Daniela Fraccarollo

Endothelial Nitric Oxide Synthase Uncoupling Impairs Endothelial Progenitor Cell Mobilization and Function in Diabetes

Uncoupling of the endothelial nitric oxide synthase (eNOS) resulting in superoxide anion (O2−) formation instead of nitric oxide (NO) causes diabetic endothelial dysfunction. eNOS regulates mobilization and function of endothelial progenitor cells (EPCs), key regulators of vascular repair. We postulate a role of eNOS uncoupling for reduced number and function of EPC in diabetes. EPC levels in diabetic patients were significantly reduced compared with those of control subjects. EPCs from diabetic patients produced excessive O2− and showed impaired migratory capacity compared with nondiabetic control subjects. NOS inhibition with NG-nitro-l-arginine attenuated O2− production and normalized functional capacity of EPCs from diabetic patients. Glucose-mediated EPC dysfunction was protein kinase C dependent, associated with reduced intracellular BH4 (tetrahydrobiopterin) concentrations, and reversible after exogenous BH4 treatment. Activation of NADPH oxidases played an additional but minor role in glucose-mediated EPC dysfunction. In rats with streptozotocin-induced diabetes, circulating EPCs were reduced to 39 ± 5% of controls and associated with uncoupled eNOS in bone marrow. Our results identify uncoupling of eNOS in diabetic bone marrow, glucose-treated EPCs, and EPCs from diabetic patients resulting in eNOS-mediated O2− production. Subsequent reduction of EPC levels and impairment of EPC function likely contributes to the pathogenesis of vascular disease in diabetes.

Addition of spironolactone to angiotensin-converting enzyme inhibition in heart failure improves endothelial vasomotor dysfunction: Role of vascular superoxide anion formation and endothelial nitric oxide synthase expression

OBJECTIVES We sought to investigate the effects of adding spironolactone (SP) to angiotensin-converting enzyme (ACE) inhibition on endothelium-dependent vasodilation in rats with chronic heart failure (CHF). BACKGROUND Adding SP to ACE inhibitors reduces mortality and morbidity in CHF. Endothelial vasomotor dysfunction contributes to increased peripheral vascular resistance and reduced myocardial perfusion in CHF. METHODS Seven days after extensive myocardial infarction (CHF) or sham operation, Wistar rats were treated with placebo, the ACE inhibitor trandolapril (TR, 0.3 mg/kg body weight per day), SP (10 mg/kg per day) or a combination of both for 11 weeks. RESULTS Maximal acetylcholine-induced, nitric oxide (NO)-dependent relaxation was significantly attenuated in aortic rings from rats with CHF as compared with sham-operated animals (R(max) 44 +/- 3% vs. 63 +/- 3%). Spironolactone alone had no influence (46 +/- 5%) and TR improved NO-mediated relaxation (55 +/- 4%), whereas treatment with both completely restored endothelium-dependent vasorelaxation (64 +/- 4%). Aortic superoxide formation was significantly increased in rats with CHF as compared with sham-operated animals, but was normalized by treatment with SP or SP plus TR. In addition, aortic messenger ribonucleic acid expression of the oxidase subunit p22(phox) in rats with CHF was significantly reduced by SP or TR plus SP. Endothelial NO synthase expression was increased in TR-treated animals. Incubation of isolated porcine coronary arteries with SP dose-dependently attenuated superoxide formation. CONCLUSIONS Spironolactone added to an ACE inhibitor normalizes NO-mediated relaxation in experimental CHF by beneficially modulating the balance of NO and superoxide anion formation.

Sustained activation of nuclear factor kappa B and activator protein 1 in chronic heart failure.

OBJECTIVE Innate immune response proteins such as inflammatory cytokines, inducible nitric oxide synthase, and toll like receptors are implicated in myocardial depression and left ventricular (LV) remodeling after myocardial infarction (MI). Although all these innate immunity proteins share the downstream activation of the transcription factor NF-kappaB (nuclear factor kappa B) and activator protein 1 (AP-1), the involvement of NF-kappaB and AP-1 in LV remodeling has not been demonstrated so far. METHODS AND RESULTS Nuclear translocation of NF-kappaB and AP-1 was studied by electrophoretic mobility shift assays and ELISA 10 weeks after large experimental MI in rats, the chronic phase of LV remodeling. In the non-infarcted myocardium of MI rats, NF-kappaB and AP-1 were significantly activated (2.5-fold) as compared to sham-operated animals. Immunohistochemistry demonstrated NF-kappaB activation mainly in cardiac myocytes. Treatment with the ACE (angiotensin converting enzyme) inhibitor trandolapril led to a further 2-fold increase in the activation of NF-kappaB and AP-1 when compared to placebo-treated animals with the same MI size (P<0.001). Human failing hearts explanted at the time of heart transplantation exhibited marked nuclear translocation of NF-kappaB in cardiac myocytes when compared to control hearts. NF-kappaB as well as AP-1 were both significantly activated in congestive heart failure due to ischemic or dilated cardiomyopathy. CONCLUSION In experimental and human heart failure, both NF-kappaB and AP-1 are chronically activated in cardiac myocytes. These findings suggest an important involvement of NF-kappaB and AP-1 in the cardiac remodeling process.

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 (MRMLCCre) that were generated with a conditional MR allele (MRflox) in combination with a transgene expressing Cre recombinase under control of the myosin light-chain (MLC2a) gene promoter. Methods and Results— Control (MRflox/flox, MRflox/wt) and MRMLCCre 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 MRMLCCre mice. Chronically infarcted MRMLCCre 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 O2·− production and upregulation of the NADPH oxidase subunits Nox2 and Nox4. At 7 days, MRMLCCre 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-&kgr;B 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.

Attenuation of Angiotensin II–Induced Vascular Dysfunction and Hypertension by Overexpression of Thioredoxin 2

Reactive oxygen species increase in the cardiovascular system during hypertension and in response to angiotensin II. Because mitochondria contribute to reactive oxygen species generation, we sought to investigate the role of thioredoxin 2, a mitochondria-specific antioxidant enzyme. Mice were created with overexpression of human thioredoxin 2 (TghTrx2 mice) and backcrossed to C57BL/6J mice for ≥6 generations. Twelve-week-old male TghTrx2 or littermate wild-type mice were made hypertensive by infusion of angiotensin II (400 ng/kg per minute) for 14 days using osmotic minipumps. Systolic arterial blood pressure was not different between TghTrx2 and wild-type animals under baseline conditions (101±1 respective 102±1 mm Hg). The angiotensin II–induced hypertension in wild-type mice (145±2 mm Hg) was significantly attenuated in TghTrx2 mice (124±1 mm Hg; P<0.001). Aortic endothelium-dependent relaxation was significantly reduced in wild-type mice after angiotensin II infusion but nearly unchanged in transgenic mice. Elevated vascular superoxide and hydrogen peroxide levels, as well as expression of NADPH oxidase subunits in response to angiotensin II infusion, were significantly attenuated in TghTrx2 mice. Mitochondrial superoxide anion levels were augmented after angiotensin II infusion in wild-type mice, and this was blunted in TghTrx2 mice. Angiotensin II infusion significantly increased myocardial superoxide formation, heart weight, and cardiomyocyte size in wild-type but not in TghTrx2 mice. These data indicate a major role for mitochondrial thioredoxin 2 in the development of cardiovascular alterations and hypertension during chronic angiotensin II infusion. Thioredoxin 2 may represent an important therapeutic target for the prevention and treatment of hypertension and oxidative stress.

Immediate Mineralocorticoid Receptor Blockade Improves Myocardial Infarct Healing by Modulation of the Inflammatory Response

Mineralocorticoid receptor (MR) blockade reduces morbidity and mortality after acute myocardial infarction; however, the underlying mechanisms are still under investigation. This study examined whether MR antagonism promotes healing of the infarcted myocardium. Starting immediately after coronary ligation, male Wistar rats were treated with the selective MR antagonist eplerenone (100 mg/kg per day by gavage) or placebo for 2 to 7 days. At 7 days, eplerenone therapy versus placebo significantly reduced thinning and dilatation of the infarcted wall, improved left ventricular function, and enhanced neovessel formation in the injured myocardium. At 2 days, eplerenone-treated rats displayed lower plasma corticosterone levels, higher circulating blood monocytes, and more macrophages infiltrating the infarcted myocardium. MR blockade led to a transient upregulation (at days 2 and 3 but not at day 7) of monocyte chemoattractant protein-1, tumor necrosis factor-&agr;, interleukin-1&bgr;, interleukin-6, interleukin-10, and interleukin-4 and an increase in factor XIIIa protein expression in the healing myocardium. Prevention of macrophage accumulation into the infarct zone by treatment with liposome-encapsulated clodronate almost abrogated the protein expression of factor XIIIa and the beneficial effects of eplerenone on infarct expansion. In conclusion, selective MR blockade immediately after myocardial infarction accelerated macrophage infiltration and transiently increased the expression of healing promoting cytokines and factor XIIIa in the injured myocardium resulting in enhanced infarct neovascularization and reduced early LV dilation and dysfunction.

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.

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 CD14+CD16+ 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.—Frantz, S., Hofmann, U., Fraccarollo, D., Schäfer, A., Kranepuhl, S., Hagedorn, I., Nieswandt, B., Nahrendorf, M., Wagner, H., Bayer, B., Pachel, C., Schön, M.P., Kneitz, S., Bobinger, T., Weidemann, F., Ertl, G., Bauersachs, J. Monocytes/macrophages prevent healing defects and left ventricular thrombus formation after myocardial infarction. FASEB J. 27, 871–881 (2013). www.fasebj.org

Novel therapeutic approaches to post-infarction remodelling

Adverse cardiac remodelling is a major cause of morbidity and mortality following acute myocardial infarction (MI). Mechanical and neurohumoral factors involved in structural and molecular post-infarction remodelling were important targets in research and treatment for years. More recently, therapeutic strategies that address myocardial regeneration and pathophysiological mechanisms of infarct wound healing appear to be useful novel tools to prevent progressive ventricular dilation, functional deterioration, life-threatening arrhythmia, and heart failure. This review provides an overview of future and emerging therapies for cardiac wound healing and remodelling after MI.

Mineralocorticoid Receptor Blockade Improves Vasomotor Dysfunction and Vascular Oxidative Stress Early After Myocardial Infarction

Mineralocorticoid receptor blockade improves mortality early after myocardial infarction (MI). This study investigated the vascular effects of mineralocorticoid receptor blockade in the early phase postinfarction in rats. Starting immediately after coronary ligation, male Wistar rats were treated with placebo or eplerenone (100 mg/kg/d). After 7 days, hemodynamic assessment was performed and endothelial function was determined. Maximum acetylcholine-induced relaxation was significantly attenuated in aortic rings from rats with heart failure after MI, and ameliorated by eplerenone treatment. Endothelium-independent relaxation by DEA-NONOate was similar among the groups. Endothelial NO synthase phosphorylation was reduced in the aorta of MI rats and restored by eplerenone therapy. Angiotensin I–induced vasoconstriction as well as angiotensin-converting enzyme protein levels were enhanced in aortas from MI placebo rats, and reduced by mineralocorticoid receptor inhibition. Aortic reactive oxygen species formation as well as the expression of the NAD(P)H oxidase subunit p22phox were increased after MI and normalized in eplerenone treated rats. In conclusion, mineralocorticoid receptor antagonism improved endothelial dysfunction in the early phase post-MI. Underlying mechanisms involve inhibition of vascular angiotensin-converting enzyme upregulation and improvement of endothelial NO synthase–derived NO bioavailability.