSC. On C-MSC, DDR impairments are linked to inflammation and fibrosis, with direct effects on C-MSC protein expression and secretome. The factors released by C-MSC could further impair CM function. Future studies will unravel the potential anti-fibrotic and anti-hypertrophic effects of DDR inhibitors in HFpEF context. [ABSTRACT FROM AUTHOR]

Aims: The aim of this study was to assess the effect of granulocyte colony-stimulating factor (G-CSF) on left ventricular (LV) function and volumes in patients with anterior ST-elevation myocardial infarction (STEMI) and depressed LV ejection fraction (EF). Methods and Results: Sixty consecutive patients with anterior STEMI, undergoing primary angioplasty percutaneous coronary intervention (PCI), with symptom-to-reperfusion time of 2-12 h and EF ≤45% after PCI, were randomized to G-CSF 5 μg/kg b.i.d. subcutaneously (n = 24) or placebo (n = 25) for 5 days, starting <12 h after PCI. The primary endpoint was an increase from baseline to 6 months of 5% in left ventricular ejection fraction (LVEF), as measured by magnetic resonance imaging (MRI). Co-primary endpoint was a ≥20 mL difference in end-diastolic volume (EDV). Infarct size and perfusion were evaluated with late gadolinium enhancement (LGE) and gated (99m)Technetium Sestamibi single-photon emission computed tomography (SPECT). Left ventricular EDV and end-systolic volume (ESV) increased from baseline to 6 months in the placebo group (81.7 ± 24.4 to 94.4 ± 26.0 mL/m(2), P < 0.00005 and 45.2 ± 20.0 to 53.2 ± 23.8 mL/m(2), P = 0.016) but were unchanged in the G-CSF group (82.2 ± 20.3 to 85.7 ± 23.7 mL/m(2), P = 0.40 and 46.0 ± 18.2 to 48.4 ± 20.8 mL/m(2), P = 0.338). There were no significant differences in EF or perfusion between groups. A significant reduction in transmural LGE segments was seen at 6 months in the G-CSF vs. placebo groups (4.38 ± 2.9 to 3.3 ± 2.6, P = 0.04 and 4.2 ± 2.6 to 3.6 ± 2.7, P = 0.301, respectively). Significantly more placebo patients had a change in left ventricular end-diastolic volume abovethe median (9.3 mL/m(2)) when reperfusion time exceeded 180 min (median time-to-reperfusion) (P = 0.0123). Severe adverse events were similar between groups. Conclusion: Early G-CSF administration attenuates ventricular remodelling in patients with anterior STEMI and EF ≤45% after successful PCI. [ABSTRACT FROM AUTHOR]

Due to differences in their knowledge base, scientists, health care professionals, and the general public have varying perceptions of the expectations and risks of using gene therapy for the treatment of cardiovascular disease. Gene therapists are aware of the importance of selecting the right vector, the right gene, and the right administration procedure for a specific application and for characterization of the heterogeneity of the physiologic response to gene expression. They also recognize the need for large-scale clinical trials to demonstrate short- and long-term safety, in addition to efficacy, for widespread acceptance. Interventional cardiologists are more likely to judge a therapy primarily on its ability to improve cardiovascular pathophysiology and function, although they too will demand safety data. Once convinced of the benefits of a gene therapy, interventional cardiologists will rely on regulatory and reimbursement authorities to approve the procedure. Attitudes of less-specialized physicians toward gene therapy and, specifically, therapeutic angiogenesis will be influenced by the level of clinical trial activity in their country and opinions expressed in the media. However, all will want safety and efficacy data as well as clear guidance on the type of patients who should be referred for treatment. Nurses' roles are set to expand with the introduction of gene-based therapies; most will need further genetics education to rise to these challenges with confidence. The success of a gene therapy will also rely on patients' perceptions and their willingness to receive the treatment. Any misconceptions based on unreliable information sources need to be addressed. [ABSTRACT FROM AUTHOR]