Rasmus Sejersten Ripa

Stem Cell Mobilization Induced by Subcutaneous Granulocyte-Colony Stimulating Factor to Improve Cardiac Regeneration After Acute ST-Elevation Myocardial Infarction Result of the Double-Blind, Randomized, Placebo-Controlled Stem Cells in Myocardial Infarction (STEMMI) Trial

Background— Phase 1 clinical trials of granulocyte-colony stimulating factor (G-CSF) treatment after myocardial infarction have indicated that G-CSF treatment is safe and may improve left ventricular function. This randomized, double-blind, placebo-controlled trial aimed to assess the efficacy of subcutaneous G-CSF injections on left ventricular function in patients with ST-elevation myocardial infarction. Methods and Results— Seventy-eight patients (62 men; average age, 56 years) with ST-elevation myocardial infarction were included after successful primary percutaneous coronary stent intervention <12 hours after symptom onset. Patients were randomized to double-blind treatment with G-CSF (10 &mgr;g/kg of body weight) or placebo for 6 days. The primary end point was change in systolic wall thickening from baseline to 6 months determined by cardiac magnetic resonance imaging (MRI). An independent core laboratory analyzed all MRI examinations. Systolic wall thickening improved 17% in the infarct area in the G-CSF group and 17% in the placebo group (P=1.0). Comparable results were found in infarct border and noninfarcted myocardium. Left ventricular ejection fraction improved similarly in the 2 groups measured by both MRI (8.5 versus 8.0; P=0.9) and echocardiography (5.7 versus 3.7; P=0.7). The risk of severe clinical adverse events was not increased by G-CSF. In addition, in-stent late lumen loss and target vessel revascularization rate in the follow-up period were similar in the 2 groups. Conclusions— Bone marrow stem cell mobilization with subcutaneous G-CSF is safe but did not lead to further improvement in ventricular function after acute myocardial infarction compared with the recovery observed in the placebo group.

Bone Marrow–Derived Mesenchymal Cell Mobilization by Granulocyte-Colony Stimulating Factor After Acute Myocardial Infarction Results From the Stem Cells in Myocardial Infarction (STEMMI) Trial

Background— Granulocyte-colony stimulating factor (G-CSF) after myocardial infarction does not affect systolic function when compared with placebo. In contrast, intracoronary infusion of bone marrow cells appears to improve ejection fraction. We aimed to evaluate the G-CSF mobilization of subsets of stem cells. Methods and Results— We included 78 patients (62 men; 56±8 years) with ST-elevation myocardial infarction treated with primary percutaneous intervention <12 hours after symptom onset. Patients were randomized to double-blind G-CSF (10 &mgr;g/kg/d) or placebo. Over 7 days, the myocardium was exposed to 25×109 G-CSF mobilized CD34+ cells, compared with 3×109 cells in placebo patients (P<0.001); and to 4.9×1011 mesenchymal stem cells, compared with 2.0×1011 in the placebo group (P<0.001). The fraction of CD34+ cells/leukocyte increased during G-CSF treatment (from 0.3±0.2 to 1.1±0.9 ×10−3, P<0.001 when compared with placebo), whereas the fraction of putative mesenchymal stem cells/leukocyte decreased (from 22±17 to 14±11 ×10−3, P=0.01 when compared with placebo). An inverse association between number of circulating mesenchymal stem cells and change in ejection fraction was found (regression coefficient −6.8, P=0.004), however none of the mesenchymal cell subtypes analyzed, were independent predictors of systolic recovery. Conclusions— The dissociated pattern for circulating CD34+ and mesenchymal stem cells could be attributable to reduced mesenchymal stem cell mobilization from the bone marrow by G-CSF, or increased homing of mesenchymal stem cells to the infarcted myocardium. The inverse association between circulating mesenchymal stem cells and systolic recovery may be of clinical importance and should be explored further.

YKL-40 a new biomarker in patients with acute coronary syndrome or stable coronary artery disease.

Background. YKL-40 is involved in remodelling and angiogenesis in non-cardiac inflammatory diseases. Aim was to quantitate plasma YKL-40 in patients with ST-elevation myocardial infarction (STEMI) or stable chronic coronary artery disease (CAD), and YKL-40 gene activation in human myocardium. Methods and results. We included 73 patients: I) 20 patients with STEMI; II) 28 patients with stable CAD; III) 15 CAD patients referred for coronary by-pass surgery. YKL-40 mRNA expression was measured in myocardium subtended by stenotic or occluded arteries and areas with no apparent disease; and IV) 10 age-matched healthy controls. Plasma YKL-40 was significantly increased in patients with STEMI (88 µg/l, median) and CAD (66 µg/l) compared to controls (16 µg/l, p<0.01 for both). Plasma YKL-40 correlated with CRP at baseline in STEMI (r=0.53, p=0.02) and CAD patients (r=0.41, p=0.031).YKL-40 gene expression was similar in ischemic and non-ischemic myocardium. Conclusions. Plasma YKL-40 was significantly increased in patients with STEMI and stable CAD. Further studies will define the role of YKL-40 as a clinically useful marker for myocardial ischemia, remodelling and maybe prognosis.

Mesenchymal stromal cell derived endothelial progenitor treatment in patients with refractory angina

Abstract Aims. We evaluated the feasibility, safety and efficacy of intra-myocardial injection of autologous mesenchymal stromal cells derived endothelial progenitor cell (MSC) in patients with stable coronary artery disease (CAD) and refractory angina in this first in man trial. Methods and results. A total of 31 patients with stable CAD, moderate to severe angina and no further revascularization options, were included. Bone marrow MSC were isolated and culture expanded for 6–8 weeks. It was feasible and safe to establish in-hospital culture expansion of autologous MSC and perform intra-myocardial injection of MSC. After six months follow-up myocardial perfusion was unaltered, but the patients increased exercise capacity (p < 0.001), reduction in CCS Class (p < 0.001), angina attacks (p < 0.001) and nitroglycerin consumption (p < 0.001), and improved Seattle Angina Questionnaire (SAQ) evaluations (p < 0.001). For all parameters there was a tendency towards improved outcome with increasing numbers of cells injected. In the MRI substudy: ejection fraction (p < 0.001), systolic wall thickness (p = 0.03) and wall thickening (p = 0.03) all improved. Conclusions. The study demonstrated that it was safe to treat patients with stable CAD with autologous culture expanded MSC. Moreover, MSC treated patients had significant improvement in left ventricular function and exercise capacity, in addition to an improvement in clinical symptoms and SAQ evaluations. Trial registration: ClinicalTrials.gov identifier: NCT00260338.

Influences of electrocardiographic ischaemia grades and symptom duration on outcomes in patients with acute myocardial infarction treated with thrombolysis versus primary percutaneous coronary intervention: results from the DANAMI‐2 trial

Objective: To determine whether ischaemia grade (GI) on the presenting ECG and duration of symptoms can identify subgroups of patients who would derive more benefit than the general population of patients with ST segment elevation acute myocardium infarction (STEMI) from primary percutaneous coronary intervention (pPCI) over thrombolytic treatment (TT) in reducing mortality or reinfarction. Methods: 1319 DANAMI-2 (Danish trial in Acute Myocardial Infarction-2) patients were classified as having grade 2 ischaemia (GI2; ST segment elevation without terminal QRS distortion) or grade 3 ischaemia (GI3; ST segment elevation with terminal QRS distortion in ⩾ 2 adjacent leads), and were divided into early and late groups split by the median time (3 h) from symptom onset to treatment. Outcomes were 30-day mortality and reinfarction. Results: Mortality was significantly higher for GI3 than for GI2 (9.7% v 4.8%, p < 0.001) and doubled for patients presenting late (GI2: 6.0% v 3.3%, p  =  0.01; GI3: 12.5% v 4.7%, p  =  0.05). Overall mortality did not differ significantly between pPCI and TT; however, a 5.5% absolute mortality reduction was seen in GI3 treated early with pPCI (1.4% v 6.9%, p  =  0.10). Reinfarction rate was particularly high among GI3 patients presenting late and treated with TT (12.2%). pPCI in such patients significantly reduced the rate of reinfarction (0%, p < 0.001). Logistic regression analysis showed that age (odds ratio (OR) 1.09, 95% confidence interval (CI) 1.06 to 1.12, p < 0.001), prior angina (OR 2.56, 95% CI 1.44 to 4.54, p  =  0.001), heart rate (OR 1.03, 95% CI 1.01 to 1.04, p  =  0.001) and GI3 (OR 1.91, 95% CI 1.06 to 3.44, p  =  0.031) were independently associated with mortality, whereas the sum of ST segment elevation was not. Conclusions: GI3 is an independent predictor of mortality among patients with STEMI. Mortality increased significantly with symptom duration in both GI2 and GI3. pPCI may be especially beneficial for patients with GI3 presenting early, whereas patients with GI3 presenting late and treated with TT are at particular risk of reinfarction.

De novo mutations in familial adenomatous polyposis (FAP).

Familial Adenomatous Polyposis (FAP) results from a germline mutation in the APC gene. A new mutation rate of 4–9×10−6 mutations/gametes/generation has been reported. In other familial cancer syndromes a bias for paternal origin of new mutations has been described. This bias is probably due to a larger number of cell divisions during spermatogenesis compared to oogenesis; giving a larger susceptibility for mutagenesis. We report here a molecular genetic analysis of 26 FAP patients with putative de novo APC mutations. In 15 families the novel origin of the mutations was confirmed by haplotyping and sequencing. Analysis of 10 of these mutations, in which the parental origin could be established, gave a 6 : 4 distribution in favour of maternal origin. This is in agreement with a 1 : 1 distribution and does not indicate an expected paternal bias. Moreover, no parental age effect was identified. We propose that APC germline mutations are not premeiotic events but more likely arise during the meiosis. This would give an equal susceptibility for mutagenesis during spermatogenesis and oogenesis, respectively. The model is in concordance with the previously established difference between APC somatic mutations, as being a mitotic event and APC germline mutations, as being a meiotic event. The confirmation of 15 de novo mutations by a molecular genetic approach is in fine agreement with previous results based on clinical records.

18F-FDG PET Imaging of Murine Atherosclerosis: Association with Gene Expression of Key Molecular Markers

Aim To study whether 18F-FDG can be used for in vivo imaging of atherogenesis by examining the correlation between 18F-FDG uptake and gene expression of key molecular markers of atherosclerosis in apoE−/− mice. Methods Nine groups of apoE−/− mice were given normal chow or high-fat diet. At different time-points, 18F-FDG PET/contrast-enhanced CT scans were performed on dedicated animal scanners. After scans, animals were euthanized, aortas removed, gamma counted, RNA extracted from the tissue, and gene expression of chemo (C-X-C motif) ligand 1 (CXCL-1), monocyte chemoattractant protein (MCP)-1, vascular cell adhesion molecule (VCAM)-1, cluster of differentiation molecule (CD)-68, osteopontin (OPN), lectin-like oxidized LDL-receptor (LOX)-1, hypoxia-inducible factor (HIF)-1α, HIF-2α, vascular endothelial growth factor A (VEGF), and tissue factor (TF) was measured by means of qPCR. Results The uptake of 18F-FDG increased over time in the groups of mice receiving high-fat diet measured by PET and ex vivo gamma counting. The gene expression of all examined markers of atherosclerosis correlated significantly with 18F-FDG uptake. The strongest correlation was seen with TF and CD68 (p<0.001). A multivariate analysis showed CD68, OPN, TF, and VCAM-1 to be the most important contributors to the uptake of 18F-FDG. Together they could explain 60% of the 18F-FDG uptake. Conclusion We have demonstrated that 18F-FDG can be used to follow the progression of atherosclerosis in apoE−/− mice. The gene expression of ten molecular markers representing different molecular processes important for atherosclerosis was shown to correlate with the uptake of 18F-FDG. Especially, the gene expressions of CD68, OPN, TF, and VCAM-1 were strong predictors for the uptake.

64Cu-DOTATATE PET/MRI for Detection of Activated Macrophages in Carotid Atherosclerotic Plaques Studies in Patients Undergoing Endarterectomy

Objective— A feature of vulnerable atherosclerotic plaques of the carotid artery is high activity and abundance of lesion macrophages. There is consensus that this is of importance for plaque vulnerability, which may lead to clinical events, such as stroke and transient ischemic attack. We used positron emission tomography (PET) and the novel PET ligand [64Cu] [1,4,7,10-tetraazacyclododecane-N,N′,N″,N‴-tetraacetic acid]-d-Phe1,Tyr3-octreotate (64Cu-DOTATATE) to specifically target macrophages via the somatostatin receptor subtype-2 in vivo. Approach and Results— Ten patients underwent simultaneous PET/MRI to measure 64Cu-DOTATATE uptake in carotid artery plaques before carotid endarterectomy. 64Cu-DOTATATE uptake was significantly higher in symptomatic plaque versus the contralateral carotid artery (P<0.001). Subsequently, a total of 62 plaque segments were assessed for gene expression of selected markers of plaque vulnerability using real-time quantitative polymerase chain reaction. These results were compared with in vivo 64Cu-DOTATATE uptake calculated as the mean standardized uptake value. Univariate analysis of real-time quantitative polymerase chain reaction and PET showed that cluster of differentiation 163 (CD163) and CD68 gene expression correlated significantly but weakly with mean standardized uptake value in scans performed 85 minutes post injection (P<0.001 and P=0.015, respectively). Subsequent multivariate analysis showed that CD163 correlated independently with 64Cu-DOTATATE uptake (P=0.031) whereas CD68 did not contribute significantly to the final model. Conclusions— The novel PET tracer 64Cu-DOTATATE accumulates in atherosclerotic plaques of the carotid artery. CD163 gene expression correlated independently with 64Cu-DOTATATE uptake measured by real-time quantitative polymerase chain reaction in the final multivariate model, indicating that 64Cu-DOTATATE PET is detecting alternatively activated macrophages. This association could potentially improve noninvasive identification and characterization of vulnerable plaques.

Plasma YKL-40 in relation to the degree of coronary artery disease in patients with stable ischemic heart disease

Abstract Background. YKL-40 is a glycoprotein secreted by macrophages and neutrophils in tissues with inflammation. Plasma YKL-40 is increased in patients with coronary artery disease (CAD) and associated with cardiovascular and all-cause mortality. Furthermore, plasma YKL-40 seems related to the number of diseased main vessels in patients with stable CAD. The aim was to further study the relation between YKL-40 and stenosis degree, stenosis type and actual ischemia in stable CAD patients. Methods. Plasma YKL-40 and hsCRP levels were determined from 206 consecutive patients with stable angina pectoris admitted for coronary angiography. Plasma YKL-40 in 245 healthy subjects was used for comparison. In addition to one to three vessel stenosis scores, two new scores for evaluating coronary angiographies were established for discriminating between focal and diffuse CAD and the extent of myocardial ischemia. Results. YKL-40 levels in CAD patients (median: 52 μg/L and quartiles: 37–85 μg/L) were significantly increased (p < 0.001) compared to the healthy controls. In univariate analyses plasma YKL-40 was significantly associated with ischemic myocardium score, age, hypertension, peripheral vascular disease and serum creatinine levels. In multivariate analyses YKL-40 was related to hsCRP, peripheral artery disease, hypertension, and statin treatment. Conclusions. Plasma YKL-40 was increased in patients with CAD compared to controls. YKL-40 was related to the ischemic myocardium, but not to degree of CAD using different scoring systems. Therefore, YKL-40 is not related to the extent of CAD, but to some other pathophysiological mechanisms of importance for the prognosis.

G-CSF therapy with mobilization of bone marrow stem cells for myocardial recovery after acute myocardial infarction—A relevant treatment?

This review of adjunctive therapy with subcutaneous granulocyte-colony stimulating factor (G-CSF) to patients with acute myocardial infarction (AMI) focus on the cardioprotective effects and potential mechanisms of G-CSF and discuss the therapeutic potential of G-CSF. All clinical trials published in peer-reviewed journals identified through PubMed are discussed. G-CSF treatment seems to be safe, and initial unblinded trials in patients with AMI were encouraging. However, larger double-blind placebo-controlled trials have not been able to demonstrate improved myocardial recovery after G-CSF treatment. Current controversies in interpretation of the results include 1) importance of direct cardiac effect of G-CSF vs indirect through bone marrow stem and progenitor cell mobilization, 2) importance of timing of G-CSF therapy, 3) importance of G-CSF dose, and 4) importance of cell types mobilized from the bone-marrow. Cell-based therapies to improve cardiac function remain promising and further experimental and clinical studies are warranted to determine the future role of G-CSF.