The adenylate cyclase activator forskolin potentiates the positive inotropic effect of the phosphodiesterase inhibitor milrinone but not of the calcium sensitizer levosimendan nor of its hemodynamically active metabolites: an apparent conundrum.

OR-1855 and OR-1896 are two hemodynamically active metabolites of the inodilator levosimendan, with calcium sensitizing activity, but their mechanism of action is still not fully understood.It has been previously reported that the positive inotropic effect of levosimendan is not potentiated by the adenylate cyclase activator forskolin whereas forskolin does potentiate the effects of the phosphodiesterase (PDE) inhibitor milrinone.To ascertain whether the active metabolites follow the same pattern of levosimendan, the positive inotropic effects of OR- 1855 and OR-1896, were studied in guinea-pig isolated papillary muscle in the presence and absence of forskolin. OR-1855 and OR-1896 were also tested as inhibitors of PDE-III and PDE-IV.Our result show that 0.1 µM forskolin did not potentiate the positive inotropic effect of either OR-1855 or OR-1896, as in the case of the parent compound levosimendan. As in previous studies, the positive inotropic effect of milrinone was markedly potentiated in the presence of forskolin.From these data we propose an explanation for the divergent behaviour of the calcium sensitizing drugs and PDE inhibitors

The inodilator levosimendan: 20 years of experience in various settings of cardiac care

Levosimendan emerged in the 1990s as a first-in-class inotrope and vasodilator that enhances cardiac contractility by sensitizing the contractile response to cardiac troponin C and causes vasodilatation by opening potassium-dependent ATP channels on vascular smooth muscle cells. Since its clinical debut in 2000, it has established itself as a valuable resource in the management of acute decompensated heart failure and is one of very few successful medical innovations of its kind in that field in recent decades. Its pharmacology is notable for delivering inotropy without an increase in myocardial oxygen consumption and for an array of secondary (‘pleiotropic’) effects that include an anti-ischemic effect, pre-conditioning and post-conditioning and cardioprotective effects and anti-oxidative effects.Proceeding from those properties it has been proposed that in addition to its use in various scenarios of low cardiac output levosimendan may be beneficial in other conditions associated with acutely decompensated heart failure, including right ventricular failure, subarachnoid hemorrhage, and cardiogenic shock with multi-organ dysfunction. The potential of levosimendan for kidney protection in situations of the cardio-renal syndrome has been identified. Additional lines of investigation include the use of levosimendan for perioperative hemodynamic support, its administration as repeated intermittent infusions to sustain patients with advanced heart failure and its application in a range of critical care settings.Levosimendan has also provided a template and a starting point for the development of a new generation of cardio-active drugs and is currently being evaluated in advanced clinical trials for the management of pulmonary hypertension in patients with heart failure with preserved ejection fraction

The inodilator levosimendan: 20 years of experience in various settings of cardiac care

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Oxidative and Nitrosative Stress in Age-Related Macular Degeneration: A Review of Their Role in Different Stages of Disease

Although the exact pathogenetic mechanisms leading to age-related macular degeneration (AMD) have not been clearly identified, oxidative damage in the retina and choroid due to an imbalance between local oxidants/anti-oxidant systems leading to chronic inflammation could represent the trigger event. Different in vitro and in vivo models have demonstrated the involvement of reactive oxygen species generated in a highly oxidative environment in the development of drusen and retinal pigment epithelium (RPE) changes in the initial pathologic processes of AMD; moreover, recent evidence has highlighted the possible association of oxidative stress and neovascular AMD. Nitric oxide (NO), which is known to play a key role in retinal physiological processes and in the regulation of choroidal blood flow, under pathologic conditions could lead to RPE/photoreceptor degeneration due to the generation of peroxynitrite, a potentially cytotoxic tyrosine-nitrating molecule. Furthermore, the altered expression of the different isoforms of NO synthases could be involved in choroidal microvascular changes leading to neovascularization. The purpose of this review was to investigate the different pathways activated by oxidative/nitrosative stress in the pathogenesis of AMD, focusing on the mechanisms leading to neovascularization and on the possible protective role of anti-vascular endothelial growth factor agents in this context

Levosimendan improves oxidative balance in cardiogenic shock/low cardiac output patients

The beneficial effects exerted by levosimendan against cardiac failure could be related to the modulation of oxidative balance. We aimed to examine the effects of levosimendan in patients with cardiogenic shock or low cardiac output on cardiac systo-diastolic function and plasma oxidants/antioxidants (glutathione, GSH; thiobarbituric acid reactive substances, TBARS). In four patients undergoing coronary artery bypass grafting or angioplasty, cardiovascular parameters and plasma GSH and TBARS were measured at T0 (before levosimendan infusion), T1 (1 h after the achievement of the therapeutic dosage of levosimendan), T2 (end of levosimendan infusion), T3 (72 h after the end of levosimendan infusion), and T4 (end of cardiogenic shock). We found an improvement in the indices of systolic (ejection fraction, cardiac output, cardiac index) and diastolic (E to early diastolic mitral annular tissue velocity, E/'; early to late diastolic transmitral flow velocity, EA) cardiac function at early T2. A reduction of central venous pressure and pulmonary wedge pressure was also observed. Plasma levels of GSH and TBARS were restored by levosimendan at T1, as well. The results obtained indicate that levosimendan administration can regulate oxidant/antioxidant balance as an early effect in cardiogenic shock/low cardiac output patients. Modulation of oxidative status on a mitochondrial level could thus play a role in exerting the cardio-protection exerted by levosimendan in these patients

Genistein improves viability, proliferation and mitochondrial function of cardiomyoblasts cultured in physiologic and peroxidative conditions

Phytoestrogens exert protective effects on the cardiovascular system through mechanisms that have yet to be clearly demonstrated. The aim of this study was to evaluate the protective effects exerted by genistein on cardiomyoblasts (H9C2) against oxidative stress, nitric oxide (NO) release, viability, proliferation/migration and mitochondrial function. H9C2 cultured in physiological or peroxidative conditions, were treated with genistein in the absence or presence of estrogen receptors (ERs), G protein\u2011\u200bcoupled\u2011estrogenic\u2011receptors (GPER), Akt, extracellular\u2011\u200bsignal\u2011regulated kinases 1/2 (ERK1/2) and p38 mitogen activated protein kinase (p38MAPK) blockers. Cell viability, proliferation, migration, mitochondrial membrane potential, mitochondrial oxygen consumption and oxidant/antioxidant system, were measured by specific assays. Western blot assay was used for the analysis of NO synthase (NOS) subtypes' and expression and activation of various kinases. In all experiments 17\u3b2\u2011estradiol was used for comparison. The results showed that phytoestrogens and estrogens can increase cell viability, proliferation/migration and improve mitochondrial membrane potential and oxygen consumption of H9C2. Furthermore, NO release was modulated by genistein and 17\u3b2\u2011estradiol. These effects were reduced or abolished by the pre\u2011treatment with ERs, GPER, Akt, ERK1/2 and p38MAPK blockers. Finally, a reduction of reactive oxygen species production and an increase of glutathione content was found in response to the two agents. In H9C2 cultured in physiological conditions, genistein induced endothelial NOS\u2011dependent NO production through the involvement of estrogenic receptors and by the modulation of intracellular signalling related to Akt, ERK1/2, and p38MAPK. Moreover, estrogens and phytoestrogens protected H9C2 against oxidative stress by reducing inducible NOS expression and through the modulation of the antioxidant system and mitochondrial functioning

The influence of mechanogastric distension via autonomic nerve on hemodynamic outputs

The meal presence of carbohydrate, particularly glucose performs a loading of intraduodenal glucose that elicits blood pressure fall; but the gastric distension cooperate counterbalancing this visceral reflex slightly increasing heart rate and blood pressure mainly via orthosympathetic nerves. However in the elderly and in patients with autonomic dysfunction postprandial hypothension, became an important clinical problem [1]. We have previously observed that several hindbrain nuclei are involved in cardiovascular reflexes elicited by mechanogastric dilatation in which vagal parasympathetic and splanchnic sympathetic component participate to define their responses [2,3]. Therefore in the present work monolateral and bilateral vagotomy and splanchnotomy have been performed to assert the involvement of visceral nervous system hindbrain nuclei in hemodynamic control following gastric mechanical dilatation at high and low pressure. The mapping of c-Fos positive neurons have shown the occurrence of cooperative pathways between vagal and splanchnic component in several nuclei under analysis, similarly an integrated response between controlateral areas of single nuclei has been observed. In particular following mechanogastric experimental protocols adopted, the physiological response on blood pressure and heart rate, shows a preponderance effect of orthosympathetic component, but its plain functional expression need of parasympathetic component involvement

17,β-estradiol inhibits hepatitis C virus mainly by interference with the release phase of its life cycle

Rationale & Aim: Estrogen and estrogen-mediated signalling protect from hepatitis C virus through incompletely understood mechanisms. We aimed to ascertain which phase(s) of HCV life cycle is/are affected by estrogens. Methods: Huh7 cells infected with the JFH1 virus (genotype 2a) were exposed to dehydroepiandrosterone, testosterone, progesterone and 17β-estradiol (tested with/without its receptor antagonist fulvestrant). Dose-response curves were established to calculate IC50 values. To dissect how 17β-estradiol interferes with phases of HCV life cycle, its effects were measured on the HCV pseudo-particle system (viral entry), the sub-genomic replicon N17/JFH1 and the replicon cell line Huh7-J17 (viral replication). Finally, in a dual-step infection model, infectious supernatants, collected from infected cells exposed to hormones, were used to infect naïve cells. Results: Progesterone and testosterone showed no inhibitory effect on HCV; dehydroepiandrosterone was only mildly inhibitory. In contrast, 17β-estradiol inhibited infection by 64-67% (IC50 values 140 to 160 nM). Fulvestrant reverted the inhibition by 17β-estradiol in a dose-dependent manner. 17β-estradiol exerted only a slight inhibition (<20%) on HCV pseudo-particles, and had no effect on cells either transiently or stably (Huh7-J17 cells) expressing the N17/JFH1 replicon. In the dual-step infection model, a significant IC50 decline occurred between primary (134 nM) and secondary (100 nM) infections (p=0.02), with extracellular HCV RNA and infectivity being reduced to a higher degree in comparison to its intracellular counterpart. Conclusions: 17β-estradiol inhibits HCV acting through its intracellular receptors, mainly interfering with late phases (assembly/release) of the HCV life cycle

Human Chorionic Gonadotropin Protects Vascular Endothelial Cells from Oxidative Stress by Apoptosis Inhibition, Cell Survival Signalling Activation and Mitochondrial Function Protection.

Background/Aim: Previous reports have made it hypothetically possible that human chorionic gonadotropin (hCG) could protect against the onset of pregnancy-related pathological conditions by acting as an antioxidant. In the present study we planned to examine the effects of hCG against oxidative stress in human umbilical vein endothelial cells (HUVEC). Methods: HUVEC were subjected to peroxidation by hydrogen peroxide. The modulation of nitric oxide (NO) release by hCG and its effects on cell viability, glutathione (GSH) levels, mitochondrial membrane potential and mitochondrial transition pore opening (MPTP) were examined by specific dyes. Endothelial and inducible NO synthase (eNOS and iNOS), Akt and extracellular -signal-regulated kinases 1/2 (ERK1/2) activation and markers of apoptosis were analyzed by Western Blot. Results: In HUVEC, hCG reduced NO release by modulating eNOS and iNOS. Moreover, hCG protected HUVEC against oxidative stress by preventing GSH reduction and apoptosis, by maintaining Akt and ERK1/2 activation and by keeping mitochondrial function. Conclusion: The present results have for the first time shown protective effects exerted by hCG on vascular endothelial function, which would be achieved by modulation of NO release, antioxidant and antiapoptotic actions and activation of cell survival signalling. These findings could have clinical implications in the management of pregnancy-related disorders

Aflibercept and Ranibizumab modulate retinal pigment epithelial cells function by acting on their cross talk with vascular endothelial cells

Background/Aims: We performed co-culture experiments between human RPE cells (ARPE-19) and human umbilical vascular endothelial cells (HUVEC) in order to evaluate how anti-VEGF drugs could affect NO release, mitochondrial function, the oxidative status, proliferation and migration of RPE cells through modulation of their cross talk with vascular endothelial cells. Materials: The co-culture HUVEC/RPE, was exposed to Ranibizumab/Aflibercept in the absence/presence of the NO synthase (NOS) inhibitor, the phosphatidylinositol 3\u2032-kinase (PI3K), the extracellular-signal-regulated kinases 1/2 (ERK1/2) and the p38 mitogen-activated protein kinase (p38 MAPK) blockers. Specific kits were used for cell viability, mitochondrial membrane potential, NO, ROS and GSH production. Western blot was performed for apoptosis markers, NOS isoforms, and others kinases detection. Cell migration was analyzed by scratch assay, whereas cell proliferation and cell cycle through xCELLigence and flow cytometry. Results: In RPE cells co-cultured with HUVEC in physiological conditions, Aflibercept/Ranibizumab increased NO release in a dose and time-dependent way. Opposite results were obtained in peroxidative conditions. Both anti-VEGF agents were able to prevent the fall of cell viability and mitochondrial membrane potential, an effect which was reduced by various inhibitors, and increased cell migration. Aflibercept/Ranibizumab counteracted the changes of apoptosis markers, NOS expression/activation, PI3K and ERK1/2 activation caused by peroxidation. These results were confirmed by cell cycle analysis. Conclusion: This study has shown new mechanisms at the basis of protective effects elicited by Aflibercept/Ranibizumab in RPE cells. HUVEC stimulated with Aflibercept/Ranibizumab, could release some paracrine factors that can modulate the RPE cells response in both physiologic and peroxidative conditions