Rationale:: The transcription factor Islet-1 is a marker of cardiovascular progenitors during embryogenesis. The isolation of Islet-1–positive (Islet-1⁺) cells from early postnatal hearts suggested that Islet-1 also marks cardiac progenitors in adult life.Objective:: We investigated the distribution and identity of Islet-1⁺cells in adult murine heart and evaluated whether their number or distribution change with age or after myocardial infarction.Methods and Results:: Distribution of Islet-1⁺cells in adult heart was investigated using gene targeted mice with nuclear β-galactosidase inserted into the Islet-1 locus. nLacZ-positive cells were only present in 3 regions of the adult heart: clusters in the interatrial septum and around the pulmonary veins, scattered within the wall of the great vessels, and a strictly delimited cluster between the right atrium and superior vena cava. Islet-1⁺cells in the first type of clusters coexpressed markers for parasympathetic neurons. Positive cells in the great arteries coexpressed smooth muscle actin and myosin heavy chain, indicating a smooth muscle cell identity. Very few Islet-1⁺cells within the outflow tract expressed the cardiomyocyte marker α-actinin. Islet-1⁺cells in the right atrium coexpressed the sinoatrial node pacemaker cell marker HCN4. Cell number and localization remained unchanged between 1 to 18 months of age. Consistently Islet-1 mRNA was detected in human sinoatrial node. Islet-1⁺cells could not be detected in the infarct zone 2 to 28 days after myocardial infarction, aside from 10 questionable cells in 1/13 hearts.Conclusions:: Our results identify Islet-1 as a novel marker of the adult sinoatrial node and do not provide evidence for Islet-1⁺cells to serve as cardiac progenitors.

Rationale:: Ventricular arrhythmias remain the leading cause of death in patients suffering myocardial ischemia. Myeloperoxidase, a heme enzyme released by polymorphonuclear neutrophils, accumulates within ischemic myocardium and has been linked to adverse left ventricular remodeling. Objective:: To reveal the role of myeloperoxidase for the development of ventricular arrhythmias. Methods and Results:: In different murine models of myocardial ischemia, myeloperoxidase deficiency profoundly decreased vulnerability for ventricular tachycardia on programmed right ventricular and burst stimulation and spontaneously as assessed by ECG telemetry after isoproterenol injection. Experiments using CD11b/CD18 integrin–deficient (CD11b ^−/− ) mice and intravenous myeloperoxidase infusion revealed that neutrophil infiltration is a prerequisite for myocardial myeloperoxidase accumulation. Ventricles from myeloperoxidase-deficient (Mpo ^−/− ) mice showed less pronounced slowing and decreased heterogeneity of electric conduction in the peri-infarct zone than wild-type mice. Expression of the redox-sensitive gap junctional protein Cx43 (Connexin 43) was reduced in the peri-infarct area of wild-type compared with Mpo ^−/− mice. In isolated wild-type cardiomyocytes, Cx43 protein content decreased on myeloperoxidase/H ₂ O ₂ incubation. Mapping of induced pluripotent stem cell–derived cardiomyocyte networks and in vivo investigations linked Cx43 breakdown to myeloperoxidase-dependent activation of matrix metalloproteinase 7. Moreover, Mpo ^−/− mice showed decreased ventricular postischemic fibrosis reflecting reduced accumulation of myofibroblasts. Ex vivo, myeloperoxidase was demonstrated to induce fibroblast-to-myofibroblast transdifferentiation by activation of p38 mitogen-activated protein kinases resulting in upregulated collagen generation. In support of our experimental findings, baseline myeloperoxidase plasma levels were independently associated with a history of ventricular arrhythmias, sudden cardiac death, or implantable cardioverter–defibrillator implantation in a cohort of 2622 stable patients with an ejection fraction >35% undergoing elective diagnostic cardiac evaluation. Conclusions:: Myeloperoxidase emerges as a crucial mediator of postischemic myocardial remodeling and may evolve as a novel pharmacological target for secondary disease prevention after myocardial ischemia.

Pulmonary arterial hypertension (PAH) remains a disease with limited therapeutic options and dismal prognosis. Despite its etiologic heterogeneity, the underlying unifying pathophysiology is characterized by increased vascular tone and adverse remodeling of the pulmonary circulation. Myeloperoxidase (MPO), an enzyme abundantly expressed in neutrophils, has potent vasoconstrictive and profibrotic properties, thus qualifying as a potential contributor to this disease. Here, we sought to investigate whether MPO is causally linked to the pathophysiology of PAH. Investigation of 2 independent clinical cohorts revealed that MPO plasma levels were elevated in subjects with PAH and predicted adverse outcome. Experimental analyses showed that, upon hypoxia, right ventricular pressure was less increased in Mpo ^–/– than in WT mice. The hypoxia-induced activation of the Rho-kinase pathway, a critical subcellular signaling pathway yielding vasoconstriction and structural vascular remodeling, was blunted in Mpo ^–/– mice. Mice subjected to i.v. infusion of MPO revealed activation of Rho-kinase and increased right ventricular pressure, which was prevented by coinfusion of the Rho-kinase inhibitor Y-27632. In the Sugen5416/hypoxia rat model, PAH was attenuated by the MPO inhibitor AZM198. The current data demonstrate a tight mechanistic link between MPO, the activation of Rho-kinase, and adverse pulmonary vascular function, thus pointing toward a potentially novel avenue of treatment.

Objective—: Recruitment of immunologic competent cells to the vessel wall is a crucial step in formation of abdominal aortic aneurysms (AAA). Innate immunity effectors (eg, macrophages), as well as mediators of adaptive immunity (eg, T cells), orchestrate a local vascular inflammatory response. IL-10 (interleukin-10) is an immune-regulatory cytokine with a crucial role in suppression of inflammatory processes. We hypothesized that an increase in systemic IL-10-levels would mitigate AAA progression. Approach and Results—: Using a single intravenous injection protocol, we transfected an IL-10 transcribing nonimmunogenic minicircle vector into the Ang II (angiotensin II)-ApoE ^−/− infusion mouse model of AAA. IL-10 minicircle transfection significantly reduced average aortic diameter measured via ultrasound at day 28 from 166.1±10.8% (control) to 131.0±5.8% (IL-10 transfected). Rates of dissecting AAA were reduced by IL-10 treatment, with an increase in freedom from dissecting AAA from 21.5% to 62.3%. Using flow cytometry of aortic tissue from minicircle IL-10-treated animals, we found a significantly higher percentage of CD4 ⁺ /CD25 ⁺ /Foxp3 (forkhead box P3) ⁺ regulatory T cells, with fewer CD8 ⁺ /GZMB ⁺ (granzyme B) cytotoxic T cells. Furthermore, isolated aortic macrophages produced less TNF-α (tumor necrosis factor-α), more IL-10, and were more likely to be MRC1 (mannose receptor, C type 1)–positive alternatively activated macrophages. These results concurred with gene expression analysis of lipopolysaccharide-stimulated and Ang II-primed human peripheral blood mononuclear cells. Conclusions—: Taken together, we provide an effective gene therapy approach to AAA in mice by enhancing antiinflammatory and dampening proinflammatory pathways through minicircle-induced augmentation of systemic IL-10 expression.

Marfan syndrome (MFS) is a connective tissue disorder caused by mutations in the Fibrillin-1 gene. It is associated with formation of thoracic aortic aneurysms that can potentially be a life-threatening condition due to aortic rupture or dissection. Excessive non-canonical transforming growth factor beta signalling, mediated by activation of extracellular signal-regulated kinases 1/2 (ERK1/2), as well as inducible nitric oxide synthase (NOS2)-dependent nitric oxide production, have been identified to drive aortic pathology in MFS through induction of elastin fragmentation and smooth muscle cell apoptosis. Despite promising results in animal studies, specific pharmacological interventions approved for clinical use in patients with MFS-related aortic disease are rare. Nitro-oleic acid (NO₂-OA) is an endogenously generated signalling modulator, which is available as an oral compound and has been shown to inhibit ERK1/2 activation and NOS2 expression in different disease models, thereby exerting promising therapeutic effects. In this study, we investigated whether NO₂-OA decreases aortic dilation in MFS. Eight-week-old MFS (Fbn1^C1041G/+) mice were treated with NO₂-OA or vehicle for 4 weeks via subcutaneously implanted osmotic minipumps. Echocardiography indicated progressive ascending aortic dilation and wall stiffening in MFS mice, which was significantly attenuated by NO₂-OA treatment. This protective effect was mediated by inhibition of aortic ERK1/2, Smad2 as well as nuclear factor kappa B overactivation and consequent attenuation of elastin fragmentation by matrix metalloproteinase 2, apoptosis, and collagen deposition. Critically, the therapeutic efficacy of NO₂-OA in MFS was further emphasized by demonstrating its capability to reduce lethal aortic complications in Fbn1^C1041G/+ mice challenged with Angiotensin II. NO₂-OA distinctly attenuates progression of aortic dilation in MFS via modulation of well-established disease-mediating pathways, thereby meriting further investigation into its application as a therapeutic agent for the treatment of this condition.