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.

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.

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.

Increased epicardial adipose tissue in young adults with congenital heart disease comorbid with major depressive disorder.

OBJECTIVE: Congenital heart disease is the most common congenital malformation. In adult congenital heart disease (ACHD), the prevalence of major depressive disorder (MDD) is increased. Beyond its immanent health risks, increased epi­ and paracardial adipose tissue has been described in MDD. Epicardial adipose tissue (EAT) is a fat depot surrounding the heart, and it is hypothesized to be associated with coronary artery disease, left-ventricular dysfunction and atrial fibrillation, being frequent problems in ACHD long-term management. We here examined whether EAT is increased in depressed patients with ACHD. METHODS: Two-hundred and ten ACHD outpatients (mean age 35.5y, 43% female) were included. MDD was diagnosed according to DSM-IV criteria using expert interviews. EAT was measured using echocardiography. Further assessments comprised NT-proBNP, left and right ventricular end-diastolic diameter, left-ventricular ejection fraction, smoking behavior and physical activity. RESULTS: Of 210 patients, 53 (25.2%) were diagnosed with MDD. EAT was increased in depressed ACHD (F = 5.04; df = 1; p = 0.026). Depressed male patients were less physically active (p < 0.05) and smoked more cigarettes (p < 0.05). EAT was positively predicted by depression severity (p = 0.039), body mass index (p < 0.001), and negatively predicted by physical activity (p = 0.019). CONCLUSIONS: The presence of MDD is associated with an increased amount of EAT in ACHD, and is dependent on depression severity. Further, the amount of EAT is at least in part mediated by a more sedentary lifestyle. Given the long-term health risks associated with increased EAT, interventions aiming at increased physical activity, smoking cessation and early identification of comorbid MDD may be recommended in ACHD.

Macrophage Mineralocorticoid Receptor Is a Pleiotropic Modulator of Myocardial Infarct Healing.

Myocardial infarction (MI) is a major cause of death worldwide. Here, we identify the macrophage MR (mineralocorticoid receptor) as a crucial pathogenic player in cardiac wound repair after MI. Seven days after left coronary artery ligation, mice with myeloid cell-restricted MR deficiency compared with WT (wild type) controls displayed improved cardiac function and remodeling associated with enhanced infarct neovascularization and scar maturation. Gene expression profiling of heart-resident and infarct macrophages revealed that MR deletion drives macrophage differentiation in the ischemic microenvironment toward a phenotype outside the M1/M2 paradigm, with regulation of multiple interrelated factors controlling wound healing and tissue repair. Mechanistic and functional data suggest that inactivation of the macrophage MR promotes myocardial infarct healing through enhanced efferocytosis of neutrophils, the suppression of free radical formation, and the modulation of fibroblast activation state. Crucially, targeted delivery of MR antagonists to macrophages, with a single administration of RU28318 or eplerenone-containing liposomes at the onset of MI, improved the healing response and protected against cardiac remodeling and functional deterioration, offering an effective and unique therapeutic strategy for cardiac repair.

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.

Intermediate CD14++CD16+ monocytes decline after transcatheter aortic valve replacement and correlate with functional capacity and left ventricular systolic function.

BACKGROUND: Transcatheter aortic valve replacement (TAVR) is the method of choice for patients with severe aortic valve stenosis, who are ineligible or at high risk for surgery. Though TAVR leads to a significant reduction in mortality, a notable amount of patients are re-hospitalized early after TAVR. Parameters or biomarkers predicting outcome are therefore needed to identify patients who benefit most. Specific monocyte subsets have been associated with cardiovascular diseases and were shown to possess prognostic value. METHODS: Peripheral blood was drawn before and after transfemoral TAVR with the self-expanding CoreValve, Boston Lotus or the balloon-expanding Edwards Sapien prosthesis. Classical (CD14++CD16-), intermediate (CD14++CD16+) and non-classical (CD14+CD16++) monocyte subsets were determined by flow cytometry. Transthoracic echocardiography was performed before, early after as well as 3 months after the TAVR procedure. RESULTS: No significant differences in the absolute monocyte counts were found after TAVR. A significant decline in the intermediate monocyte population was though observed early after TAVR (pre 4.01±0.38%, post 2.803±0.34%, p≤0.05). Creatinine levels stayed stable after TAVR procedure and intermediate monocytes were associated with worse renal function. Monocyte decline was not related to changes in CRP-, noradrenaline, cortisol or aldosterone-levels. The amount of intermediate monocytes correlated with worse cardiac function and predicted the possibility to reach an improvement in NYHA functional class at 3 months after TAVR. CONCLUSIONS: A significant decline of intermediate monocytes occurs shortly after TAVR. High levels of intermediate monocytes were associated with worse cardiac function and predicted poor functional capacity, hinting at a possible prognostic value.

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.

Modeling Cardiac Fibrosis in Mice: (Myo)Fibroblast Phenotype After Ischemia.

Cardiac (myo)fibroblasts play a key role in the regulation of wound healing and pathogenic remodeling after myocardial infarction. Impaired scar formation and alterations of the extracellular matrix network precipitate cardiac dysfunction leading to increased morbidity and mortality. Therapeutic approaches addressing (myo)fibroblast phenotype appear to be useful in preventing progressive structural, electrical, and functional impairment and heart failure.Permanent ligation of the left anterior descending coronary artery has proven to be a valuable experimental model to investigate the arrays of pathways/mechanisms involved in cardiac repair and extracellular matrix remodeling in ischemic heart failure. Here we describe the surgical procedure to occlude the left coronary artery in mice. Moreover, we present an accurate method to isolate (myo)fibroblasts from ischemic myocardium, with maintenance of the functional phenotype, using the specific marker for mouse cardiac fibroblasts mEF-SK4. The protocol can be completed within a few hours, and the isolated fibroblasts/myofibroblasts are suitable for downstream molecular biology applications, like gene expression profiling and cell culture.

Changes in concentrations of circulating fibroblast activation protein alpha are associated with myocardial damage in patients with acute ST-elevation MI.

BACKGROUND: Fibroblast activation protein alpha (FAP) is a membrane-bound serine protease expressed by activated fibroblasts after myocardial infarction (MI). Reduced circulating FAP levels were associated with increased mortality in patients with acute coronary syndrome. We hypothesized that FAP concentrations are altered after acute ST-elevation MI (STEMI), and related to myocardial damage. METHODS: We measured circulating FAP concentrations in blood plasma of 60 patients on admission, day 1, day 3 and day 5 after STEMI, and in 25 apparently healthy blood donors as controls. RESULTS: Plasma FAP concentrations were lower in STEMI patients on admission (71ng/mL) than in blood donors (101ng/mL, P<0.0001). FAP concentrations declined in STEMI patients from admission to day 3 (66ng/mL, P<0.05) and day 5 (57ng/mL, P<0.05). FAP concentrations on day 5 were inversely correlated with maximum CK and maximum CRP levels. In a multiple linear regression analysis, maximum CRP was independently associated with low FAP concentrations on day 5 after STEMI. When stratified according to the absolute amount of FAP change from admission to day 5 (ΔFAP), patients with high ΔFAP (-22ng/mL) had worse left ventricular function, higher levels of hs-cTnT, CK on admission, maximum CK and CRP than patients with low ΔFAP (-3ng/mL). CONCLUSIONS: Our study first demonstrates alterations of circulating FAP concentrations acutely after STEMI. A greater decline of circulating FAP concentrations in the first 5days after STEMI is associated with increased myocardial damage and inflammation. Measurement of circulating FAP might help to better understand the relation of myocardial injury and inflammatory response in the individual patient.

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.

Mental disorders in adults with congenital heart disease: Unmet needs and impact on quality of life.

OBJECTIVE: In adult congenital heart disease (ACHD), mental health status and quality of life become important issues due to improved life expectancy. Current literature provides conflicting data regarding mental health status in ACHD. Furthermore, none of the studies so far compared prevalence rates with a matched control group. METHODS: The prevalence of mental disorders was assessed in 150 ACHD using a structured interview, and compared to 12-months estimates of the general German population. Quality of life (QoL) was measured with World Health Organization Quality of Life instrument. Furthermore, we related the diagnostic results of widely used screening instruments for depression (Beck Depression Inventory-2; BDI-2; Hospital Anxiety and Depression Scale; HADS) with clinical diagnoses, to receive optimal sensitivity and specificity values. RESULTS: The prevalence of psychiatric disorders was significantly higher in ACHD than in the general population (48.0%; CI: 44.7-60.0 vs. 35.7%; CI: 33.5-37.9). Mood (30.7%; CI: 24.0-38.0 vs. 10.7%;CI:9.4-12.0) and anxiety disorders (28.0%; CI:22.0-36.7 vs. 16.8%; CI: 15.0-18.6) were the leading causes of psychiatric illness. Sixteen of 150 ACHD patients (10.7%) received specific treatment for psychiatric disorders before entering the study. Overall quality of life was independently and negatively associated with a diagnosis of major depression (p<0.001), alcohol dependency (p=0.004), nicotine dependency (p=0.036), and NYHA class (p=0.007). Accuracy of the HADS-D and BDI-2 as screening instruments was moderate (AUC 0.60-0.81), depending on the cut-off score used. CONCLUSIONS: Psychiatric disorders, particularly mood and anxiety disorders are significantly more frequent in ACHD compared to the general population. However, these disorders are rarely diagnosed resulting in under treatment and loss of quality of life. Quality of life is independently associated with the existence of mood, anxiety and substance use disorders. When self-rating instruments (BDI-2, HADS) are used as screening instruments in ACHD care, lower cut-off values are recommended.

Multidrug resistance associated protein-1 (MRP1) deficiency attenuates endothelial dysfunction in diabetes.

AIM: The multidrug resistance associated protein-1 (MRP1) is the main transporter of oxidized glutathione in endothelial cells, and blockade of MRP1 improves endothelial cell dysfunction induced by reactive oxygen species. We therefore investigated the role of MRP1 in hyperglycemia-induced endothelial dysfunction and ROS production. METHODS AND RESULTS: Diabetes was induced in 12 week old male MRP1(-/-)- or corresponding FVB wild-type (wt) mice by injection of streptozotocin (50mg/kg for 5 days). Eight weeks thereafter acetylcholine-induced endothelium-dependent vasorelaxation was blunted in aortic rings from diabetic wt mice (blood glucose levels >250 mg/dl) compared with nondiabetic animals (Rmax 74 ± 2% vs. 94 ± 2%, p<0.001). However in aortae from diabetic mice lacking MRP1, endothelium-dependent vasorelaxation was only mildly impaired (Rmax 87 ± 3%, p<0.001 vs. wt). Endothelium-independent relaxation induced by DEA-NONOate was not different among the groups. Streptozotocin-induced diabetes significantly increased aortic superoxide anion and hydrogen peroxide production in wild-type but not in MRP1(-/-) mice. Aortic levels of glutathione were significantly diminished in STZ-treated FVB mice, while preserved in MRP1(-/-) mice. Further, in cultured human aortic endothelial cells, high glucose levels (30 mmol/l) over 5 days significantly increased superoxide production which was inhibited by downregulation of MRP1 via siRNA. CONCLUSIONS: These data indicate that MRP1 plays an important role for endothelial dysfunction and reactive oxygen species production in diabetes and under conditions of hyperglycemia. MRP1 therefore may represent a therapeutic target in treatment of diabetes induced vascular dysfunction.

Pentaerythritol Tetranitrate Targeting Myocardial Reactive Oxygen Species Production Improves Left Ventricular Remodeling and Function in Rats With Ischemic Heart Failure.

Reduced nitric oxide bioavailability contributes to progression of cardiac dysfunction and remodeling in ischemic heart failure. Clinical use of organic nitrates as nitric oxide donors is limited by development of nitrate tolerance and reactive oxygen species formation. We investigated the effects of long-term therapy with pentaerythritol tetranitrate (PETN), an organic nitrate devoid of tolerance, in rats with congestive heart failure after extensive myocardial infarction. Seven days after coronary artery ligation, rats were randomly allocated to treatment with PETN (80 mg/kg BID) or placebo for 9 weeks. Long-term PETN therapy prevented the progressive left ventricular dilatation and improved left ventricular contractile function and relaxation in rats with congestive heart failure. Mitochondrial superoxide anion production was markedly increased in the failing left ventricular myocardium and nearly normalized by PETN treatment. Gene set enrichment analysis revealed that PETN beneficially modulated the dysregulation of mitochondrial genes involved in energy metabolism, paralleled by prevention of uncoupling protein-3, thioredoxin-2, and superoxide dismutase-2 downregulation. Moreover, PETN provided a remarkable protective effect against reactive fibrosis in chronically failing hearts. Mechanistically, induction of heme oxygenase-1 by PETN prevented mitochondrial superoxide generation, NOX4 upregulation, and ensuing formation of extracellular matrix proteins in fibroblasts from failing hearts. In summary, PETN targeting reactive oxygen species generation prevented the changes of mitochondrial antioxidant enzymes and progressive fibrotic remodeling, leading to amelioration of cardiac functional performance. Therefore, PETN might be a promising therapeutic option in the treatment of ischemic heart diseases involving oxidative stress and impairment in nitric oxide bioactivity.

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.

The nitric oxide donor pentaerythritol tetranitrate reduces platelet activation in congestive heart failure.

BACKGROUND: Platelet activation associated with endothelial dysfunction and impaired endogenous platelet inhibition is part of the cardiovascular phenotype of congestive heart failure (CHF) and contributes to the increased risk for thromboembolic complications. Pentaerythritol tetranitrate (PETN) has been shown to release nitric oxide without development of nitrate tolerance. We investigated the effect of chronic PETN treatment on platelet activation and aggregation in an experimental CHF model. METHODS AND RESULTS: Chronic ischemic heart failure was induced in male Wistar rats by coronary artery ligation. Starting 7 days thereafter, rats were randomised to placebo or PETN (80 mg/kg twice daily). After 9 weeks, activation of circulating platelets was determined measuring platelet bound fibrinogen, which requires activated glycoprotein IIb/IIIa on the platelet surface. Binding was quantified by flow-cytometry using a FITC-labelled anti-fibrinogen antibody. Platelet-bound fibrinogen was significantly increased in CHF-Placebo (mean fluorescence intensity: Sham 88±4, CHF-Placebo 104±6, p<0.05) and reduced following treatment with PETN (89±7, p<0.05 vs. CHF-Placebo). Maximal and final ADP-induced aggregation was significantly enhanced in CHF-Placebo vs. Sham-operated animals and normalized / decreased following chronic PETN treatment. Moreover, platelet adhesion was significantly reduced (number of adherent platelets: control: 85.6±5.5, PETN: 40±3.3; p<0.001) and VASP phosphorylation significantly enhanced following in vitro PETN treatment. CONCLUSION: Chronic NO supplementation using PETN reduces platelet activation in CHF rats. Thus, PETN may constitute a useful approach to prevent thromboembolic complications in CHF.

Efficacy of mineralocorticoid receptor antagonism in the acute myocardial infarction phase: eplerenone versus spironolactone.

AIMS: The selective mineralocorticoid receptor (MR) antagonist eplerenone given early in patients with acute myocardial infarction (MI) improves clinical outcome, whereas little is known about the effectiveness of early spironolactone therapy. We aimed to compare the ability of eplerenone and spironolactone to promote cardiac repair after experimental MI. METHODS AND RESULTS: Starting immediately after coronary artery ligation, C57BL/6J mice were treated with placebo, eplerenone, or spironolactone. At 7 days, treatment with eplerenone or spironolactone reduced thinning and expansion of healing infarct and improved early left ventricular chamber enlargement. Remarkably, eplerenone therapy resulted in significantly greater improvement than spironolactone of left ventricular contractile function and relaxation, associated with a more considerable leftward and downward shift of the pressure volume curve. Seven-day survival rate was significantly increased only in eplerenone treated mice. Moreover, eplerenone was superior to spironolactone in ameliorating neovessel formation in the injured myocardium. Optimized flow cytometry analysis of the monocyte differentiation marker Ly6C revealed predominant accumulation of Ly6Chigh monocytes/macrophages at the site of ischemic injury during the early inflammatory phase in placebo-treated mice. In contrast, MR antagonism, especially by eplerenone, led to a skewing of the monocyte/macrophage population toward a higher frequency of healing promoting Ly6Clow cells. CONCLUSION: The MR antagonist eplerenone versus spironolactone showed superior efficacy during the acute MI phase with more beneficial effects on survival, early cardiac dilation, and functional decline. Modulation of monocyte maturation and enhanced infarct neovessel formation appears to play a pivotal role.