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Background An increasing number of patients living with chronic ischemic cardiomyopathy (ICM) are potential candidates for cell therapy, including adipose tissue-derived mesenchymal stromal cells (ASC). However, proper assessment of clinical effects in relation to cellular effector mechanisms, such as angiogenesis, is currently lacking. Purpose To investigate the prognostic value of assessing angiogenesis non-invasively using novel PET imaging in terms of functional outcome after cell therapy in a homologous animal model of ICM. Methods Myocardial infarction was induced by permanent ligation of the left anterior descending coronary artery. Four weeks after infarction, the rats were scanned with [18F]-FDG and echocardiography, and based on left ventricular ejection fraction (LVEF) and infarct size randomized to allogeneic ASC treatment (n=14) or saline (n=9). Animals were treated using echo-guided trans-thoracic intramyocardial injections. Follow-up echocardiography was performed four weeks after treatment. Angiogenesis was assessed non-invasively using the [68Ga]-NODAGA-E[(cRGDyK)]2 (RGD) PET-tracer before treatment and two weeks after the treatment (Figure). The output was RGD-uptake by maximum standardized uptake value (SUVmax). Results RGD-uptake in the infarct area significantly decreased in the saline group (p=0.04), while this decline was not significant in the ASC group (p=0.58). There was no effect on LVEF of the cell therapy in this study (p=0.70). When the rats were grouped by RGD-uptake post treatment, the high RGD-uptake group (n=8) improved LVEF compared to the rats with medium or low RGD-uptake (n=15, p=0.04) irrespective of the treatment. This could indicate that non-invasive detection of a high degree of myocardial angiogenesis measured using RGD is predictive of improvement in cardiac pump function in ICM. Conclusions Following injection of ASC or saline, a high RGD-uptake precluded improvement in pump function. This is the first evidence of how uptake of the novel RGD tracer relates to changes in pump function in ICM and the results could affect the design of future clinical trials using regenerative therapy for ICM patients. Funding Acknowledgement Type of funding sources: Private grant(s) and/or Sponsorship. Main funding source(s): Aase and Ejnar Danielsens FondDoctor Sofus Carl Emil Friis and wife Olga Doris Friis' Scholarship Study design, RGD-uptake, and results

Background The diagnosis of ST elevation myocardial infarction (STEMI) is challenging when the culprit is in the left circumflex coronary artery (CX) territory because ST elevations are often not captured by the standard 12-lead electrocardiogram (ECG). Although, guidelines recommend the acquisition of the additional posterior leads V7-V9 (pECG) when the suspicion of acute coronary syndrome (ACS) is high and the ECG non-diagnostic, this is not routinely done. Purpose The purpose of the FLAWLESS trial, was to improve the prehospital CX STEMI diagnostic. The study consisted of 2 parts: a) a training and implementation study, and b) an outcomes study after implementation. In the implementation study we evaluated the FLAWLESS process from the paramedic's point of view on experiences, implementation of pECG lead recordings and its barriers. Methods Before initiating the trial, all active paramedics in 2 health care regions were educated via a specifically designed and mandatory online 30 min course and all 250 ambulances equipped with a SMART-CARD (instructing how to record pECG leads) and FAQ-sheet. All paramedics were invited by email to anonymously answer an online questionnaire (OQ) designed in REDCap® and interviewed. Utility-score and difficulty-score, ranging from 0 (not useful at all/very easy) to 100 (very useful/very difficult), were introduced for quantitative assessments. Results A total of 1268 paramedics were invited to answer the OQ. The response rate was intermediate at 35%. Among responders, 89% had completed the OEP. On duty 80% had used FAQ-sheet and 74% SMART-CARD in the field. The median utility scores were 80 (25th and 75th quartiles 67–90) for OEP, 79 (61–90) for FAQ-sheet and 85 (75–97) for SMART-CARD, respectively. The implementation of pECG leads recordings was fairly high – 54% reported always recording V7-V9 in ACS patients and 36% reported doing it frequently. Difficulty-score for recording V7-V9 leads in the prehospital setting was 50 (19–70). Finally, 43% reported difficulties that were related to technicalities i.e. defibrillators not having dedicated V7, V8 and V9 cables, hence ambulance staff is forced to record and transmit a second ECG after moving the V4, V5 and V6 cables to the V7-V9 positioned electrodes. Conclusion We demonstrated that large-scale online training of paramedics in the recording of prehospital 15-lead ECG is feasible. The evaluation was positive regarding training and support tools in the ambulances but almost 50% of paramedics found the recording very difficult in the field. Future ECG machines used in emergency settings should be constructed with 13 instead of 10 cables to allow simultaneously recording of 15 leads (standard, precordial and the V7-V9 posterior). This would ease acquisition, facilitate implementation of guideline recommendation. Funding Acknowledgement Type of funding sources: None.

Background It has been proposed that multiple comorbidities including diabetes, hypertension and dyslipidemia contribute to a pro-inflammatory state that induces oxidative stress in the microvascular endothelium, leading to coronary microvascular dysfunction (CMD) and cardiac diastolic dysfunction. Purpose We tested the hypothesis that subclinical inflammation is an underlying cause of both CMD and diastolic dysfunction and that the effect of inflammation on diastolic function is partly mediated by CMD. Methods In a cross-sectional study design, we included women with angina but no flow limiting coronary artery stenosis (180 with diabetes, 156 without diabetes) and 95 asymptomatic controls. Blood samples were analysed for 91 mainly inflammatory cardiovascular biomarkers. Coronary microvascular function was assessed as coronary flow velocity reserve (CFVR) by transthoracic Doppler echocardiography. Correlation of each biomarker with CFVR and E/e' as a marker of diastolic function, was examined by age adjusted linear regression and mediation analysis was conducted to assess if any effect of inflammation on E/e' was mediated by impaired CFVR. Results CFVR was lowest in patients with diabetes and highest in controls and, conversely, E/e' was highest in patients with diabetes (both test for trend p Population charachteristics compared across groups with trend test (unadjusted linear regression) Diabetes (n=180) No diabetes (n=156) Control (n=95) p for trend Age, mean (min, max) 64 (36,80) 62 (32,80) 61 (32,81)* 0.003 BMI, mean (min, max) 30.7 (15.2, 45.5) 26.1 (18.6,40.0)* 24.7 (18.6,38.4)* Generic directed acyclic graph Conclusion This is the first study to link a large number of mainly inflammatory biomarkers to CMD and diastolic dysfunction in patients suspected of microvascular angina. Direct association between CMD and diastolic function was only seen in those with impaired CFVR and poorest diastolic function. Acknowledgement/Funding The Danish Council for Independent Research and the Danish Heart Foundation

Background Improvements in medical and interventional therapies have transformed ischemic heart disease into a chronic illness for lot of patients. The disease is in progress and by time patients suffer from cardiac symptoms, reduced work capacity and decline in quality of life. Stem cell therapy is investigated as a treatment option for these patients. Purpose In this study, long-term safety and efficacy of autologous intra-myocardial injections of adipose-derived stromal cells (ASCs) were studied in patients with refractory angina. Methods Sixty patients were double-blinded 2:1 randomised to ASC or saline injections and followed for three years. The patients had significant angina due to ≥1 coronary artery stenosis but preserved left ventricular ejection fraction. ASCs were obtained from abdomen, ex vivo culture expanded and VEGF-A165 stimulated before delivery into the ischemic myocardium. Results The cardiac symptoms, CCS and NYHA classification, were significantly reduced in the ASC group during the three years follow-up period (2.5±0.9 to 1.8±1.2, P=0.002 and 2.4±0.6 to 2.2±0.8, P=0.007, respectively). However, no significant change was observed in CCS or NYHA in the placebo group during the follow-up period (2.5±0.8 to 2.1±1.3, P=0.186 and 2.7±0.6 to 2.4±0.8, P=0.314, respectively). Moreover, the number of weekly angina attacks reported was significantly reduced in the ASC group (P=0.017), but not in the placebo group (P=0.425). For patients in the ASC group, the bicycle exercise time (383±30s to 370±44s, P=0.052) and the exercise performance in watt were un-changed (81±6 to 78±10, P=0.123), but the performance in METs was reduced significantly (4.2±0.3 to 4.0±0.4, P=0.027) during the follow-up period. At the same time in the placebo group, there was a significant decline in bicycle exercise time (437±53s to 383±58s, P=0.001), the exercise performance measured in watt (87±12 watt to 80±12 watt, P=0.019) and in METs (4.5±0.4 to 4.1±0.4, P=0.002). In both groups, significant improved quality-of-life, angina stability, angina frequency and physical limitation score was observed but not for overall satisfaction score. Conclusion Patients receiving ASCs had improved cardiac symptoms during the three years follow-up period, which was not the case for patients in the placebo group. Moreover, patients receiving ASCs had unchanged exercise capacity, in opposition to deterioration in the placebo group. Acknowledgement/Funding Arvid Nilssons Foundation; Rigshospitalets Research Foundation; Aase and Ejnar Danielsens Foundation

This editorial refers to ‘Randomized trial of combination cytokine and adult autologous bone marrow progenitor cell administration in patients with non-ischaemic dilated cardiomyopathy: the REGENERATE-DCM clinical trial’[†][1], by S. Hamshere et al ., on page 3061. Dilated cardiomyopathy (DCM) and heart failure is a serious disease with a high morbidity and mortality rate in spite of optimal medical therapy. Although coronary artery disease is the main aetiology for the condition, a large group of patients have non-ischaemic DCM. Regenerative therapy with stem cells has been a focus to improve cardiac function, physical performance, and quality of life, and to reduce morbidity and mortality in these severely diseased patients. The focus was initially on cell sources from the bone marrow, but recent studies have extended interest to cells from many different tissues such as adipose tissue, heart, umbilical cord, etc., ( Figure 1 ). Cytokine therapy with granulocyte colony-stimulating factor (G-CSF) has for many years been used in haematology to mobilize stem cells from the bone marrow into the blood for transplantation. The use of G-CSF for regenerative stem cell therapy in cardiac disease was intensively studied pre-clinically and clinically a decade ago ( Figure 2 ). Figure 1 Illustration of combined therapy with granulocyte colony-stimulating factor (G-CSF) mobilization of cells from the bone marrow to the bloodstream and intracoronary injection of different subsets of cell populations from the bone marrow. LAD, left anterior descending; MNC, mononuclear cells, CD34+, mononuclear cells with the surface marker CD34; MSC, mesenchymal stromal/stem cells. Figure 2 Subcutaneous injection of G-CSF … [1]: #fn-2

Aims: Clinical trials suggest that intracoronary delivery of autologous bone marrow-derived cells (BMCs) 1-7 days post-Acute myocardial infarction (AMI) may improve left ventricular (LV) function. Earlier time points have not been evaluated. We sought to determine the effect of intracoronary autologous BMC on LV function when delivered within 24 h of successful reperfusion therapy. Methods and results: A multi-centre phase II randomized, double-blind, and placebo-controlled trial. One hundred patients with anterior AMI and significant regional wall motion abnormality were randomized to receive either intracoronary infusion of BMC or placebo (1:1) within 24 h of successful primary percutaneous intervention (PPCI). The primary endpoint was the change in left ventricular ejection fraction (LVEF) between baseline and 1 year as determined by advanced cardiac imaging. At 1 year, although LVEF increased compared with baseline in both groups, the between-group difference favouring BMC was small (2.2%; 95% confidence interval, CI: -0.5 to 5.0; P = 0.10). However, there was a significantly greater myocardial salvage index in the BMC-treated group compared with placebo (0.1%; 95% CI: 0.0-0.20; P = 0.048). Major adverse events were rare in both treatment groups. Conclusion: The early infusion of intracoronary BMC following PPCI for patients with AMI and regional wall motion abnormality leads to a small non-significant improvement in LVEF when compared with placebo; however, it may play an important role in infarct remodelling and myocardial salvage. Clinical trial registration: Clinicaltrials.gov NCT00765453 and EudraCT 2007-002144-16.

Aims Regenerative treatment with mesenchymal stromal cells (MSCs) has been promising in patients with ischaemic heart failure but needs confirmation in larger randomized trials. We aimed to study effects of intra-myocardial autologous bone marrow-derived MSC treatment in patients with severe ischaemic heart failure. Methods and results The MSC-HF trial is a randomized, double-blind, placebo-controlled trial. Patients were randomized 2 : 1 to intra-myocardial injections of MSC or placebo, respectively. The primary endpoint was change in left ventricular end-systolic volume (LVESV), measured by magnetic resonance imaging or computed tomography at 6 months follow-up. Sixty patients aged 30–80 years with severe ischaemic heart failure, New York Heart Association (NYHA) classes II–III, left ventricular ejection fraction (LVEF)

Aims We aimed to quantify the release of biomarkers of myocardial damage in relation to direct intramyocardial injections of genes and stem cells in patients with severe coronary artery disease. Methods and results We studied 71 patients with ‘no-option’ coronary artery disease. Patients had, via the percutaneous transluminal route, a total of 11 ± 1 (mean ± SD) intramyocardial injections of vascular endothelial growth factor genes ( n = 56) or mesenchymal stromal cells ( n = 15). Injections were guided to an ischaemic area by electromechanical mapping, using the NOGA™/Myostar™ catheter system. Plasma CKMB (upper normal laboratory limit = 5 µg/L) was 2 µg/L (2–3) at baseline; increased to 6 (5–9) after 8 h ( P < 0.0001) and normalized to 4 (3–5) after 24 h. A total of eight patients (17%), receiving a volume of 0.3 mL per injection, had CKMB rises exceeding three times the upper limit, whereas no patient in the group receiving 0.2 mL had a more than two-fold CKMB increase. No patient developed new ECG changes. There were no clinically ventricular arrhythmias and no death. Conclusion NOGA mapping followed by direct intramyocardial injections of stem cells or genes lead to measurable release of cardiac biomarkers compared with NOGA mapping alone. The increase in biomarkers was related to the injected volume.

Aims Increased plasma concentrations of B-type natriuretic peptide (BNP) and its precursor (proBNP) provide important prognostic information in patients presenting with acute coronary syndromes. Although a majority of these patients undergo early invasive assessment, the effects of coronary angiography per se on plasma BNP and proBNP concentrations are not known. We therefore sought to determine whether coronary angiography and ventriculography affect the cardiac secretion of these prognostic markers. Methods and results Blood samples were collected before and two minutes after coronary angiography and ventriculography in patients with or without coronary artery disease (CAD) and normal left ventricular ejection fraction. In patients with suspected CAD and normal left ventricular ejection fraction, the plasma proBNP concentration transiently increased from 11 pmol/l (range 1–67 pmol/l) to 19 pmol/l (range 5–102 pmol/l, \batchmode \documentclass[fleqn,10pt,legalpaper]{article} \usepackage{amssymb} \usepackage{amsfonts} \usepackage{amsmath} \pagestyle{empty} \begin{document} \(n=29,P{

Mobilization of stem cells into the peripheral circulation for myocardial regeneration using subcutaneous injections of granulocyte-colony-stimulating factor (G-CSF) has been tested in both patients with acute myocardial infarction (AMI) and patients with chronic myocardial ischaemia. G-CSF treatment seems to be safe and unblinded trials in patients with AMI were encouraging. However, larger double-blind placebo-controlled trials have not been able to demonstrate effect of G-CSF treatment. In patients with chronic myocardial ischaemia, small-unblinded G-CSF trials did not show effect on myocardial perfusion and function. In both patient populations, G-CSF did mobilize stem cells of known importance to myocardial regeneration, but there seemed to be a general lack of homing of the stem cells into the ischaemic myocardium. In AMI, factors of importance to homing of stem cells, stem cell derived factor-1, are maximally elevated in plasma 3 weeks after infarction, suggesting that this time point could be the optimal time for stem cell mobilization treatment. The known complex interaction of stem cells and cytokines for induction of vasculogenesis should be implemented in future clinical trials, to elucidate whether G-CSF mobilization of stem cells might be useful as a new regenerative treatment in patients with ischaemic heart disease.

Aims To assess the safety and effects of combined treatment with vascular endothelial growth factor-A165 plasmid (VEGF-A165) and granulocyte- colony stimulating factor (G-CSF) mobilization of bone marrow stem cells in patients with severe chronic ischaemic heart disease (IHD). Methods and results Sixteen patients with severe chronic IHD were treated with intramyocardial injections of VEGF-A165 plasmid followed 1 week later by G-CSF (10 mg/kg/day for 6 days). Two control groups included (i) sixteen patients treated with intramyocardial injections of VEGF-A165 plasmid and (ii) sixteen patients treated with intramyocardial injections of placebo. In the G-CSF group, circulating CD34þ stem cells increased almost 10-fold compared with the control groups (P , 0.0001). After 3 months, there was no improvement in myocardial perfusion at single photon emission computerized tomography in the VEGF-A165 and G-CSF treated group, and clinical symptoms were unchanged. There were no side effects to the gene and G-CSF therapy. Conclusion Intramyocardial VEGF-A165 gene transfer followed by bone marrow stem cell mobilization with G-CSF seemed safe. However, a significant increase in circulating stem cells did not lead to improved myocardial perfusion or clinical effects suggesting a neutral effect of the treatment. To improve homing of stem cells, higher doses of VEGF-A165 and/or use of SDF-1 transfer might be considered.

The normal heart secretes Atrial Natriuretic Peptide (ANP) and Brain Natriuretic Peptide (BNP) from the atria, which counteract cardiac overload by stimulating renal natriuresis and lowering systemic blood pressure.1On a cellular level, the endocrine capacity of the atria is reflected by the presence of secretory granules for peptide storage, which almost 50 years ago gave the first clue to the discovery of the endocrine heart.2Cardiac natriuretic peptides are stored in secretory granules and secreted upon relevant stimuli such as stretch of the atrial myocytes.3When the function of the left ventricle deteriorates, however, the ventricular myocytes also begin to express the ANP and BNP genes and progressively become the major site of peptide secretion. This endocrine feature of ventricular peptide expression andsecretion has now proven its diagnostic and prognostic value in congestive heart failure patients. Cardiac conditions with impaired function of the left ventricle can be detected by measurement of the circulating concentrations of natriuretic peptides and their precursors.4–6The European Society of Cardiology has consequently included plasma measurement of cardiac natriuretic peptides as diagnostic markers of congestive …

by S. Hamed et al., on page 1876Erythropoietin (EPO) is a cytokine for erythrocyte precursorsin the bone marrow, and the glycoprotein hormone thatregulates red blood cell production. EPO is mainly producedin the kidney and production is induced by hypoxia/ischae-mia. The main transcriptional factor is HIF-1, which is alsoinduced by hypoxia/ischaemia in myocardial cells. EPO isalso expressed in the heart. However, its expression mayonly be brief, it has in fact only been shown 24–48 h afterpermanent coronary artery occlusion in mice hearts.

Purpose: Regenerative stem cell therapy is an emerging treatment modality for patients with coronary artery disease (CAD). The study assessed long-term safety and efficacy of percutaneous intramyocardial injection of autologous bone-marrow derived mesenchymal stromal cells (MSC) in patients with severe stable CAD and refractory angina. Methods: Thirty-one patients with severe stable CAD and refractory angina were included. Patients had reversible myocardial ischemia and no further revascularization options. Autologous bone-marrow MSCs were isolated, culture expanded and stimulated with vascular endothelial growth-factor to facilitate endothelial differentiation. MSCs were injected into an ischemic, viable region of the myocardium using a electromechanical guided percutaneous catheter system. Patients were followed for 3 years. Results: We found significant and sustained clinical improvements in exercise time from 6:23±1:43 to 7:00±2:12 minutes (p = 0.0016), angina class (CCS) from 3.0±0.3 to 0.9±1.0 (p < 0.0001)), weekly number of angina attacks from 13.8±13.7 to 4.3±7.3 (p < 0.0001) and weekly uses of nitro-glycerine from 10.7±10.0 to 4.5±6.8 (p = 0.0017). In the Seattle Angina Questionnaire there were significant improvements in physical limitation score, angina stability score, angina frequency score and quality of life score (all p < 0.0001). When comparing all hospital admissions from 3 years before treatment and 3 years after treatment, we observed highly reduced admission rates for stable angina (p < 0.0001), revascularization (p = 0.003) and overall cardiovascular disease (p < 0.0001). No early or late side-effects of the treatment were observed. Conclusions: The final 3-year follow-up data after percutaneous intramyocardial injection of autologous MSCs, in patients with severe CAD and refractory angina, demonstrated sustained and significantly positive clinical effects, reduced hospital admissions for cardiovascular disease and excellent safety. The results indicate that the treatment does not only improve clinical symptoms but also slows down disease progression.

In eight patients with atrial fibrillation of less than 3 months duration and without congestive heart failure the plasma concentration of atrial natriuretic peptide was determined one day before, the day after and again 30 days after electrical cardioversion therapy. The pretreatment plasma concentration of the peptide was 99 pg mg−1 (23–480, median and range). The day after cardioversion to sinus rhythm the peptide concentration had normalized to 36 pg ml−1 (18–151). The plasma concentration of atrial natriuretic peptide remained stable in all but one patient for a period of 30 days (46 pg ml−1, 16–695) ( P = 0·03). In conclusion, the plasma concentration of atrial natriuretic peptide in patients with atrial fibrillation was significantly reduced after electrical cardioversion to sinus rhythm and remained stable for a period of 30 days.