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The pro-angiogenic abilities of adipose-derived stromal cells (ASCs) make them attractive candidates for cellular therapy, especially for ischemic disease indications. However, details regarding the underlying mechanisms remain elusive. Therefore, this study aimed to investigate paracrine and juxtacrine abilities of ASCs in angiogenesis triple cell co-cultures by detailed image analysis of the vascular-like structures. Fibroblast-endothelial cell co-cultures were established, and ASCs were added directly or indirectly through inserts. The cultures were treated with antibodies or subjected to analyses using ELISA and RT2 PCR Arrays. The model consistently generated vascular-like structures. ASCs increased the total branch lengths equally well in paracrine and juxtacrine conditions, by increasing the number of branches and average branch lengths (ABL). In contrast, addition of VEGF to the model increased the number of branches, but not the ABL. Still, ASCs increased the VEGF levels in supernatants of paracrine and juxtacrine co-cultures, and anti-VEGF treatment decreased the sprouting. ASCs themselves up-regulated collagen type V in response to paracrine signals from the co-cultures. The results suggest that ASCs initiate sprouting through secretion of several paracrine factors, among which VEGF is identified, but VEGF alone does not recapitulate the paracrine actions of ASCs. By employing neutralizing antibodies and dismantling common model outputs using image analysis, the triple cell co-culture is an attractive tool for discovery of the paracrine factors in ASCs’ secretome which act in concert with VEGF to improve angiogenesis.

Abstract Background A predominant side effect of radiotherapy for head and neck cancer is salivary gland hypofunction and xerostomia leading to debilitating oral disorders and impaired quality of life (QoL). Intraglandular mesenchymal stem cell therapy has shown promising results as a treatment for xerostomia. Methods This is a randomised, double-blinded, placebo-controlled, parallel-group, prospective, single-centre trial investigating the safety, tolerability, and effectiveness of allogeneic stem cells as a treatment for radiation-induced hyposalivation and xerostomia for previous head and neck cancer patients. We will include a total of 120 patients who previously have been treated with radiotherapy for a head and neck cancer in Denmark. Participants will be randomly assigned using block randomisation to one of two parallel groups in a 1:1 ratio to receive ultrasound-guided injection of allogeneic adipose-derived mesenchymal stem cell (ASC) (n = 60) or placebo (n = 60) into the submandibular glands. Placebo will consist of CryoStor10 (BiolifeSolutions), the freeze media for ASCs containing 10% dimethyl sulfoxide (DMSO). The primary endpoint is change in unstimulated whole saliva flow rate. The secondary endpoints are change in stimulated whole saliva flow rate, QoL, and composition of saliva. Further secondary endpoints are safety and immune response (human leukocyte antigen (HLA) response) to the stem cells will be assessed. Patients are evaluated at baseline (before treatment), after 4 months, and after 12 months. All study personnel, except study personnel thawing and preparing the treatment for injection, and participants will be blinded to group assignment. Unblinded study personnel will not participate in the outcome assessment. Discussion The trials will investigate the efficacy and safety of ASC injection to the submandibular gland as a potential new treatment for post-radiation xerostomia. We hope the results will pave the way for a clinically relevant treatment to ameliorate patients with xerostomia, a severely hampering condition. Trial registration The study is approved by the Danish Data Protection Agency (protocol number P-2020-1164), the National Ethics Committee protocol number: (Protocol number: 1802872), and the Danish Medical Agency (2018-000348-24). The protocol was registered at the ClinicalTrials.gov database (NCT04776538).

Abstract Aims Patients suffering from chronic ischaemic heart failure with reduced left ventricular ejection fraction (HFrEF) have reduced quality‐of‐life, repetitive hospital admissions, and reduced life expectancy. Allogeneic cell therapy is currently investigated as a potential treatment option after initially encouraging results from clinical autologous and allogeneic trials in patients with HFrEF. We aimed to investigate the allogeneic Cardiology Stem Cell Centre Adipose tissue derived mesenchymal Stromal Cell product (CSCC_ASC) as an add‐on therapy in patients with chronic HFrEF. Methods and results This is a Danish multi‐centre double‐blinded placebo‐controlled phase II study with direct intra‐myocardial injections of allogeneic CSCC_ASC. A total of 81 HFrEF patients were included and randomized 2:1 to CSCC_ASC or placebo injections. The inclusion criteria were reduced left ventricular ejection fraction (LVEF ≤ 45%), New York Heart Association (NYHA) class II‐III despite optimal anti‐congestive heart failure medication and no further revascularization options. Injections of 0.3 mL CSCC_ASC (total cell dose 100 × 106 ASCs) (n = 54) or isotonic saline (n = 27) were performed into the viable myocardium in the border zone of infarcted tissue using the NOGA Myostar® catheter (Biological Delivery System, Cordis, Johnson & Johnson, USA). The primary endpoint, left ventricular end systolic volume (LVESV), was evaluated at 6‐month follow‐up. The safety was measured during a 3‐years follow‐up period. Results Mean age was 67.0 ± 9.0 years and 66.6 ± 8.1 years in the ASC and placebo groups, respectively. LVESV was unchanged from baseline to 6‐month follow‐up in the ASC (125.7 ± 68.8 mL and 126.3 ± 72.5 mL, P = 0.827) and placebo (134.6 ± 45.8 mL and 135.3 ± 49.6 mL, P = 0.855) group without any differences between the groups (0.0 mL (95% CI −9.1 to 9.0 mL, P = 0.992). Neither were there significant changes in left ventricular end diastolic volume or LVEF within the two groups or between groups −5.7 mL (95% CI −16.7 to 5.3 mL, P = 0.306) and −1.7% (95% CI −4.4. to 1.0, P = 0.212), respectively). NYHA classification and 6‐min walk test did not alter significantly in the two groups (P > 0.05). The quality‐of‐life, total symptom, and overall summary score improved significantly only in the ASC group but not between groups. There were 24 serious adverse events (SAEs) in the ASC group and 11 SAEs in the placebo group without any significant differences between the two groups at 1‐year follow‐up. Kaplan–Meier plot using log‐rank test of combined cardiac events showed an overall mean time to event of 30 ± 2 months in the ASC group and 29 ± 2 months in the placebo group without any differences between the groups during the 3 years follow‐up period (P = 0.994). Conclusions Intramyocardial CSCC_ASC injections in patients with chronic HFrEF were safe but did not improve myocardial function or structure, nor clinical symptoms.

Lynggaard et al. profile the salivary proteome and metaproteome in patients with head and neck cancer who have received radiation therapy and an intraglandular mesenchymal stem cell (MSC) treatment for radiation-induced xerostomia and in healthy controls. MSC therapy impacts the composition of the salivary proteome in the longer-term.

Abstract Background Adipose-derived stromal cells (ASCs) possess a multitude of regenerative capabilities, which include immunomodulation, angiogenesis, and stimulation of extracellular matrix (ECM) remodeling. However, the underlying mechanisms leading to ECM remodeling remain largely elusive and highlight the need for functional in vitro models for mode of action studies. Therefore, the purpose of this study was to develop an in vitro co-culture model to investigate the capabilities of ASCs to modulate fibroblasts and ECM. Methods An ECM in vitro model with ASCs and normal human dermal fibroblasts (NHDFs) was established utilizing macromolecular crowding, ascorbic acid, and TGF-β stimulation. Paracrine and juxtacrine co-cultures were created using transwell inserts and cell cultures with direct cell–cell contacts. The cultures were screened using RT2 PCR Profiler Arrays; the protein levels of myofibroblast differentiation marker alpha smooth muscle actin (αSMA) and ECM remodeling enzymes were analyzed using western blot on cell lysates; the formation of collagen type I, III, VI, and fibronectin was investigated using ELISA on culture supernatants; and the deposition of collagens was analyzed using immunocytochemistry. Results TGF-β stimulation of NHDF monocultures increased the expression of 18 transcripts relevant for ECM formation and remodeling, the protein levels of αSMA and matrix metalloproteinase-2 (MMP-2), the formation of collagen type I, III, VI, and fibronectin, and the deposition of collagen type I and VI and decreased the protein levels of MMP-14. Inclusion of ASCs in the ECM co-culture model increased the formation of collagen type I and III through paracrine mechanisms and the formation of collagen type VI through juxtacrine mechanisms. Conclusions The co-culture model provides effective stimulation of NHDF monocultures by TGF-β for enhanced formation and deposition of ECM. In the model, ASCs induce changes in ECM by increasing formation of collagen type I, III and VI. The obtained results could guide further investigations of ASCs’ capabilities and underlying mechanisms related to ECM formation and remodeling.

Abstract Aims The long‐term outcomes of the intracoronary delivery of autologous bone marrow‐derived cells (BMCs) after acute myocardial infarction are not well established. Following the promising 1 year results of the REGENERATE‐AMI trial (despite it not achieving its primary endpoint), this paper presents the analysis of the 5 year clinical outcomes of these acute myocardial infarction patients who were treated with an early intracoronary autologous BMC infusion or placebo. Methods and results A 5 year follow‐up of major adverse cardiac events (defined as the composite of all‐cause death, recurrent myocardial infarction, and all coronary revascularization) and of rehospitalization for heart failure was completed in 85 patients (BMC n = 46 and placebo n = 39). The incidence of major adverse cardiac events was similar between the BMC‐treated patients and the placebo group (26.1% vs. 18.0%, P = 0.41). There were no cases of cardiac death in either group, but an increase in non‐cardiac death was seen in the BMC group (6.5% vs. 0%, P = 0.11). The rates of recurrent myocardial infarction and repeat revascularization were similar between the two groups. There were no cases of rehospitalization for heart failure in either group. Conclusion This 5 year follow‐up analysis of the REGENERATE‐AMI trial did not show an improvement in clinical outcomes for patients treated with cell therapy. This contrasts with the 1 year results which showed improvements in the surrogate outcome measures of ejection fraction and myocardial salvage index.

The expeditious progress of Mesenchymal Stromal Cells (MSC) for therapeutic intervention calls for means to compare differences in potency of cell products. The differences may be attributed to innumerable sources including tissue origin, production methods, or even between batches. While the immunomodulatory potential of MSC is recognized and well-documented by an expansive body of evidence, the methodologies and findings vary markedly. In this study, we utilized flowcytometric analysis of lymphocyte proliferation based on cryopreserved peripheral blood mononuclear cells for quantification of the inhibitory effect of MSC. Technical aspects of fluorescent staining and cryopreservation of peripheral blood mononuclear cells were evaluated to obtain optimal results and increase feasibility. A range of common specific and unspecific mitogens was titrated to identify the conditions, in which the effects of Adipose tissue-derived Stromal Cells (ASC; a type of MSC) were most pronounced. Specific stimulation by antibody-mediated activation of CD3 and CD28 via TransAct and Dynabeads lead to substantial proliferation of lymphocytes, which was inhibited by ASC. These results were closely mirrored when applying unspecific stimulation in form of phytohemagglutinin (PHA), but not concanavalin A or pokeweed mitogen. The mixed lymphocyte reaction is a common assay which exploits alloreactivity between donors. While arguably more physiologic, the output of the assay often varies substantially, and the extent of proliferation is limited since the frequency of alloreactive cells is low, as opposed to the mitogens. To heighten the proliferative response and robustness, combinations of 2-5 donors were tested. Maximum proliferation was observed when combining 4 or more donors, which was efficiently suppressed by ASC. Several desirable and unfavorable traits can be attributed to the tested stimuli in the form of keywords. The importance of these traits should be scored on a laboratory-level to identify the ideal mitogen. In our case the ranking listed PHA as the most suited candidate. Developing robust assays is no trivial feat. By disclosing the full methodological framework in the present study, we hope to aid others in establishing functional metrics on the road to potency assays.

Small extracellular vesicles (EVs) and their cargo are an important component of cell-to-cell communication in cardiac disease. Allogeneic adipose derived stem cells (ADSCs) are thought to be a potential approach for cardiac regenerative therapy in ischemic heart disease. The SCIENCE study investigated the effect of ADSCs administered via intramyocardial injection on cardiac function in patients with ischemic heart disease. The aim of this substudy, based on samples from 15 patients, was to explore small EV miRNA dynamics after treatment with ADSCs compared to a placebo. Small EVs were isolated at several timepoints after the percutaneous intramyocardial application of ADSCs. No significant effect of ADSC treatment on small EV concentration was detected. After 12 months, the expression of miR-126 decreased significantly in ADSC patients, but not in the placebo-treated group. However, all cardiac miRNAs correlated with plasma cardiac biomarkers. In line with the overall negative results of the SCIENCE study, with the exception of one miR, we did not detect any significant regulation of small EV miRNAs in this patient collective.

Abstract The concept that cell‐based repair of myocardial injury might be possible was introduced almost two decades ago; however, the field of cardiovascular reparative medicine has been criticized as translation to clinically effective approaches has been slow. The recent retraction of a series of papers has further impacted perception of this area of research. As researchers, clinicians, and teachers in this field, we felt it incumbent to critically appraise the current state of cardiac cell repair, determine what can be learned from past mistakes, and formulate best practices for future work. This special communication summarizes an introspective assessment of what has fallen short, how to prevent similar issues, and how the field might best move forward in the service of science and patients.

An increasing number of patients are living with chronic ischemic cardiomyopathy (ICM) and/or heart failure. Treatment options and prognostic tools are lacking for many of these patients. Our aim was to investigate the prognostic value of imaging angiogenesis and macrophage activation via positron emission tomography (PET) in terms of functional improvement after cell therapy. Myocardial infarction was induced in rats. Animals were scanned with [18F]FDG PET and echocardiography after four weeks and randomized to allogeneic adipose tissue-derived stromal cells (ASCs, n = 18) or saline (n = 9). Angiogenesis and macrophage activation were assessed before and after treatment by [68Ga]Ga-RGD and [64Cu]Cu-DOTATATE. There was no overall effect of the treatment. Rats that improved left ventricular ejection fraction (LVEF) had higher uptake of both [68Ga]Ga-RGD and [64Cu]Cu-DOTATATE at follow-up (p = 0.006 and p = 0.008, respectively). The uptake of the two tracers correlated with each other (r = 0.683, p = 0.003 pre-treatment and r = 0.666, p = 0.004 post-treatment). SUVmax at follow-up could predict improvement in LVEF (p = 0.016 for [68Ga]Ga-RGD and p = 0.045 for [64Cu]Cu-DOTATATE). High uptake of [68Ga]Ga-RGD and [64Cu]Cu-DOTATATE PET after injection of ASCs or saline preceded improvement in LVEF. The use of these tracers could improve the monitoring of heart failure patients in treatment.

The emerging field of advanced therapy medicinal products (ATMP) holds promise of treating a variety of diseases. Adipose-derived stromal cells (ASCs) are currently being marketed or tested as cell-based therapies in numerous clinical trials. To ensure safety and efficacy of treatments, high-quality products must be manufactured. A good manufacturing practice (GMP) compliant and consistent manufacturing process including validated quality control methods is critical. Product design and formulation are equally important to ensure clinical feasibility. Here, we present a GMP-compliant, xeno-free, and semiautomated manufacturing process and quality controls, used for large-scale production of a cryopreserved off-the-shelf ASC product and tested in several phase I and II allogeneic clinical applications.

Abstract Background Stem cell therapy is investigated as a treatment option for patients with ischemic heart disease. In this study, long-term safety and efficacy of autologous intra-myocardial injections of adipose-derived stromal cells (ASCs) was studied in patients with refractory angina. Methods Sixty patients with coronary artery stenosis and preserved left ventricular ejection fraction were 2:1 randomised to intramyocardial injections of ASCs or saline and followed for 3 years. Results For patients in the ASC group, the bicycle exercise time and the exercise performance in watt were un-changed (383 ± 30 s to 370 ± 44 s, P = 0.052 and 81 ± 6 to 78 ± 10, P = 0.123, respectively), 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. However, in the same period, there was in the placebo group a significant decline in bicycle exercise time (437 ± 53 s to 383 ± 58 s, P = 0.001), the exercise performance measured in watt (87 ± 12 W to 80 ± 12 W, P = 0.019) and in METs (4.5 ± 0.4 to 4.1 ± 0.4, P = 0.002). Moreover, angina measured as CCS class was significantly reduced in the ASC group but not in the placebo group (2.5 ± 0.9 to 1.8 ± 1.2, P = 0.002 and 2.5 ± 0.8 to 2.1 ± 1.3, P = 0.186, respectively). However, no significant change was observed between the two groups. Conclusions Patients receiving ASCs had improved cardiac symptoms and unchanged exercise capacity, in opposition to deterioration in the placebo group. Trial registration ClinicalTrials.gov Identifier: NCT01449032. Registered 7 October 2011—Retrospectively registered, https://www.clinicaltrials.gov/ct2/show/NCT01449032?term=jens+kastrup&rank=7

Individual patient data (IPD)-based meta-analysis (ACCRUE, meta-analysis of cell-based cardiac studies, NCT01098591) revealed an insufficient effect of intracoronary cell-based therapy in acute myocardial infarction. Patients with ischemic heart failure (iHF) have been treated with reparative cells using percutaneous endocardial, surgical, transvenous or intracoronary cell delivery methods, with variable effects in small randomized or cohort studies. The objective of this meta-analysis was to investigate the safety and efficacy of percutaneous transendocardial cell therapy in patients with iHF. Two investigators extracted the data. Individual patient data (IPD) (n = 8 studies) and publication-based (n = 10 studies) aggregate data were combined for the meta-analysis, including patients (n = 1715) with chronic iHF. The data are reported in accordance with PRISMA guidelines. The primary safety and efficacy endpoints were all-cause mortality and changes in global ejection fraction. The secondary safety and efficacy endpoints were major adverse events, hospitalization and changes in end-diastolic and end-systolic volumes. Post hoc analyses were performed using the IPD of eight studies to find predictive factors for treatment safety and efficacy. Cell therapy was significantly (p < 0.001) in favor of survival, major adverse events and hospitalization during follow-up. A forest plot analysis showed that cell therapy presents a significant benefit of increasing ejection fraction with a mean change of 2.51% (95% CI: 0.48; 4.54) between groups and of significantly decreasing end-systolic volume. The analysis of IPD data showed an improvement in the NYHA and CCS classes. Cell therapy significantly decreased the end-systolic volume in male patients; in patients with diabetes mellitus, hypertension or hyperlipidemia; and in those with previous myocardial infarction and baseline ejection fraction ≤ 45%. The catheter-based transendocardial delivery of regenerative cells proved to be safe and effective for improving mortality and cardiac performance. The greatest benefit was observed in male patients with significant atherosclerotic co-morbidities.

Objective To assess if 12 novel circulating biomarkers, when added to ‘standard predictors’ available in general practice, could improve the 10-year prediction of cardiovascular events and mortality in patients with stable coronary heart disease.Design The patients participated as placebo receiving patients in the randomised clarithromycin for patients with stable coronary artery disease (CLARICOR) trial at a random time in their disease trajectory.Setting Five Copenhagen University cardiology departments and a coordinating centre.Participants 1998 participants with stable coronary artery disease.Outcomes Death and composite of myocardial infarction, unstable angina pectoris, cerebrovascular disease and death.Results When only ‘standard predictors’ were included, 83.4% of all-cause death predictions and 68.4% of composite outcome predictions were correct. Log(calprotectin) and log(cathepsin-S) were not associated (p≥0.01) with the outcomes, not even as single predictors. Adding the remaining 10 biomarkers (high-sensitive assay cardiac troponin T; neutrophil gelatinase-associated lipocalin; osteoprotegerin; N-terminal pro-B-type natriuretic peptide; tumour necrosis factor receptor 1 and 2; pregnancy-associated plasma protein A; endostatin; YKL40; cathepsin-B), which were all individually significantly associated with the prediction of the two outcomes, increased the figures to 84.7% and 69.7%.Conclusion When ‘standard predictors’ routinely available in general practices are used for risk assessment in consecutively sampled patients with stable coronary artery disease, the addition of 10 novel biomarkers to the prediction model improved the correct prediction of all-cause death and the composite outcome by

Background The inflammatory biomarker YKL‐40 has previously been studied as a potential risk marker in cardiovascular disease. We aimed to assess the prognostic reclassification potential of serum YKL‐40 in patients with stable coronary artery disease. Methods and Results The main study population was the placebo group of the CLARICOR (Effect of Clarithromycin on Mortality and Morbidity in Patients With Ischemic Heart Disease) trial. The primary outcome was a composite of acute myocardial infarction, unstable angina pectoris, cerebrovascular disease, and all‐cause mortality. We used Cox proportional hazards regression models adjusted for C‐reactive protein level and baseline cardiovascular risk factors. Improvement in prediction by adding serum YKL‐40 to the risk factors was calculated using the Cox‐Breslow method and c‐statistic. A total of 2200 patients were randomized to placebo, with a follow‐up duration of 10 years. YKL‐40 was associated with an increased risk of the composite outcome (hazard ratio per unit increase in (YKL‐40) 1.13, 95% CI 1.03–1.24, P=0.013) and all‐cause mortality (hazard ratio 1.32, 95% CI 1.17–1.49, P

PurposeSystemic inflammation and coronary microvascular dysfunction (CMD) may be causal drivers of heart failure with preserved ejection fraction (HFpEF). We tested the hypothesis that subclinical inflammation is associated with non-endothelial dependent CMD and diastolic dysfunction.MethodsIn a cross-sectional study of 336 women with angina but no flow limiting coronary artery stenosis (180 with diabetes) and 95 asymptomatic controls, blood samples were analysed for 90 biomarkers of which 34 were part of inflammatory pathways. CMD was assessed as coronary flow velocity reserve (CFVR) by transthoracic Doppler echocardiography and defined as CFVRResultsCMD was found in 59% of participants whereas only 4% fulfilled strict criteria for diastolic dysfunction. Thirty-five biomarkers, 17 of them inflammatory, were negatively correlated with CFVR and 25, 15 inflammatory, were positively correlated with E/e'. A total of 13 biomarkers, 9 inflammatory, were associated with both CFVR and E/e'. CFVR and E/e' were only correlated in the subgroup of patients with CMD and signs of increased filling pressure (E/e'>10) (p = 0.012).ConclusionThis is the first study to link a large number of mainly inflammatory biomarkers to both CMD and E/e', thus confirming a role of inflammation in both conditions. However, despite a high prevalence of CMD, few patients had diastolic dysfunction and the data do not support a major pathophysiologic role of non-endothelial dependent CMD in diastolic dysfunction.

Allogeneic cell-based therapies using adipose tissue-derived stromal cells (ASCs) offer an off-the-shelf alternative to autologous therapy. An underlying assumption is that ASC can modulate the immune response of the recipient. However, in vitro models are required to explore and identify cell interactions and mechanisms of action, to ensure sufficient and sustained effects, and to document these. In this study, we shed light on the effect of ASC manufactured for clinical use on monocyte-derived dendritic cells and an inflammatory microenvironment. ASCs were isolated from healthy voluntary donors, expanded using a human platelet lysate in bioreactors, and cryopreserved as per clinical use. Monocyte-derived dendritic cells were generated by isolation of monocytes and differentiation with GM-CSF and IL-4. Dendritic cells were cocultured with different ratios of ASC and matured with LPS and IFN-γ. Dexamethasone was included as an immunosuppressive control. Dendritic cells were analyzed by flow cytometry for CD11c, CD40, CD80, CD83, CD86, PD-L1, and HLA-DR, and supernatants were analyzed for FGF2, HGF, IL-10, IL-12p70, LIF, MIF, PDGF, PlGF, and IDO. Reduced expression of maturation markers was observed on ASC-treated dendritic cells, while high levels of PD-L1 were maintained. Interestingly, the expression of CD83 was elevated. Escalating ratios of ASC did not affect the concentration of IL-10 considerably, whereas the presence of IL-12 was reduced in a dose-dependent manner. Besides offsetting the IL-12/IL-10 balance, the concentrations of IDO and MIF were elevated in cocultures. Concentrations of FGF2, HGF, LIF, and PIGF were high in ASC cocultures, whereas PDGF was depleted. In a robust coculture model, the addition of ASC to dendritic cells inhibited the dendritic maturation substantially, while inducing a less inflammatory and more tolerogenic milieu. Despite the exposure to dendritic cells and inflammatory stimuli, ASC resulted in supernatants with trophic factors relevant for regeneration. Thus, ASC can perform immunomodulation while providing a regenerative environment.

Adipose-derived stromal cell (ASC) therapy is currently investigated as a new treatment option for patients with ischemic heart disease (IHD). The aim of this study was to evaluate the effect of ASC therapy in patients with chronic IHD measuring myocardial perfusion and cardiac function using cardiac magnetic resonance imaging (CMRI). Patients were included in MyStromalCell trial, a phase II, randomized, double-blinded, placebo-controlled study investigated the effect of ASCs in patients with chronic IHD with preserved left ventricular ejection fraction (LVEF). In total, 41 of 60 patients underwent cine, late enhancement, rest and stress imaging with CMRI. There was a non-significant difference between stress and rest values in maximal signal intensity, a measure of myocardial perfusion, from baseline to follow-up comparing placebo with ASC group (–52.52 ± 88.61 and 3.05 ± 63.17, p = 0.061, respectively). LVEF, myocardial mass, stroke volume, left ventricle end-diastolic volume and end-systolic volume changed non-significantly (–0.5 ± 4.7%, –3.5 ± 13.1 g, –0.7 ± 8.6 mL, 1.9 ± 25.1 mL and 2.6 ± 16.5 mL, respectively) in the placebo group and in the ASC group (0.7 ± 8.6%, 0.9 ± 10.8 g, –0.3 ± 26.1 mL, –3.0 ± 31.5 mL and –2.7 ± 20.4 mL, respectively) from baseline to 6 months follow-up. The amount of scar tissue was unchanged in the placebo group by 0.0 ± 1.6 g, p = 1.0 and in the ASC group with –0.3 ± 2.3 g, p = 0.540. There was no difference between the groups. There was a non-significant trend toward increased myocardial perfusion but no significant changes in functional parameters or amount of scar tissue in patients treated with ASCs compared with patients allocated into the placebo group.

Mesenchymal stromal cells have proven capable of improving cardiac pump function in patients with chronic heart failure, yet little is known about their mode of action. The aim of the study was to investigate the short-term effect of cryopreserved allogeneic rat adipose tissue-derived stromal cells (ASC) on cardiac composition, cellular subpopulations, and gene transcription in a rat model of chronic ischemic cardiomyopathy (ICM). Myocardial infarction (MI) was induced by permanent ligation of the left anterior descending coronary artery. After 6 weeks, the rats were treated with ASCs, saline, or no injection, using echo-guided trans-thoracic intramyocardial injections. The cardiac tissue was subsequently collected for analysis of cellular subpopulations and gene transcription 3 and 7 days after treatment. At day 3, an upregulation of genes associated with angiogenesis were present in the ASC group. On day 7, increases in CCR2+ and CD38+ macrophages (p = 0.047 and p = 0.021), as well as in the CD4/CD8 lymphocyte ratio (p = 0.021), were found in the ASC group compared to the saline group. This was supported by an upregulation of genes associated with monocytes/macrophages. In conclusion, ASC treatment initiated an immune response involving monocytes/macrophages and T-cells and induced a gene expression pattern associated with angiogenesis and monocyte/macrophage differentiation.

Abstract Background The purpose of the predictors for major cardiovascular outcomes in stable ischaemic heart disease (PREMAC) study is exploratory and hypothesis generating. We want to identify biochemical quantities which—conditionally on the values of available standard demographic, anamnestic, and biochemical data—may improve the prediction of cardiovascular outcomes and/or death in patients suffering from stable ischaemic heart disease. The candidate biochemical quantities include N-terminal pro-B-type natriuretic peptide, YKL-40, osteoprotegerin, high-sensitive assay cardiac troponin T (hs-cTnT), pregnancy-associated plasma protein-A (PAPP-A), cathepsin B, cathepsin S, soluble TNF receptor 1 and 2, neutrophil gelatinase-associated lipocalin, endostatin, and calprotectin. As an extra objective, we also want to assess if skewness in these predictors may explain why the clarithromycin for patients with stable coronary heart disease (CLARICOR) trial found increased all-cause and cardiovascular (CV) mortality on a brief clarithromycin regimen compared with placebo. Methods Baseline data were obtained from the hospital files at five cardiology clinics covering the Copenhagen area. The CLARICOR trial included data from 4372 stable coronary artery disease patients recruited among such patients alive and diagnosed with acute myocardial infarction or unstable angina pectoris during 1993 to 1999 in Copenhagen and randomised during October 1999 to April 2000 to the CLARICOR trial of 14 days clarithromycin versus placebo. Initial follow-up lasted for 2.6 years, during which outcomes were collected through hospital and death registries and assessed by an adjudication committee. Corresponding register data later showed to produce similar results. The adjudicated outcomes were therefore replaced and augmented by register data on outcomes to cover 10 years of follow-up. Biochemical marker data were obtained from analysis of serum from the CLARICOR bio-bank collected at randomisation and stored at −80° C. Using Cox proportional hazard method, we will identify among the candidate biochemical quantities those which are significant predictors when used alone and in combination with the standard predictors as defined in the present study. Discussion Patients who became stable during the period 1993 to 1999 and died before October 1999 are missing. The data from the placebo patients are nevertheless useful to identify new prognostic biomarkers in patients with stable coronary artery disease, and data from both trial groups are useful to assess important potential skewness between randomised groups. However, due to the potential selection bias, we do not feel that it is advisable to try to rank identified biochemical predictors relative to each other nor to use the results for predictive purposes. Trial registration ClinicalTrials.gov, NCT00121550 Date of registration 13 July 2005 Date of enrolment of first participant 12 October 1999

Abstract Background Adipose derived stromal cells (ASCs) are a rich and convenient source of cells for clinical regenerative therapeutic approaches. However, applications of ASCs often require cell expansion to reach the needed dose. In this study, cultivation of ASCs from stromal vascular fraction (SVF) over two passages in the automated and functionally closed Quantum Cell Expansion System (Quantum system) is compared with traditional manual cultivation. Methods Stromal vascular fraction was isolated from abdominal fat, suspended in α-MEM supplemented with 10% Fetal Bovine Serum and seeded into either T75 flasks or a Quantum system that had been coated with cryoprecipitate. The cultivation of ASCs from SVF was performed in 3 ways: flask to flask; flask to Quantum system; and Quantum system to Quantum system. In all cases, quality controls were conducted for sterility, mycoplasmas, and endotoxins, in addition to the assessment of cell counts, viability, immunophenotype, and differentiation potential. Results The viability of ASCs passage 0 (P0) and P1 was above 96%, regardless of cultivation in flasks or Quantum system. Expression of surface markers and differentiation potential was consistent with ISCT/IFATS standards for the ASC phenotype. Sterility, mycoplasma, and endotoxin tests were consistently negative. An average of 8.0 × 107 SVF cells loaded into a Quantum system yielded 8.96 × 107 ASCs P0, while 4.5 × 106 SVF cells seeded per T75 flask yielded an average of 2.37 × 106 ASCs—less than the number of SVF cells seeded. ASCs P1 expanded in the Quantum system demonstrated a population doubling (PD) around 2.2 regardless of whether P0 was previously cultured in flasks or Quantum, while ASCs P1 in flasks only reached a PD of 1.0. Conclusion: Manufacturing of ASCs in a Quantum system enhances ASC expansion rate and yield significantly relative to manual processing in T-flasks, while maintaining the purity and quality essential to safe and robust cell production. Notably, the use of the Quantum system entails significantly reduced working hours and thereby costs.

Background: Studies that evaluate larger numbers of protein biomarkers in patients with coronary microvascular dysfunction (CMD) have not previously been performed, and very little is known concerning the pathogenetic mechanisms leading to CMD.Our objective was to analyze associations between a broad cardiovascular disease (CVD) protein biomarker assay and CMD, and further explore internal biomarker relations in order to identify possible targets for future treatment interventions. Methods: In 174 women with angina pectoris and no significant obstructive coronary artery disease (

BACKGROUND:A link between angina with no obstructive coronary artery disease (CAD) and heart failure with preserved left ventricular ejection fraction has been proposed, but evidence in support of this is lacking. In a cross-sectional study, we investigated whether left ventricular diastolic function in women with angina pectoris and no obstructive CAD differed from a reference population. METHODS:We included 956 women with angina and

Background. While regenerative stem cell therapy for ischemic heart disease has moved into phase 3 studies, little is still known about retention and migration of cell posttransplantation. In human studies, the ability to track transplanted cells has been limited to labeling with radioisotopes and tracking using nuclear imaging. This method is limited by low resolution and short half-lives of available radioisotopes. Longitudinal tracking using magnetic resonance imaging (MRI) of myocardial injected cells labeled with iron oxide nanoparticles has shown promising results in numerous preclinical studies but has yet to be evaluated in human studies. We aimed to evaluate MRI tracking of mesenchymal stromal cells (MSCs) labeled with ultrasmall paramagnetic iron oxide (USPIO) nanoparticles after intramyocardial transplantation in patients with ischemic heart disease (IHD). Methods. Five no-option patients with chronic symptomatic IHD underwent NOGA-guided intramyocardial transplantation of USPIO-labeled MSCs. Serial MRI scans were performed to track labeled cells both visually and using semiautomated T2∗ relaxation time analysis. For safety, we followed symptoms, quality of life, and myocardial function for 6 months. Results. USPIO-labeled MSCs were tracked for up to 14 days after transplantation at injection sites both visually and using semiautomated regional T2∗ relaxation time analysis. Labeling of MSCs did not impair long-term safety of treatment. Conclusion. This was a first-in-man clinical experience aimed at evaluating the utility of MRI tracking of USPIO-labeled bone marrow-derived autologous MSCs after intramyocardial injection in patients with chronic IHD. The treatment was safe, and cells were detectable at injection sites up to 14 days after transplantation. Further studies are needed to clarify if MSCs migrate out of the injection area into other areas of the myocardium or if injected cells are washed out into the peripheral circulation. The trial is registered with ClinicalTrials.gov NCT03651791.

Non-ischemic dilated cardiomyopathy (NIDCM) constitutes one of the most common causes to non-ischemic heart failure. Despite treatment, the disease often progresses, causing severe morbidity and mortality, making novel treatment strategies necessary. Due to the regenerative actions of mesenchymal stem cells (MSCs), they have been proposed as a treatment for NIDCM. This systematic review aims to evaluate efficacy and mode of action (MoA) of MSC-based therapies in NIDCM. A systematic literature search was conducted in Medline (Pubmed) and Embase. A total of 27 studies were included (3 clinical trials and 24 preclinical studies). MSCs from different tissues and routes of delivery were reported, with bone marrow-derived MSCs and direct intramyocardial injections being the most frequent. All included clinical trials and 22 preclinical trials reported an improvement in cardiac function following MSC treatment. Furthermore, preclinical studies demonstrated alterations in tissue structure, gene, and protein expression patterns, primarily related to fibrosis and angiogenesis. Consequently, MSC treatment can improve cardiac function in NIDCM patients. The MoA underlying this effect involves anti-fibrosis, angiogenesis, immunomodulation, and anti-apoptosis, though these processes seem to be interdependent. These encouraging results calls for larger confirmatory clinical studies, as well as preclinical studies utilizing unbiased investigation of the potential MoA.

Elevated pregnancy-associated plasma protein A (PAPP-A) is associated with mortality in acute coronary syndromes. Few studies have assessed PAPP-A in stable coronary artery disease (CAD) and results are conflicting. We assessed the 10-year prognostic relevance of PAPP-A levels in stable CAD. The CLARICOR trial was a randomized controlled clinical trial including outpatients with stable CAD, randomized to clarithromycin versus placebo. The placebo group constituted our discovery cohort (n = 1.996) and the clarithromycin group the replication cohort (n = 1.975). The composite primary outcome was first occurrence of cardiovascular event or death. In the discovery cohort, incidence rates (IR) for the composite outcome were higher in those with elevated PAPP-A (IR 12.72, 95% Confidence Interval (CI) 11.0−14.7 events/100 years) compared to lower PAPP-A (IR 8.78, 8.25−9.34), with comparable results in the replication cohort. Elevated PAPP-A was associated with increased risk of the composite outcome in both cohorts (discovery Hazard Ratio (HR) 1.45, 95% CI 1.24−1.70; replication HR 1.29, 95% CI 1.10−1.52). In models adjusted for established risk factors, these trends were attenuated. Elevated PAPP-A was associated with higher all-cause mortality in both cohorts. We conclude that elevated PAPP-A levels are associated with increased long-term mortality in stable CAD, but do not improve long-term prediction of death or cardiovascular events when added to established predictors.

BackgroundWe aimed to assess the associations and predictive powers between the soluble receptors for tumor necrosis factor (TNF)‐α (TNFR1 and TNFR2) and cardiovascular outcomes in patients with stable coronary heart disease. Methods and ResultsCLARICOR (Effect of Clarithromycin on Mortality and Morbidity in Patients With Ischemic Heart Disease) is a randomized clinical trial comparing clarithromycin with placebo in patients with stable coronary heart disease. The primary outcome was a composite of nonfatal acute myocardial infarction, unstable angina pectoris, cerebrovascular disease, and all‐cause mortality. Patients were followed up for 10 years; discovery sample, those assigned placebo (1204 events in n=1998); and replication sample, those assigned clarithromycin (1220 events in n=1979). We used Cox regression adjusted for C‐reactive protein level, established cardiovascular risk factors, kidney function, and cardiovascular drugs. After adjustments, higher serum levels of TNFR1 and TNFR2 were associated with the composite outcome in the discovery sample (hazard ratio per SD increase, 1.13; 95% confidence interval, 1.05–1.22; P=0.001 for TNFR1; hazard ratio, 1.16; 95% confidence interval, 1.08–1.24; P

Background. Cell therapy for heart disease has been proven safe and efficacious, despite poor cell retention in the injected area. Improving cell retention is hypothesized to increase the treatment effect. In the present study, human adipose-derived stromal cells (ASCs) were delivered in an in situ forming alginate hydrogel following acute myocardial infarction (AMI) in rats. Methods. ASCs were transduced with luciferase and tested for ASC phenotype. AMI was inducted in nude rats, with subsequent injection of saline (controls), 1 × 106 ASCs in saline or 1 × 106 ASCs in 1% (w/v) alginate hydrogel. ASCs were tracked by bioluminescence and functional measurements were assessed by magnetic resonance imaging (MRI) and 82rubidium positron emission tomography (PET). Results. ASCs in both saline and alginate hydrogel significantly increased the ejection fraction (7.2% and 7.8% at 14 days and 7.2% and 8.0% at 28 days, resp.). After 28 days, there was a tendency for decreased infarct area and increased perfusion, compared to controls. No significant differences were observed between ASCs in saline or alginate hydrogel, in terms of retention and functional salvage. Conclusion. ASCs improved the myocardial function after AMI, but administration in the alginate hydrogel did not further improve retention of the cells or myocardial function.

We aimed to evaluate the effect of intramyocardial injections of autologous VEGF-A165-stimulated adipose-derived stromal cells (ASCs) in patients with refractory angina. MyStromalCell trial is a randomized double-blind placebo-controlled study including sixty patients with CCS/NYHA class II-III, left ventricular ejection fraction > 40%, and at least one significant coronary artery stenosis. Patients were treated with ASC or placebo in a 2 : 1 ratio. ASCs from the abdomen were culture expanded and stimulated with VEGF-A165. At 6 months follow-up, bicycle exercise tolerance increased significantly in time duration 22 s (95%CI −164 to 208 s) (P=0.034), in watt 4 (95%CI −33 to 41, 0.048), and in METs 0.2 (95%CI −1.4 to 1.8) (P=0.048) in the ASC group while there was a nonsignificant increase in the placebo group in time duration 9 s (95%CI −203 to 221 s) (P=0.053), in watt 7 (95%CI −40 to 54) (P=0.41), and in METs 0.1 (95%CI −1.7 to 1.9) (P=0.757). The difference between the groups was not significant (P=0.680, P=0.608, and P=0.720 for time duration, watt, and METs, resp.). Intramyocardial delivered VEGF-A165-stimulated ASC treatment was safe but did not improve exercise capacity compared to placebo. However, exercise capacity increased in the ASC but not in the placebo group. This trial is registered with ClinicalTrials.gov NCT01449032.

Background. Ischemic heart failure (IHF) has a poor prognosis in spite of optimal therapy. We have established a new allogeneic Cardiology Stem Cell Centre adipose-derived stromal cell (CSCC_ASC) product from healthy donors. It is produced without animal products, in closed bioreactor systems and cryopreserved as an off-the-shelf product ready to use. Study Design. A multicentre, double-blind, placebo-controlled phase II study with direct intramyocardial injections of allogeneic CSCC_ASC in patients with chronic IHF. A total of 81 patients will be randomised at 2 : 1 to CSCC_ASC or placebo. There is no HLA tissue type matching needed between the patients and the donors. Methods. The treatment will be delivered by direct injections into the myocardium. The primary endpoint is change in the left ventricle endsystolic volume at 6-month follow-up. Secondary endpoints are safety and changes in left ventricle ejection fraction, myocardial mass, stroke volume, and cardiac output. Other secondary endpoints are change in clinical symptoms, 6-minute walking test, and the quality of life after 6 and 12 months. Conclusion. The aim of the present study is to demonstrate safety and the regenerative efficacy of the allogeneic CSCC_ASC product from healthy donors in a double-blind, placebo-controlled, multicentre study in patients with IHF.

BackgroundThe majority of women with angina‐like chest pain have no obstructive coronary artery disease when evaluated with coronary angiography. Coronary microvascular dysfunction is a possible explanation and associated with a poor prognosis. This study evaluated the prevalence of coronary microvascular dysfunction and the association with symptoms, cardiovascular risk factors, psychosocial factors, and results from diagnostic stress testing. Methods and ResultsAfter screening 3568 women, 963 women with angina‐like chest pain and a diagnostic coronary angiogram without significant coronary artery stenosis (

Angiogenesis is considered integral to the reparative process after ischemic injury. The αvβ3 integrin is a critical modulator of angiogenesis and highly expressed in activated endothelial cells. 68Ga-NODAGA-E[(cRGDyK)]2 (RGD) is a positron-emission-tomography (PET) ligand targeted towards αvβ3 integrin. The aim was to present data for the uptake of RGD and correlate it with histology and to further illustrate the differences in angiogenesis due to porcine adipose-derived mesenchymal stromal cell (pASC) or saline treatment in minipigs after induction of myocardial infarction (MI). Three minipigs were treated with direct intra-myocardial injection of pASCs and two minipigs with saline. MI was confirmed by 82Rubidium (82Rb) dipyridamole stress PET. Mean Standardized Uptake Values (SUVmean) of RGD were higher in the infarct compared to non-infarct area one week and one month after MI in both pASC-treated (SUVmean: 1.23 vs. 0.88 and 1.02 vs. 0.86, p < 0.05 for both) and non-pASC-treated minipigs (SUVmean: 1.44 vs. 1.07 and 1.26 vs. 1.04, p < 0.05 for both). However, there was no difference in RGD uptake, ejection fractions, coronary flow reserves or capillary density in histology between the two groups. In summary, indications of angiogenesis were present in the infarcted myocardium. However, no differences between pASC-treated and non-pASC-treated minipigs could be demonstrated.

Treatment of myocardial infarction (MI) with bone marrow-derived mesenchymal stem cells and recently also adipose-derived stem cells has shown promising results. In contrast to clinical trials and their use of autologous bone marrow-derived cells from the ischemic patient, the animal MI models are often using young donors and young, often immune-compromised, recipient animals. Our objective was to compare bone marrow-derived mesenchymal stem cells with adipose-derived stem cells from an elderly ischemic patient in the treatment of MI using a fully grown non-immune-compromised rat model. Mesenchymal stem cells were isolated from adipose tissue and bone marrow and compared with respect to surface markers and proliferative capability. To compare the regenerative potential of the two stem cell populations, male Sprague–Dawley rats were randomized to receive intramyocardial injections of adipose-derived stem cells, bone marrow-derived mesenchymal stem cells, or phosphate-buffered saline 1 week following induction of MI. After 4 weeks, left ventricular ejection fraction (LVEF) was improved in the adipose-derived stem cell group, and scar wall thickness was greater compared with the saline group. Adipose-derived as well as bone marrow-derived mesenchymal stem cells prevented left ventricular end diastolic dilation. Neither of the cell groups displayed increased angiogenesis in the myocardium compared with the saline group. Adipose-derived stem cells from a human ischemic patient preserved cardiac function following MI, whereas this could not be demonstrated for bone marrow-derived mesenchymal stem cells, with only adipose-derived stem cells leading to an improvement in LVEF. Neither of the stem cell types induced myocardial angiogenesis, raising the question whether donor age and health have an effect on the efficacy of stem cells used in the treatment of MI.

In patients with stable coronary artery disease (CAD) and refractory angina, we performed direct intramyocardial injections of autologous mesenchymal stromal cells (MSC) and followed the safety and efficacy of the treatment for 12 months. A total of 31 patients with stable CAD, moderate to severe angina, normal left ventricular ejection fraction, and no further revascularization options were included. Bone marrow MSCs were isolated and culture expanded for 6–8 weeks and then stimulated with vascular endothelial growth factor (VEGF) for 1 week. The 12-month follow-up demonstrated that it was safe to culture expand MSCs and use the cells for clinical treatment. The patients' maximal metabolic equivalent (MET) during exercise increased from 4.23 MET at baseline to 4.72 MET at 12-month follow-up ( p < 0.001), Canadian Cardiovascular Society Class (CCS) was reduced from 3.0 to 0.8 ( p < 0.001), angina attacks per week from 13.8 to 3.2 ( p < 0.001), and nitroglycerin consumption from 10.7 to 3.4 per week ( p < 0.001). In addition, Seattle Angina Questionnaire (SAQ) evaluations demonstrated highly significant improvements in physical limitation, angina stability, angina frequency, and quality of life ( p < 0.001 for all). It is safe in the intermediate/long term to treat patients with stable CAD using autologous culture expanded MSCs. Previously reported, early and highly significant improvements in exercise capacity and clinical symptoms persist after 12 months. The results are encouraging, and a larger controlled study is warranted.

Background. Regenerative therapy is an emerging treatment modality. To determine migration and retention of implanted cells, it is crucial to develop noninvasive tracking methods. The aim was to determine ex vivo magnetic resonance imaging (MRI) detection limits of ultrasmall superparamagnetic iron-oxide (USPIO) labeled mesenchymal stromal cells (MSCs). Materials and Methods. 248 gel-phantoms were constructed and scanned on a 1.5T MRI-scanner. Phantoms contained human MSCs preincubated with USPIO nanoparticles for 2, 6, or 21 hours using 5 or 10 μg USPIO/105 MSCs. In addition, porcine hearts were scanned after injection of USPIO labeled MSCs. Results. Using 21 h incubation time and 10 μg USPIO/105 MSCs, labeled cells were clearly separated from unlabeled cells on MRI using 250.000 (P

Angiogenesis is part of the healing process following an ischemic injury and is vital for the post-ischemic repair of the myocardium. Therefore, it is of particular interest to be able to noninvasively monitor angiogenesis. This might, not only permit risk stratification of patients following myocardial infarction, but could also facilitate development and improvement of new therapies directed towards stimulation of the angiogenic response. During angiogenesis endothelial cells must adhere to one another to form new microvessels. αvβ3 integrin has been found to be highly expressed in activated endothelial cells and has been identified as a critical modulator of angiogenesis. 68Ga-NODAGA-E[c(RGDyK)]2 (RGD) has recently been developed by us as an angiogenesis positron-emission-tomography (PET) ligand targeted towards αvβ3 integrin. In the present study, we induced myocardial infarction in Göttingen minipigs. Successful infarction was documented by 82Rubidium-dipyridamole stress PET and computed tomography. RGD uptake was demonstrated in the infarcted myocardium one week and one month after induction of infarction by RGD-PET. In conclusion, we demonstrated angiogenesis by noninvasive imaging using RGD-PET in minipigs hearts, which resemble human hearts. The perspectives are very intriguing and might permit the evaluation of new treatment strategies targeted towards increasing the angiogenetic response, e.g., stem-cell treatment.

BackgroundHuman adipose stem cells (hASCs) can promote angiogenesis through secretion of proangiogenic factors such as vascular endothelial growth factor (VEGF). In other cell types, it has been shown that induction of VEGF is mediated by both protease activated receptor 2 (PAR2) and hypoxia inducible factor 1(HIF-1). The present study hypothesized that PAR2 stimulation through activation of kinase signaling cascades lead to induction of HIF-1 and secretion of VEGF.Methodology/principal findingsImmunohistochemistry revealed the expression of PAR2 receptors on the surface of hASCs. Blocking the PAR2 receptors with a specific antibody prior to trypsin treatment showed these receptors are involved in trypsin-evoked increase in VEGF secretion from hASCs. Blocking with specific kinase inhibitors suggested that that activation of MEK/ERK and PI3-kinase/Akt pathways are involved in trypsin-eveoked induction of VEGF. The effect of the trypsin treatment on the transcription of VEGF peaked at 6 hours after the treatment and was comparable to the activation observed after keeping hASCs for 24 hours at 1% oxygen. In contrast to hypoxia, trypsin alone failed to induce HIF-1 measured with ELISA, while the combination of trypsin and hypoxia had an additive effect on both VEGF transcription and secretion, results which were confirmed by Western blot.ConclusionIn hASCs trypsin and hypoxia induce VEGF expression through separate pathways.

BACKGROUND: Cysteinyl-leukotrienes (cys-LT) are powerful spasmogenic and immune modulating lipid mediators involved in inflammatory diseases, in particular asthma. Here, we investigated whether cys-LT signaling, in the context of atherosclerotic heart disease, compromises the myocardial microcirculation and its response to hypoxic stress. To this end, we examined Apoe(-/-) mice fed a hypercholesterolemic diet and analysed the expression of key enzymes of the cys-LT pathway and their receptors (CysLT1/CysLT2) in normal and hypoxic myocardium as well as the potential contribution of cys-LT signaling to the acute myocardial response to hypoxia. METHODS AND PRINCIPAL FINDINGS: Myocardial biopsies from Apoe(-/-) mice demonstrated signs of chronic inflammation with fibrosis, increased apoptosis and expression of IL-6, as compared to biopsies from C57BL/6J control mice. In addition, we found increased leukotriene C(4) synthase (LTC(4)S) and CysLT1 expression in the myocardium of Apoe(-/-) mice. Acute bouts of hypoxia further induced LTC(4)S expression, increased LTC(4)S enzyme activity and CysLT1 expression, and were associated with increased extension of hypoxic areas within the myocardium. Inhibition of cys-LT signaling by treatment with montelukast, a selective CysLT1 receptor antagonist, during acute bouts of hypoxic stress reduced myocardial hypoxic areas in Apoe(-/-) mice to levels equal to those observed under normoxic conditions. In human heart biopsies from 14 patients with chronic coronary artery disease mRNA expression levels of LTC(4)S and CysLT1 were increased in chronic ischemic compared to non-ischemic myocardium, constituting a molecular basis for increased cys-LT signaling. CONCLUSION: Our results suggest that CysLT1 antagonists may have protective effects on the hypoxic heart, and improve the oxygen supply to areas of myocardial ischemia, for instance during episodes of sleep apnea.