Mineralocorticoid receptor promotes cardiac macrophage inflammaging.

Inflammaging, a pro-inflammatory status that characterizes aging and primarily involving macrophages, is a master driver of age-related diseases. Mineralocorticoid receptor (MR) activation in macrophages critically regulates inflammatory and fibrotic processes. However, macrophage-specific mechanisms and the role of the macrophage MR for the regulation of inflammation and fibrotic remodeling in the aging heart have not yet been elucidated. Transcriptome profiling of cardiac macrophages from male/female young (4 months-old), middle (12 months-old) and old (18 and 24 months-old) mice revealed that myeloid cell-restricted MR deficiency prevents macrophage differentiation toward a pro-inflammatory phenotype. Pathway enrichment analysis showed that several biological processes related to inflammation and cell metabolism were modulated by the MR in aged macrophages. Further, transcriptome analysis of aged cardiac fibroblasts revealed that macrophage MR deficiency reduced the activation of pathways related to inflammation and upregulation of ZBTB16, a transcription factor involved in fibrosis. Phenotypic characterization of macrophages showed a progressive replacement of the TIMD4+MHC-IIneg/low macrophage population by TIMD4+MHC-IIint/high and TIMD4-MHC-IIint/high macrophages in the aging heart. By integrating cell sorting and transwell experiments with TIMD4+/TIMD4-macrophages and fibroblasts from old MRflox/MRLysMCre hearts, we showed that the inflammatory crosstalk between TIMD4- macrophages and fibroblasts may imply the macrophage MR and the release of mitochondrial superoxide anions. Macrophage MR deficiency reduced the expansion of the TIMD4- macrophage population and the emergence of fibrotic niches in the aging heart, thereby protecting against cardiac inflammation, fibrosis, and dysfunction. This study highlights the MR as an important mediator of cardiac macrophage inflammaging and age-related fibrotic remodeling.

C1q-TNF-Related Protein-9 Promotes Cardiac Hypertrophy and Failure.

RATIONALE: Myocardial endothelial cells promote cardiomyocyte hypertrophy, possibly through the release of growth factors. The identity of these factors, however, remains largely unknown, and we hypothesized here that the secreted CTRP9 (C1q-tumor necrosis factor-related protein-9) might act as endothelial-derived protein to modulate heart remodeling in response to pressure overload. OBJECTIVE: To examine the source of cardiac CTRP9 and its function during pressure overload. METHODS AND RESULTS: CTRP9 was mainly derived from myocardial capillary endothelial cells. CTRP9 mRNA expression was enhanced in hypertrophic human hearts and in mouse hearts after transverse aortic constriction (TAC). CTRP9 protein was more abundant in the serum of patients with severe aortic stenosis and in murine hearts after TAC. Interestingly, heterozygous and especially homozygous knock-out C1qtnf9 (CTRP9) gene-deleted mice were protected from the development of cardiac hypertrophy, left ventricular dilatation, and dysfunction during TAC. CTRP9 overexpression, in turn, promoted hypertrophic cardiac remodeling and dysfunction after TAC in mice and induced hypertrophy in isolated adult cardiomyocytes. Mechanistically, CTRP9 knock-out mice showed strongly reduced levels of activated prohypertrophic ERK5 (extracellular signal-regulated kinase 5) during TAC compared with wild-type mice, while CTRP9 overexpression entailed increased ERK5 activation in response to pressure overload. Inhibition of ERK5 by a dominant negative MEK5 mutant or by the ERK5/MEK5 inhibitor BIX02189 blunted CTRP9 triggered hypertrophy in isolated adult cardiomyocytes in vitro and attenuated mouse cardiomyocyte hypertrophy and cardiac dysfunction in vivo, respectively. Downstream of ERK5, we identified the prohypertrophic transcription factor GATA4, which was directly activated through ERK5-dependent phosphorylation. CONCLUSIONS: The upregulation of CTRP9 during hypertrophic heart disease facilitates maladaptive cardiac remodeling and left ventricular dysfunction and might constitute a therapeutic target in the future.

Gut Microbiota-Dependent Trimethylamine N-Oxide Predicts Risk of Cardiovascular Events in Patients With Stroke and Is Related to Proinflammatory Monocytes.

Objective- Gut microbiota-dependent metabolites, in particular trimethylamine N-oxide (TMAO), have recently been reported to promote atherosclerosis and thrombosis. Here, we examined for the first time the relation of TMAO and the risk of incident cardiovascular events in patients with recent first-ever ischemic stroke in 2 independent prospective cohorts. Moreover, the link between TMAO and proinflammatory monocytes as a potential contributing factor for cardiovascular risk in stroke patients was studied. Approach and Results- In a first study (n=78), higher TMAO plasma levels were linked with an increased risk of incident cardiovascular events including myocardial infarction, recurrent stroke, and cardiovascular death (fourth quartile versus first quartile; hazard ratio, 2.31; 95% CI, 1.25-4.23; P<0.01). In the second independent validation cohort (n=593), high TMAO levels again heralded marked increased risk of adverse cardiovascular events (fourth quartile versus first quartile; hazard ratio, 5.0; 95% CI, 1.7-14.8; P<0.01), and also after adjustments for cardiovascular risk factors including hypertension, diabetes mellitus, LDL (low-density lipoprotein) cholesterol, and estimated glomerular filtration rate (hazard ratio, 3.3; 95% CI, 1.2-10.9; P=0.04). A significant correlation was also found between TMAO levels and percentage of proinflammatory intermediate CD14++CD16+ monocytes ( r=0.70; P<0.01). Moreover, in mice fed a diet enriched with choline to increase TMAO synthesis, levels of proinflammatory murine Ly6Chigh monocytes were higher than in the chow-fed control group (choline: 9.2±0.5×103 per mL versus control: 6.5±0.5×103 per mL; P<0.01). This increase was abolished in mice with depleted gut microbiota (choline+antibiotics: 5.4±0.7×103 per mL; P<0.001 versus choline). Conclusions- The present study demonstrates for the first time a graded relation between TMAO levels and the risk of subsequent cardiovascular events in patients with recent prior ischemic stroke. Our data support the notion that TMAO-related increase of proinflammatory monocytes may add to elevated cardiovascular risk of patients with increased TMAO levels.

The glucocorticoid receptor in monocyte-derived macrophages is critical for cardiac infarct repair and remodeling.

Cell- and tissue-specific actions of glucocorticoids are mediated by the glucocorticoid receptor. Here, we demonstrate that the glucocorticoid receptor (GR) in macrophages is essential for cardiac healing after myocardial infarction. Compared with GRflox (wild-type controls), GRLysMCre mice that lacked GR in myeloid cells showed increased acute mortality as a result of cardiac rupture. Seven days after left coronary artery ligation, GRLysMCre mice exhibited worse cardiac function and adverse remodeling associated with impaired scar formation and angiogenic response to ischemic injury. Inactivation of GR altered the functional differentiation/maturation of monocyte-derived macrophages in the infarcted myocardium. Mechanistically, CD45+/CD11b+/Ly6G-/F4/80+ macrophages isolated from GRLysMCre infarcts showed deregulation of factors that control inflammation, neovascularization, collagen degradation, and scar tissue formation. Moreover, we demonstrate that cardiac fibroblasts sorted from the ischemic myocardium of GRLysMCre mice compared with cells isolated from injured GRflox hearts displayed higher matrix metalloproteinase 2 expression, and we provide evidence that the macrophage GR regulates myofibroblast differentiation in the infarct microenvironment during the early phase of wound healing. In summary, GR signaling in macrophages, playing a crucial role in tissue-repairing mechanisms, could be a potential therapeutic target during wound healing after ischemic myocardial injury.-Galuppo, P., Vettorazzi, S., Hövelmann, J., Scholz, C.-J., Tuckermann, J. P., Bauersachs, J., Fraccarollo, D. The glucocorticoid receptor in monocyte-derived macrophages is critical for cardiac infarct repair and remodeling.

Fibroblast activation protein alpha expression identifies activated fibroblasts after myocardial infarction.

INTRODUCTION: Fibroblast activation protein α (FAP) is a membrane-bound serine protease expressed by activated fibroblasts during wound healing in the skin. Expression of FAP after myocardial infarction (MI) and potential effects on cardiac wound healing are largely unknown. METHODS: MI was induced in rats and FAP expression was analyzed at 3, 7 and 28 days post-MI by microarray, Western blot and immunohistochemistry. In human hearts after MI, a FAP(+) fibroblast population was identified, and characterized by immunohistochemistry for prolyl-4-hydroxylase ß, α-smooth muscle actin, Thy-1 and vimentin. Signaling pathways leading to FAP expression were studied in human cardiac fibroblasts by Western blot and ELISA using TGFß1, TGF-beta type I-receptor (TGFbR1)-inhibitor SB431542 or the MAPK-inhibitor U0126 as well as siRNA targeting SMAD2 and SMAD3. Finally, fibroblasts were assayed for FAP-dependent migration (modified Boyden-chamber), proliferation (BrdU-assay) and gelatinolytic activity by gelatin zymography. RESULTS: In rats, FAP expression was increased after MI especially in the peri-infarct area peaking at 7 days post-MI. Co-localization analysis identified the majority of FAP(+) cells as activated proto-myofibroblasts and myofibroblasts. Concordantly, FAP(+) fibroblasts were abundant in ischemic tissue of human hearts after MI, but not in healthy control hearts. In vitro, FAP was induced by TGFß1 via the canonical SMAD2/SMAD3 pathway. Depletion of FAP in fibroblasts reduced migratory capacity, while proliferation was not affected. Gelatin zymography revealed gelatinase activity by fibroblast-derived FAP. CONCLUSION: In this study, we show for the first time the expression of FAP in activated fibroblasts after MI and its activation by TGFß1. Effects of FAP on fibroblast migration and gelatinolytic activity indicate a potential role in cardiac wound healing and remodeling.

Inflammatory monocytes determine endothelial nitric-oxide synthase uncoupling and nitro-oxidative stress induced by angiotensin II.

Endothelial nitric-oxide synthase (eNOS) uncoupling and increased inducible NOS (iNOS) activity amplify vascular oxidative stress. The role of inflammatory myelomonocytic cells as mediators of these processes and their impact on tetrahydrobiopterin availability and function have not yet been defined. Angiotensin II (ATII, 1 mg/kg/day for 7 days) increased Ly6C(high) and CD11b(+)/iNOS(high) leukocytes and up-regulated levels of eNOS glutathionylation in aortas of C57BL/6 mice. Vascular iNOS-dependent NO formation was increased, whereas eNOS-dependent NO formation was decreased in aortas of ATII-infused mice as assessed by electron paramagnetic resonance (EPR) spectroscopy. Diphtheria toxin-mediated ablation of lysozyme M-positive (LysM(+)) monocytes in ATII-infused LysM(iDTR) transgenic mice prevented eNOS glutathionylation and eNOS-derived N(ω)-nitro-L-arginine methyl ester-sensitive superoxide formation in the endothelial layer. ATII increased vascular guanosine triphosphate cyclohydrolase I expression and biopterin synthesis in parallel, which was reduced in monocyte-depleted LysM(iDTR) mice. Vascular tetrahydrobiopterin was increased by ATII infusion but was even higher in monocyte-depleted ATII-infused mice, which was paralleled by a strong up-regulation of dihydrofolate reductase expression. EPR spectroscopy revealed that both vascular iNOS- and eNOS-dependent NO formation were normalized in ATII-infused mice following monocyte depletion. Additionally, deletion as well as pharmacologic inhibition of iNOS prevented ATII-induced endothelial dysfunction. In summary, ATII induces an inflammatory cell-dependent increase of iNOS, guanosine triphosphate cyclohydrolase I, tetrahydrobiopterin, NO formation, and nitro-oxidative stress as well as eNOS uncoupling in the vessel wall, which can be prevented by ablation of LysM(+) monocytes.

Soluble guanylyl cyclase activation improves progressive cardiac remodeling and failure after myocardial infarction. Cardioprotection over ACE inhibition.

Impaired nitric oxide (NO)-soluble guanylate cyclase (sGC)-cGMP signaling is involved in the pathogenesis of ischemic heart diseases, yet the impact of long-term sGC activation on progressive cardiac remodeling and heart failure after myocardial infarction (MI) has not been explored. Moreover, it is unknown whether stimulating the NO/heme-independent sGC provides additional benefits to ACE inhibition in chronic ischemic heart failure. Starting 10 days after MI, rats were treated with placebo, the sGC activator ataciguat (10 mg/kg/twice daily), ramipril (1 mg/kg/day), or a combination of both for 9 weeks. Long-term ataciguat therapy reduced left ventricular (LV) diastolic filling pressure and pulmonary edema, improved the rightward shift of the pressure-volume curve, LV contractile function and diastolic stiffness, without lowering blood pressure. NO/heme-independent sGC activation provided protection over ACE inhibition against mitochondrial superoxide production and progressive fibrotic remodeling, ultimately leading to a further improvement of cardiac performance, hypertrophic growth and heart failure. We found that ataciguat stimulating sGC activity was potentiated in (myo)fibroblasts during hypoxia-induced oxidative stress and that NO/heme-independent sGC activation modulated fibroblast-cardiomyocyte crosstalk in the context of heart failure and hypoxia. In addition, ataciguat inhibited human cardiac fibroblast differentiation and extracellular matrix protein production in response to TGF-ß1. Overall, long-term sGC activation targeting extracellular matrix homeostasis conferred cardioprotection against progressive cardiac dysfunction, pathological remodeling and heart failure after myocardial infarction. NO/heme-independent sGC activation may prove to be a useful therapeutic target in patients with chronic heart failure and ongoing fibrotic remodeling.

Monocytes/macrophages prevent healing defects and left ventricular thrombus formation after myocardial infarction.

Myocardial infarction (MI) leads to rapid necrosis of cardiac myocytes. To achieve tissue integrity and function, inflammatory cells are activated, including monocytes/macrophages. However, the effect of monocyte/macrophage recruitment after MI remains poorly defined. After experimental MI, monocytes and macrophages were depleted through serial injections of clodronate-containing liposomes. Monocyte/macrophage infiltration was reduced in the myocardium after MI by active treatment. Mortality was increased due to thromboembolic events in monocyte- and macrophage-depleted animals (92 vs. 33%; P<0.01). Left ventricular thrombi were detectable as early as 24 h after MI; this was reproduced in a genetic model of monocyte/macrophage ablation. A general prothrombotic state, increased infarct expansion, and deficient neovascularization were not observed. Severely compromised extracellular matrix remodeling (collagen I, placebo liposome vs. clodronate liposome, 2.4 ± 0.2 vs. 0.8 ± 0.2 arbitrary units; P<0.001) and locally lost integrity of the endocardium after MI are potential mechanisms. Patients with a left ventricular thrombus had a relative decrease of CD14CD16 monocyte/macrophage subsets in the peripheral blood after MI (no thrombus vs. thrombus, 14.2 ± 0.9 vs. 7.80 ± 0.4%; P<0.05). In summary, monocytes/macrophages are of central importance for healing after MI. Impaired monocyte/macrophage function appears to be an unrecognized new pathophysiological mechanism for left ventricular thrombus development after MI.

The direct factor Xa inhibitor Rivaroxaban reduces platelet activation in congestive heart failure.

BACKGROUND: Platelet activation in congestive heart failure (CHF) contributes to an increased risk for thromboembolic complications. Rivaroxaban, the first oral direct FXa inhibitor is approved in Europe for prevention and treatment of venous thrombosis, pulmonary embolism, and prevention of thromboembolic events in atrial fibrillation. As heart failure is an important risk factor for thromboembolism and increased platelet activation is common in heart failure, we investigated the potential effect of Rivaroxaban treatment on platelets in an experimental CHF model. METHODS AND RESULTS: Chronic myocardial infarction was induced in male Wistar rats by coronary ligation. Rats were randomized to placebo or Rivaroxaban (3 and 10mg/kg once daily). After 10 weeks platelet activation was assessed. Platelet-bound fibrinogen, detected by flow-cytometry, was significantly increased in CHF-Placebo (p<0.05) and reduced following treatment with Rivaroxaban (p<0.05 vs. CHF-Placebo). ADP-induced aggregation was significantly enhanced in CHF-Placebo vs. sham-operated animals (p<0.05) and normalized following chronic FXa inhibition (p<0.05 vs. CHF-Placebo). In separate in vitro experiments, attenuated platelet aggregation was present after incubating whole blood directly with Rivaroxaban but absent when the experiment was performed in platelet-rich plasma only. Thus, a direct effect on platelets could be excluded. CONCLUSION: Chronic direct factor Xa inhibition using Rivaroxaban reduces platelet activation in CHF rats by attenuating the secondary phase of ADP-induced platelet aggregation. Thus, Rivaroxaban may constitute a useful approach to prevent thromboembolic complications and reduce platelet activation in CHF at the same time.

Fractalkine activates a signal transduction pathway similar to P2Y12 and is associated with impaired clopidogrel responsiveness.

OBJECTIVE: Fractalkine (FKN) activates a G(αi) protein-coupled signaling pathway similar to the one activated by ADP via P2Y(12), which is the drug target of clopidogrel. FKN levels are increased under several disease conditions associated with impaired clopidogrel responsiveness. METHODS AND RESULTS: Blood samples were obtained from healthy volunteers and from 40 patients under chronic clopidogrel treatment. FKN reduced prostaglandin E1-induced vasodilator-stimulated phosphoprotein phosphorylation by ≈ 25% (P<0.01) at least partially mimicking the effect of ADP via P2Y(12). In vitro, FKN increased platelet reactivity index in clopidogrel-treated patients indicating potential activation of downstream targets of P2Y(12). When stratifying patients by their FKN levels, patients within the highest quartile of FKN (2042 ± 25 pg/mL) had the weakest response to clopidogrel (platelet reactivity index, 68 ± 4%), and patients within the lowest quartile (479 ± 50 pg/mL) had the strongest response (platelet reactivity index, 48 ± 7%; P=0.0106). FKN by itself induced phosphoinositide 3-kinase activation leading to Akt phosphorylation at Ser(473) (P<0.01 versus basal). CONCLUSIONS: In addition to desensitizing platelets to prostaglandin E1 via G(αi), FKN induces phosphoinositide 3-kinase-dependent Akt phosphorylation via a G(ßγ) protein similar to ADP signaling through P2Y(12). FKN increased the platelet ADP response in clopidogrel-treated patients. Once released from an atherosclerotic lesion, this mechanism could contribute locally to impaired clopidogrel responsiveness at the vulnerable plaque.

Deletion of cardiomyocyte mineralocorticoid receptor ameliorates adverse remodeling after myocardial infarction.

BACKGROUND: Mineralocorticoid receptor (MR) blockade improves morbidity and mortality among patients with heart failure; however, the underlying mechanisms are still under investigation. We studied left ventricular remodeling after myocardial infarction in mice with cardiomyocyte-specific inactivation of the MR gene (MR(MLCCre)) that were generated with a conditional MR allele (MR(flox)) in combination with a transgene expressing Cre recombinase under control of the myosin light-chain (MLC2a) gene promoter. METHODS AND RESULTS: Control (MR(flox/flox), MR(flox/wt)) and MR(MLCCre) mice underwent coronary artery ligation. MR ablation had no detectable baseline effect on cardiac morphology and function. The progressive left ventricular chamber enlargement and functional deterioration in infarcted control mice, detected by echocardiography and conductance catheter analysis during the 8-week observation period, were substantially attenuated in MR(MLCCre) mice. Chronically infarcted MR(MLCCre) mice displayed attenuated pulmonary edema, reduced cardiac hypertrophy, increased capillary density, and reduced accumulation of extracellular matrix proteins in the surviving left ventricular myocardium. Moreover, cardiomyocyte-specific MR ablation prevented the increases in myocardial and mitochondrial O(2)(·-) production and upregulation of the NADPH oxidase subunits Nox2 and Nox4. At 7 days, MR(MLCCre) mice exhibited enhanced infarct neovessel formation and collagen structural organization associated with reduced infarct expansion. Mechanistically, cardiomyocytes lacking MR displayed accelerated stress-induced activation and subsequent suppression of nuclear factor-κB and reduced apoptosis early after myocardial infarction. CONCLUSION: Cardiomyocyte-specific MR deficiency improved infarct healing and prevented progressive adverse cardiac remodeling, contractile dysfunction, and molecular alterations in ischemic heart failure, highlighting the importance of cardiomyocyte MR for heart failure development and progression.

Behavioral pathway to a broken heart: The link between adverse childhood experiences, depression, physical exercise and cardiovascular health.

Background and aim: Adverse childhood experiences (ACEs) are a major risk factor for unfavorable behavioral, mental and health outcomes later in life. However, the precise pathway via which ACEs convey these risks, in particular regarding health outcomes such as cardiovascular disease, remains unknown. Here, we combined psychiatric and cardiac methods to investigate the pathway via which childhood adversities may lead to adult adverse cardiovascular health, with a focus on epicardial adipose tissue (EAT) as a risk marker. Methods: 210 adult congenital heart disease outpatients (mean age 35.5 y, 43% female) completed a thorough cardiac and psychiatric evaluation. Psychiatric measurements included an expert interview, the childhood trauma questionnaire (CTQ), Beck's depression inventory II (BDI-II), quality of life and the global scale of functioning, amongst others. All patients completed a full cardiac workup including EAT assessment using echocardiography. We then computed bootstrapping mediation models using ACEs as a predictor, depression and physical activity as mediators and EAT as dependent variable in PROCESS. Results: CTQ scores had a significant indirect effect on EAT via a serial mediation of BDI and physical activity [a*b2*d = 0.0260, 95% BCa CI [0.0047, 0.0619]]. Conclusion: Using mediation analyses, we show that adverse childhood events are linked to increased depressive symptoms, which are linked to decreased physical activity, which in turn are linked to a higher amount of epicardial adipose tissue. While other pathways most certainly exist and replication is needed, this suggests a meaningful pathway via which ACEs lead to adverse cardiovascular health, with several potential targets for health interventions across time.

Impact of fasting on stress systems and depressive symptoms in patients with major depressive disorder: a cross-sectional study.

Major depressive disorder (MDD) is frequently associated with poor response to treatment. Common antidepressants target neurotransmission and neuronal plasticity, which require adequate energy supply. As imaging studies indicate disturbances in central energy metabolism, and caloric restriction improves neuroplasticity and impacts mood and cognition, correction of energy status might increase the effectiveness of antidepressant treatments and reduce the psychopathological symptoms of depression. Metabolic parameters, stress hormones, and brain-derived neurotrophic factor (BDNF) levels were assessed in serum of depressed inpatients (MDD, N = 21) and healthy volunteers (Ctrl, N = 28) before and after a 72 h fasting period during which only water was consumed. Depression severity was assessed by Beck's Depression Inventory (BDI)-2 sum-score and cognitive-affective and somatic sub-scores. Fasting similarly impacted metabolic parameters and stress systems in both groups. Fasting elevated BDI-2 sum-scores and somatic sub-scores in Ctrl. In MDD, fasting increased somatic-, but decreased cognitive-affective symptoms. Sub-group analyses based on BDI-2 sum-scores pre-fasting showed that cognitive-affective symptoms decreased in patients with moderate/severe but not in those with mild symptoms. This was associated with differential changes in BDNF levels. In conclusion, fasting improved cognitive-affective sub-scores in MDD patients with moderate/severe symptoms that had not responded to prior therapy. Interventions that modulate energy metabolism might directly improve cognitive-affective symptoms and/or augment therapeutic efficacy in moderate-to-severely depressed patients.

Conditional overexpression of neuronal nitric oxide synthase is cardioprotective in ischemia/reperfusion.

BACKGROUND: We previously demonstrated that conditional overexpression of neuronal nitric oxide synthase (nNOS) inhibited L-type Ca2+ channels and decreased myocardial contractility. However, nNOS has multiple targets within the cardiac myocyte. We now hypothesize that nNOS overexpression is cardioprotective after ischemia/reperfusion because of inhibition of mitochondrial function and a reduction in reactive oxygen species generation. METHODS AND RESULTS: Ischemia/reperfusion injury in wild-type mice resulted in nNOS accumulation in the mitochondria. Similarly, transgenic nNOS overexpression caused nNOS abundance in mitochondria. nNOS translocation into the mitochondria was dependent on heat shock protein 90. Ischemia/reperfusion experiments in isolated hearts showed a cardioprotective effect of nNOS overexpression. Infarct size in vivo was also significantly reduced. nNOS overexpression also caused a significant increase in mitochondrial nitrite levels accompanied by a decrease of cytochrome c oxidase activity. Accordingly, O(2) consumption in isolated heart muscle strips was decreased in nNOS-overexpressing nNOS(+)/αMHC-tTA(+) mice already under resting conditions. Additionally, we found that the reactive oxygen species concentration was significantly decreased in hearts of nNOS-overexpressing nNOS(+)/αMHC-tTA(+) mice compared with noninduced nNOS(+)/αMHC-tTA(+) animals. CONCLUSION: We demonstrated that conditional transgenic overexpression of nNOS resulted in myocardial protection after ischemia/reperfusion injury. Besides a reduction in reactive oxygen species generation, this might be caused by nitrite-mediated inhibition of mitochondrial function, which reduced myocardial oxygen consumption already under baseline conditions.

Clopidogrel improves endothelial function and NO bioavailability by sensitizing adenylyl cyclase in rats with congestive heart failure.

Clopidogrel treatment in patients with coronary artery disease not only inhibits platelet activation but also improves endothelial function and nitric oxide (NO) bioavailability. Congestive heart failure (CHF) is associated with endothelial dysfunction and increased platelet activation. In rats with CHF following myocardial infarction (MI), we investigated whether treatment with clopidogrel modifies endothelial function. Eight weeks after coronary artery ligation, rats with CHF were randomized to placebo or the P2Y(12) receptor antagonist clopidogrel (5 mg/kg twice daily, given by gavage) for another 2 weeks. Afterwards, endothelial function was assessed in isolated aortic rings in organ bath experiments. Acetylcholine-induced, endothelium-dependent, nitric oxide-mediated vasorelaxation was significantly attenuated in CHF rats compared to sham-operated animals, and was significantly improved by treatment with clopidogrel. Adenosine-induced vasorelaxation via adenylyl cyclase stimulation was attenuated in CHF and significantly improved by clopidogrel. Increased vasoconstriction to phenylephrine was observed in CHF, particularly evident under cyclooxygenase inhibition, but prevented by clopidogrel treatment. Vasoconstriction by the P2Y(12) activator 2MeS-ADP was increased in CHF. Clopidogrel-treated CHF animals displayed enhanced phosphorylation of AKT and eNOS. In conclusion, clopidogrel improved endothelial function and NO bioavailability in heart failure. During CHF, sensitivity to P2Y(12) signaling was increased leading to impaired adenylyl cyclase-mediated signaling. Chronic P2Y(12)-blockade with clopidogrel improved adenylyl cyclase-mediated signaling including increased AKT- and eNOS-phosphorylation contributing to improved NO-mediated vasorelaxation.

Expansion of CD10neg neutrophils and CD14+HLA-DRneg/low monocytes driving proinflammatory responses in patients with acute myocardial infarction.

Immature neutrophils and HLA-DRneg/low monocytes expand in cancer, autoimmune diseases and viral infections, but their appearance and immunoregulatory effects on T-cells after acute myocardial infarction (AMI) remain underexplored. We found an expansion of circulating immature CD16+CD66b+CD10neg neutrophils and CD14+HLA-DRneg/low monocytes in AMI patients, correlating with cardiac damage, function and levels of immune-inflammation markers. Immature CD10neg neutrophils expressed high amounts of MMP-9 and S100A9, and displayed resistance to apoptosis. Moreover, we found that increased frequency of CD10neg neutrophils and elevated circulating IFN-γ levels were linked, mainly in patients with expanded CD4+CD28null T-cells. Notably, the expansion of circulating CD4+CD28null T-cells was associated with cytomegalovirus (CMV) seropositivity. Using bioinformatic tools, we identified a tight relationship among the peripheral expansion of immature CD10neg neutrophils, CMV IgG titers, and circulating levels of IFN-γ and IL-12 in patients with AMI. At a mechanistic level, CD10neg neutrophils enhanced IFN-γ production by CD4+ T-cells through a contact-independent mechanism involving IL-12. In vitro experiments also highlighted that HLA-DRneg/low monocytes do not suppress T-cell proliferation but secrete high levels of pro-inflammatory cytokines after differentiation to macrophages and IFN-γ stimulation. Lastly, using a mouse model of AMI, we showed that immature neutrophils (CD11bposLy6GposCD101neg cells) are recruited to the injured myocardium and migrate to mediastinal lymph nodes shortly after reperfusion. In conclusion, immunoregulatory functions of CD10neg neutrophils play a dynamic role in mechanisms linking myeloid cell compartment dysregulation, Th1-type immune responses and inflammation after AMI.

Genetic ablation of fibroblast activation protein alpha attenuates left ventricular dilation after myocardial infarction.

INTRODUCTION: Regulating excessive activation of fibroblasts may be a promising target to optimize extracellular matrix deposition and myocardial stiffness. Fibroblast activation protein alpha (FAP) is upregulated in activated fibroblasts after myocardial infarction (MI), and alters fibroblast migration in vitro. We hypothesized that FAP depletion may have a protective effect on left ventricular (LV) remodeling after MI. MATERIALS AND METHODS: We used the model of chronic MI in homozygous FAP deficient mice (FAP-KO, n = 51) and wild type mice (WT, n = 55) to analyze wound healing by monocyte and myofibroblast infiltration. Heart function and remodeling was studied by echocardiography, morphometric analyses including capillary density and myocyte size, collagen content and in vivo cell-proliferation. In non-operated healthy mice up to 6 months of age, morphometric analyses and collagen content was assessed (WT n = 10, FAP-KO n = 19). RESULTS: Healthy FAP-deficient mice did not show changes in LV structure or differences in collagen content or cardiac morphology. Infarct size, survival and cardiac function were not different between FAP-KO and wildtype mice. FAP-KO animals showed less LV-dilation and a thicker scar, accompanied by a trend towards lower collagen content. Wound healing, assessed by infiltration with inflammatory cells and myofibroblasts were not different between groups. CONCLUSION: We show that genetic ablation of FAP does not impair cardiac wound healing, and attenuates LV dilation after MI in mice. FAP seems dispensable for normal cardiac function and homeostasis.

Guanylyl cyclase activator ataciguat improves vascular function and reduces platelet activation in heart failure.

INTRODUCTION: Endothelial dysfunction and platelet activation due to impaired endogenous platelet inhibition by nitric oxide (NO) are part of the cardiovascular phenotype in congestive heart failure (CHF). We investigated whether chronic activation of the NO target enzyme soluble guanylyl cyclase (sGC) would beneficially modulate vascular function and platelet activation in experimental CHF. MATERIALS AND METHODS: Chronic myocardial infarction was induced by coronary ligation in male Wistar rats. Animals were either treated with placebo or the sGC activator ataciguat (10 mg/kg/twice daily by gavage). After 10 weeks, hemodynamic assessment was performed and only animals with impaired left-ventricular end-diastolic pressures of more than 15 mmHg were included in the analysis. Vasomotor function was determined in organ bath studies. NO bioavailability was assessed by in vivo platelet vasodilator-stimulated phosphoprotein (VASP) phosphorylation. P-selectin was determined as a marker of platelet degranulation. RESULTS: Endothelium-dependent, NO-mediated vasorelaxation as well as vascular sensitivity to exogenous NO were significantly impaired in aortic rings from CHF rats and normalised by ataciguat. In parallel, in vivo VASP phosphorylation reflecting NO bioavailability was significantly attenuated in platelets from CHF rats and normalised by ataciguat. Platelet activation, which was increased in CHF, was reduced by treatment with ataciguat. CONCLUSION: Chronic sGC activation improved vasomotor function and reduced platelet activation in CHF rats.

Improvement in left ventricular remodeling by the endothelial nitric oxide synthase enhancer AVE9488 after experimental myocardial infarction.

BACKGROUND: Reduced endothelial nitric oxide (NO) bioavailability contributes to the progression of heart failure. In this study, we investigated whether the transcription enhancer of endothelial NO synthase (eNOS) AVE9488 improves cardiac remodeling and heart failure after experimental myocardial infarction (MI). METHODS AND RESULTS: Starting 7 days after coronary artery ligation, rats with MI were treated with placebo or AVE9488 (25 ppm) as a dietary supplement for 9 weeks. AVE9488 therapy versus placebo substantially improved left ventricular (LV) function, reduced LV filling pressure, and prevented the rightward shift of the pressure-volume curve. AVE9488 also attenuated the extent of pulmonary edema, reduced LV fibrosis and myocyte cross-sectional area, and prevented the increases in LV gene expression of atrial natriuretic factor, brain natriuretic peptide, and endothelin-1. eNOS protein levels and calcium-dependent NOS activity were decreased in the surviving LV myocardium from placebo MI rats and normalized by AVE9488. The beneficial effects of AVE9488 on LV dysfunction and remodeling after MI were abrogated in eNOS-deficient mice. Aortic eNOS protein expression and endothelium-dependent NO-mediated vasorelaxation were significantly enhanced by AVE9488 treatment after infarction, whereas increased vascular superoxide anion formation was reduced. Moreover, AVE9488 prevented the marked depression of circulating endothelial progenitor cell levels in rats with heart failure after MI. CONCLUSIONS: Long-term treatment with the eNOS enhancer AVE9488 improved LV remodeling and contractile dysfunction after MI. Molecular alterations, circulating endothelial progenitor cell levels, and endothelial vasomotor dysfunction were improved by AVE9488. Pharmacological interventions designed to increase eNOS-derived NO constitute a promising therapeutic approach for the amelioration of postinfarction ventricular remodeling and heart failure.

Inhibition of platelet activation in rats with severe congestive heart failure by a novel endothelial nitric oxide synthase transcription enhancer.

AIMS: Increased risk of thrombo-embolic events in congestive heart failure (CHF) has been attributed to a hypercoagulable state including vascular endothelial dysfunction and reduced bioavailability of nitric oxide (NO) as well as platelet activation. We investigated whether treatment with a novel endothelial NO synthase (eNOS)-transcription enhancer positively modulates systemic NO bioavailability and reduces platelet activation in rats with CHF. METHODS AND RESULTS: After experimental myocardial infarction, male Wistar rats were treated with either placebo or the eNOS-transcription enhancer, AVE9488 (25 ppm/day) for 10 weeks. In rats with severe CHF (left ventricular end-diastolic pressure >15 mmHg), platelet vasodilator-stimulated phosphoprotein (VASP)-phosphorylation reflecting the integrity of the NO/cGMP pathway was significantly reduced (mean immunofluorescence at Ser(157): Sham, 61.4 +/- 9.1; CHF-Placebo, 37.4 +/- 4.9; P < 0.05; Ser(239): Sham, 18.1 +/- 2.5; CHF-Placebo, 13.2 +/- 0.6; P < 0.05). Platelet surface expression of P-selectin and glycoprotein 53 were increased in CHF rats compared with sham-operated animals. Chronic treatment with AVE9488 significantly enhanced platelet VASP-phosphorylation in CHF rats (Ser(157): 70.4 +/- 16.2; Ser(239): 19.3 +/- 1.8). In parallel, platelet surface expression of P-selectin and glycoprotein 53 was reduced in the treatment group. CONCLUSION: Platelet activation was evident in CHF rats. Therapy with the eNOS-transcription enhancer, AVE9488, reduced platelet activation in parallel to normalization of platelet NO bioavailability.