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. METHODS: The co-culture HUVEC/RPE, was exposed to Ranibizumab/Aflibercept in the absence/presence of the NO synthase (NOS) inhibitor, the phosphatidylinositol 3'-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.

Anti-Vascular Endothelial Growth Factors Protect Retinal Pigment Epithelium Cells Against Oxidation by Modulating Nitric Oxide Release and Autophagy.

BACKGROUND/AIMS: the anti-vascular endothelial growth factors (VEGF), Aflibercept and Ranibizumab, are used for the treatment of macular degeneration. Here we examined the involvement of nitric oxide (NO), mitochondria function and of apoptosis/autophagy in their antioxidant effects in human retinal pigment epithelium cells (RPE). METHODS: RPE were exposed to Ranibizumab/Aflibercept in the absence or presence of NO synthase (NOS) inhibitor and of autophagy activator/blocker, rapamicyn/3-methyladenine. Specific kits were used for cell viability, NO and reactive oxygen species detection and mitochondrial membrane potential measurement, whereas Western Blot was performed for apoptosis/ autophagy markers and other kinases detection. RESULTS: In RPE cultured in physiological conditions, Aflibercept/Ranibizumab increased NO release in a dose and time-dependent way. Opposite results were obtained in RPE pretreated with hydrogen peroxide. Moreover, both the anti-VEGF agents were able to prevent the fall of cell viability and of mitochondrial membrane potential. Those effects were reduced by the NOS inhibitor and 3-methyladenine and were potentiated by rapamycin. Finally, Aflibercept and Ranibizumab counteracted the changes of apoptosis/autophagy markers, NOS, Phosphatidylinositol-3-Kinase/Protein Kinase B and Extracellular signal-regulated kinases 1/2 caused by peroxidation. CONCLUSION: Aflibercept and Ranibizumab protect RPE against peroxidation through the modulation of NO release, apoptosis and autophagy.

Modulation of Oxidative Stress by 17 ß-Estradiol and Genistein in Human Hepatic Cell Lines In Vitro.

BACKGROUND/AIMS: estrogens and phytoestrogens exert hepatoprotection through mechanisms not clearly examined yet. Here, we investigated the protective effects exerted by 17ß-estradiol and genistein against oxidative stress in hepatocytes and hepatic stellate cells (HSCs) and the involvement of specific receptors and the intracellular signalling. METHODS: Huh7.5 and LX-2, alone or in co-culture with Huh7.5, were treated with 17ß-estradiol and genistein alone or in the presence of menadione and of estrogen receptors (ERs) and G protein-coupled-estrogenic-receptors (GPER) blockers. Cell viability, mitochondrial membrane potential and oxidant/antioxidant system were measured by specific kits. Western Blot was used for the analysis of Akt and p38-mitogen-activated-protein kinases (MAPK) activation and α-smooth-muscle actin expression. RESULTS: In Huh7.5, 17ß-estradiol and genistein prevented the effects of peroxidation by modulating Akt and p38MAPK activation. Similar antioxidant and protective findings were obtained in LX-2 of co-culture experiments, only. ERs and GPER blockers were able to prevent the effects of 17ß-estradiol and genistein. CONCLUSION: In Huh7.5 and LX-2, 17ß-estradiol and genistein counteract the effects of peroxidation through the involvement of ERs and GPER and by an intracellular signalling related to Akt and p38MAPK. As concerning LX-2, paracrine factors released by Huh7.5 play a key role in protection against oxidative stress.

Gastric distension causes changes in heart rate and arterial blood pressure by affecting the crosstalk between vagal and splanchnic systems in anesthetised rats.

Various hindbrain nuclei have been demonstrated to be involved in the control of the cardiovascular reflexes elicited by both non-noxious and noxious gastric distension, through parasympathetic and sympathetic activation. The different role played by the branches of autonomic nervous system in exerting these effects and their crosstalk in relation to low-/high-pressure distension rate has not been examined yet. Therefore, in the present work, monolateral and bilateral vagotomy and splanchnicotomy were performed in anesthetised rats to analyse the involvement of hindbrain nuclei in haemodynamic changes caused by gastric distension at high (80 mmHg) and low (15 mmHg) pressure. The analysis of c-Fos expression in neuronal areas involved in cardiovascular control allowed us to examine their recruitment in response to various patterns of gastric distension and the crosstalk between vagal and splanchnic systems. The results obtained show that the low-pressure (non-noxious) gastric distension increases both heart rate and arterial blood pressure. In addition, the vagus nerve and hindbrain nuclei, such as nucleus ambiguous, ventrolateral medulla and lateral reticular nucleus, appear to be primarily involved in observed responses. In particular, we have found that although vagus nerve plays a central role in exerting those cardiovascular reflex changes at low gastric distension, for its functional expression an intact splanchnic system is mandatory. Hence, the absence of splanchnic input attenuates pressor responses or turns them into depressor responses. Instead at high-pressure (noxious) gastric distension, the splanchnic nerve represents the primary component in regulating the reflex cardiovascular effects.

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.

Effects of Artemetin on Nitric Oxide Release and Protection against Peroxidative Injuries in Porcine Coronary Artery Endothelial Cells.

Artemetin is one of the main components of Achillea millefolium L. and Artemisia absinthium, which have long been used for the treatment of various diseases. To date, however, available information about protective effects of their extracts on the cardiovascular system is scarce. Therefore, we planned to analyze the effects of artemetin on nitric oxide (NO) release and the protection exerted against oxidation in porcine aortic endothelial (PAE) cells. In PAE, we examined the modulation of NO release caused by artemetin and the involvement of muscarinic receptors, ß2-adrenoreceptors, estrogenic receptors (ER), protein-kinase A, phospholipase-C, endothelial-NO-synthase (eNOS), Akt, extracellular-signal-regulated kinases 1/2 (ERK1/2) and p38 mitogen activated protein kinase (p38 MAPK). Moreover, in cells treated with hydrogen peroxide, the effects of artemetin were examined on cell survival, glutathione (GSH) levels, apoptosis, mitochondrial membrane potential and transition pore opening. Artemetin increased eNOS-dependent NO production by the involvement of muscarinic receptors, ß2-adrenoreceptors, ER and all the aforementioned kinases. Furthermore, artemetin improved cell viability in PAE that were subjected to peroxidation by counteracting GSH depletion and apoptosis and through the modulation of mitochondrial function. In conclusion, artemetin protected endothelial function by acting as antioxidant and antiapoptotic agent and through the activation of ERK1/2 and Akt. Copyright © 2015 John Wiley & Sons, Ltd.

Protective effects elicited by levosimendan against liver ischemia/reperfusion injury in anesthetized rats.

As in other organs, oxidative stress-induced injury and cell death may result from free oxygen radical-dependent mechanisms and alterations in signal transduction pathways leading to apoptosis. Among the new suggested therapies for injuries caused by oxidative stress, the use of levosimendan has been reported to be quite promising. In the present study, we aimed to examine the protective effects of levosimendan against liver oxidative stress in anesthetized rats and to analyze the involvement of mitochondrial adenosine triphosphate-dependent potassium (mitoK(ATP)) channels and nitric oxide (NO). In 50 anesthetized rats, liver ischemia/reperfusion (I/R) was performed via nontraumatic portal occlusion. In some animals, levosimendan was infused into the portal vein at the onset of reperfusion, whereas other rats received the vehicle only. Moreover, in some rats, levosimendan was given after the intraportal administration of L-Nω-nitro-arginine methyl ester (L-NAME) or 5-hydroxydecanoate (5HD). The portal vein blood flow was measured, and blood samples were taken for the determination of transaminases, thiobarbituric acid reactive substances (TBARS), and reduced glutathione (GSH); liver biopsy samples were used for B cell lymphoma 2-associated X protein, caspase-9, Akt, and endothelial nitric oxide synthase (eNOS) activation through western blotting. Also, caspase-3 activity was measured. In rats, I/R caused an increase in apoptotic markers, transaminases, and TBARS and a decrease in GSH and Akt activation. Levosimendan administration was able to counteract oxidative damage and apoptosis in a dose-dependent way and to increase GSH, Akt, and eNOS activation. All effects of levosimendan were abolished by pretreatment with L-NAME and 5HD. In conclusion, the results of the present study show that levosimendan can exert protection against ischemic liver damage through mechanisms related to NO production and mitoKATP channel function. These data provide interesting perspectives into the use of levosimendan in hepatic surgery and transplantation.

PAINTER: a spatiospectral image reconstruction algorithm for optical interferometry.

Astronomical optical interferometers sample the Fourier transform of the intensity distribution of a source at the observation wavelength. Because of rapid perturbations caused by atmospheric turbulence, the phases of the complex Fourier samples (visibilities) cannot be directly exploited. Consequently, specific image reconstruction methods have been devised in the last few decades. Modern polychromatic optical interferometric instruments are now paving the way to multiwavelength imaging. This paper is devoted to the derivation of a spatiospectral (3D) image reconstruction algorithm, coined Polychromatic opticAl INTErferometric Reconstruction software (PAINTER). The algorithm relies on an iterative process, which alternates estimation of polychromatic images and complex visibilities. The complex visibilities are not only estimated from squared moduli and closure phases, but also differential phases, which helps to better constrain the polychromatic reconstruction. Simulations on synthetic data illustrate the efficiency of the algorithm and, in particular, the relevance of injecting a differential phases model in the reconstruction.

Intracoronary secretin increases cardiac perfusion and function in anaesthetized pigs through pathways involving ß-adrenoceptors and nitric oxide.

Secretin has been implicated in cardiovascular regulation through its specific receptors, as well as through ß-adrenoceptors and nitric oxide, although data on its direct effect on coronary blood flow and cardiac function have remained scarce. The present study aimed to determine the primary in vivo effect of secretin on cardiac function and perfusion and the mechanisms related to the autonomic nervous system, secretin receptors and NO. In addition, in coronary endothelial cells the intracellular pathways involved in the effects of secretin on NO release were also examined. In 30 pigs, intracoronary secretin infusion at 2.97 pg for each millilitre per minute of coronary blood flow at constant heart rate and aortic blood pressure increased coronary blood flow, maximal rate of change of left ventricular pressure, segmental shortening, cardiac output and coronary NO release (P<0.05). These responses were graded in a further five pigs. Moreover, while blockade of muscarinic cholinoreceptors (n=5) and of α-adrenoceptors (n=5) did not abolish the observed responses to secretin, blockade of ß1-adrenoceptors (n=5) prevented the effects of secretin on cardiac function. In addition, blockade of ß2-adrenoceptors (n=5) and NO synthase inhibition (n=5) prevented the coronary response and the effect of secretin on NO release. All these effects were abolished by a secretin receptor inhibitor (n=5). In coronary endothelial cells, the increased NO production caused by secretin was found to be related to cAMP/protein kinase A signalling activated as downstream effectors of stimulation of secretin receptors and ß2-adrenoceptors. In conclusion, in anaesthetized pigs secretin primarily increased cardiac function and perfusion through the involvement of specific receptors, ß-adrenoceptors and NO release.

Levosimendan protection against kidney ischemia/reperfusion injuries in anesthetized pigs.

Ischemia/reperfusion (I/R) injury is an important cause of acute renal failure because of oxidative, inflammatory, and apoptotic mechanisms. The aim of the present study was to examine any possible protective effects of levosimendan in an in vivo pig model of renal I/R injury. In 40 anesthetized pigs (eight groups of five pigs each), I/R was induced by clamping-reopening the left renal artery. During ischemia, in three groups of pigs, levosimendan and the multiorgan preservation solution Custodiol, alone or in combination with levosimendan, were infused in the renal artery. In two other groups of animals, levosimendan in combination with Custodiol was administered after the intrarenal nitric-oxide (NO) synthase blocker N(ω)-nitro-L-arginine methyl ester (L-NAME) or the mitochondrial ATP-sensitive K(+) channel (K(ATP) channel) inhibitor 5-hydroxydecanoate (5-HD). In the other animals, saline, L-NAME, or 5-HD were administered alone. Throughout the experiments, urinary N-acetyl-ß-glucosaminidase (NAG) release was measured, and renal function was assessed. Moreover, renal biopsy samples were taken for the detection of apoptosis and tissue peroxidation. In pigs treated with levosimendan or the combination of levosimendan and Custodiol, NAG, peroxidation, and apoptotic markers were lower than in animals treated with Custodiol alone. In addition, renal function was better preserved, and cell survival and antioxidant systems were more activated. All beneficial effects were prevented by L-NAME and 5-HD. In conclusion, levosimendan alone or in combination with Custodiol exerted better protection against renal I/R injuries than Custodiol alone through antioxidant, antiapoptotic, and prosurvival actions depending on mitochondrial K(ATP) channels and NO-related mechanisms.

Levosimendan modulates programmed forms of cell death through K(ATP) channels and nitric oxide.

Levosimendan exerts cardioprotection through mitochondrial K(ATP) (mitoK(ATP)) channels opening. In addition, intracoronary levosimendan was found to modulate programmed forms of cell death by nitric oxide (NO) involvement. The aim of this study was to examine the role of mitoK(ATP) channels and NO in the effects of levosimendan on apoptosis/autophagy. In H9c2 cells treated with hydrogen peroxide apoptosis/autophagy, survival signaling, cell viability, mitochondrial membrane potential, and permeability transition pore opening were analyzed through Western blot and colorimetric and fluorescence assays. Pretreatment of H9c2 cells with levosimendan was able to counteract the oxidative injuries caused by hydrogen peroxide. The effects of levosimendan were potentiated by diazoxide and were similar to those elicited by the autophagic activator rapamycin. The autophagic inhibitor 3-methyladenine reduced the effects of levosimendan, whereas after the pan-caspases inhibitor N-Acetyl-Asp-Glu-Val-Asp-al (Z-VAD.FMK), cell survival and autophagy in response to levosimendan increased. Both the mitoK(ATP) channels inhibition and the NO synthase blocking attenuated the cardioprotection elicited by levosimendan. The results have shown that levosimendan protects H9c2 cells against oxidative injuries through the modulation of the interplay between autophagy and apoptosis and the activation of survival signaling. The mitoK(ATP) channels and NO may be involved in such cardioprotection through interference with mitochondrial functioning.

Intracoronary melatonin increases coronary blood flow and cardiac function through ß-adrenoreceptors, MT1/MT2 receptors, and nitric oxide in anesthetized pigs.

Melatonin is involved in the regulation of the cardiovascular system through the modulation of sympathetic function and the nitric oxide (NO)-related pathway and interaction with MT1/MT2 receptors. However, information regarding its direct actions on coronary blood flow and cardiac function is scarce. This study therefore determined the primary in vivo effect of melatonin on cardiac function and perfusion and the involvement of the autonomic nervous system, MT1/MT2 receptors, and NO. In 35 pigs, melatonin infused into the coronary artery at 70 pg for each mL/min of coronary blood flow while preventing changes in heart rate and arterial pressure increased coronary blood flow, dP/dt(max), segmental shortening, and cardiac output by about 12%, 14%, 8%, and 23% of control values (P < 0.05), respectively. These effects were accompanied by an increase in coronary NO release of about 46% (P < 0.05) of control values. The aforementioned responses were graded in a further five pigs. Moreover, the blockade of muscarinic cholinoreceptors (n = 5) and α-adrenoreceptors (n = 5) did not abolish the observed responses to melatonin. After ß(1)-adrenoreceptors blocking (n = 5), melatonin failed to affect cardiac function, whereas ß(2)-adrenoreceptors (n = 5) and NO synthase inhibition (n = 5) prevented the coronary response and the effect of melatonin on NO release. Finally, all effects were prevented by MT1/MT2 receptor inhibitors (n = 10). In conclusion, melatonin primarily increased coronary blood flow and cardiac function through the involvement of MT1/MT2 receptors, ß-adrenoreceptors, and NO release. These findings add new information about the mechanisms through which melatonin physiologically modulates cardiovascular function and exerts cardioprotective effects.

Modulation of calcium movements by urocortin II in endothelial cells.

BACKGROUND: In endothelial cells urocortin II has recently been found to activate nitric oxide synthase through cAMP-dependent and Ca(2+)-related pathway. AIM: The present study was therefore planned to determine the mechanisms of urocortin II effect on Ca(2+) movements. METHODS: In Fura-2 loaded porcine aortic endothelial cells (PAE), the effects of urocortin II on [Ca(2+)]c were analyzed and compared with those of various K(+) channels agonists/antagonists. RESULTS: In Fura-2 loaded PAE, urocortin II promoted a transient increase of [Ca(2+)]c mainly originating from an intracellular pool sensitive to thapsigargin and slightly from the extracellular space. In addition, urocortin II caused the hyperpolarization of plasma membrane through the opening of K(+) channels, which contributed to the increased [Ca(2+)]c. These effects were abolished by the corticotropin releasing factor receptors (CRFR2) blocker, the adenylyl cyclase and Ca(2+)-calmodulin-kinase (CaMKII) inhibitors and by blockers of K(+) channels. In addition, in PAE cultured in Na(+)-free medium or loaded with the plasma-membrane Ca(2+) pump inhibitor the urocortin II-evoked Ca(2+) transient was slower. CONCLUSION: The results obtained show that urocortin II affects intracellular Ca(2+) homeostasis in PAE by both promoting a discharge of intracellular pool and by interfering with the operation of store-dependent channels through CRFR2-cAMP-CaMKII related signalling and K(+) channels opening.

Modulation of programmed forms of cell death by intracoronary levosimendan during regional myocardial ischemia in anesthetized pigs.

PURPOSE: Powerful mediators of programmed cell death, such as apoptosis and autophagy, can contribute to myocyte cell loss during pathological cardiac conditions. Levosimendan has been shown to exert beneficial hemodynamic effects in presence of global myocardial ischemia and heart failure through vasodilatation and increase of cardiac contractility. Recently, the intracoronary administration of a bolus levosimendan was found to exert favourable cardiac anti-stunning effects without lowering arterial pressure, which limits the use of levosimendan mainly in coronary artery disease. Here we tested whether the intracoronary administration of levosimendan can beneficially modulate programmed cell death in acute regional myocardial ischemia. METHODS: Acute regional myocardial ischemia was induced in 20 anaesthetized pigs and intracoronary levosimendan 15 min bolus administration was started 4 h afterwards. The effects of levosimendan on coronary blood flow and cardiac function were evaluated and myocardial biopsies were examined for criteria of autophagy and apoptosis. RESULTS: The administration of levosimendan caused a significant increase of coronary blood flow (p < 0.05) in absence of changes in cardiac function. Moreover, levosimendan prevented the down-regulation of the anti-apoptotic gene, Bcl-2, and the up-regulation of the apoptotic markers Bax and cytochrome c, which resulted in a reduced expression of TUNEL fragmented nuclei (p < 0.05). Furthermore, levosimendan maintained Beclin 1 at 4 h and potentiated LC3 II expression, these results being consistent with autophagy activation. CONCLUSIONS: Such effects of intracoronary levosimendan bolus administration during regional myocardial ischemia indicate the occurrence of cardio-protection by modulation of programmed form of cell death.

Gender differences in sympathetic neural activation following uncomplicated acute myocardial infarction.

AIMS: To determine whether the magnitude of post-acute myocardial infarction (AMI) sympathetic activation is greater in women (F-AMI) than men (M-AMI). METHODS AND RESULTS: Both sympatho-humoral activation and female gender are associated with worse outcome in the early phase following AMI. However, women have lower sympathetic output than men. We therefore examined matched groups of F-AMI (18) and M-AMI (18) patients 2-4 days following uncomplicated AMI, then 3 monthly to 9 months; matched normal control (NC) groups comprised M-NC (18) and F-NC (18). Muscle sympathetic nerve activity (MSNA) was measured by microneurography. Muscle sympathetic nerve activity was lower in the F-NC than M-NC (at least P < 0.05) and greater in the two AMI groups than their corresponding NC groups (at least P < 0.001). Muscle sympathetic nerve activity was similar in the F-AMI and M-AMI groups indicating a post-AMI increase in women of about twice that in men (P < 0.0001). Both AMI groups returned to corresponding NC (lower in women) levels by 9 months. CONCLUSION: Following uncomplicated AMI, women developed a relatively greater magnitude of sympathetic activation lasting until its resolution at 9 months. This is consistent with reports of their worse prognosis observed during this time period, with important potential clinical implications.

Urocortin II induces nitric oxide production through cAMP and Ca2+ related pathways in endothelial cells.

BACKGROUND: Urocortin II has previously been shown in anesthetized pigs to increase coronary blood flow through activation of the endothelial nitric oxide synthase (eNOS) pathway and involvement of the subtype 2 of corticotropin releasing factor receptors (CRFR2). However, little information has been available regarding the intracellular signalling through these receptors and leading to the release of nitric oxide (NO). AIM: The present study was therefore planned to determine the mechanism involved in such signalling. METHODS: In porcine aortic endothelial cells (PAE) the effects of urocortin II on NO production and ERK, Akt, p38 and eNOS phosphorylation were examined in absence or presence of the adenylyl cyclase agonist forskolin and antagonist 2'5' dideoxyadenosine, the Ca2+ ionophore A23187, the Ca2+-calmodulin-kinase inhibitor KN93, the CRFR2 blocker astressin 2B and of the protein kinases specific inhibitors UO126, wortmannin and SB203580. RESULTS: Urocortin II caused a significant increase of NO production, which was amplified by forskolin and A23187 (P <0.05). All effects of urocortin II were abolished by l-NAME, 2'5' dideoxyadenosine, KN93, astressin 2B and by pre-treatment of cells with UO126, wortmannin and SB203580. Western Blot analysis confirmed the involvement of ERK, Akt and p38 in the eNOS activation. CONCLUSION: In PAE urocortin II interaction with CRFR2 caused a cAMP-dependent and Ca2+-related phoshorylation of ERK, Akt and p38 leading to eNOS activation.

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 protein­â€‹coupled­estrogenic­receptors (GPER), Akt, extracellular­â€‹signal­regulated 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 17ß­estradiol 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 17ß­estradiol. These effects were reduced or abolished by the pre­treatment 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 NOS­dependent 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.

Preeclampsia and intrauterine growth restriction: Role of human umbilical cord mesenchymal stem cells-trophoblast cross-talk.

BACKGROUND: Oxidative stress is involved in the pathogenesis and maintenance of pregnancy-related disorders, such as intrauterine growth restriction (IUGR) and preeclampsia (PE). Human umbilical cord mesenchymal stem cells (hUMSCs) have been suggested as a possible therapeutic tool for the treatment of pregnancy-related disorders in view of their paracrine actions on trophoblast cells. OBJECTIVES: To quantify the plasma markers of peroxidation in patients affected by PE and IUGR and to examine the role of oxidative stress in the pathophysiology of PE and IUGR in vitro by using hUMSCs from physiological and pathological pregnancies and a trophoblast cell line (HTR-8/SVneo). STUDY DESIGN: In pathological and physiological pregnancies the plasma markers of oxidative stress, arterial blood pressure, serum uric acid, 24h proteinuria, weight gain and body mass index (BMI) were examined. Furthermore, the pulsatility index (PI) of uterine and umbilical arteries, and of fetal middle cerebral artery was measured. In vitro, the different responses of hUMSCs, taken from physiological and pathological pregnancies, and of HTR-8/SVneo to pregnancy-related hormones in terms of viability and nitric oxide (NO) release were investigated. In some experiments, the above measurements were performed on co-cultures between HTR-8/SVneo and hUMSCs. RESULTS: The results obtained have shown that in pathological pregnancies, body mass index, serum acid uric, pulsatility index in uterine and umbilical arteries and markers of oxidative stress were higher than those found in physiological ones. Moreover, in PE and IUGR, a relation was observed between laboratory and clinical findings and the increased levels of oxidative stress. HTR-8/SVneo and hUMSCs showed reduced viability and increased NO production when stressed with H2O2. Finally, HTR-8/SVneo cultured in cross-talk with hUMSCs from pathological pregnancies showed a deterioration of cell viability and NO release when treated with pregnancy-related hormones. CONCLUSION: Our findings support that hUMSCs taken from patients affected by PE and IUGR have significant features in comparison with those from physiologic pregnancies. Moreover, the cross-talk between hUMSCs and trophoblast cells might be involved in the etiopathology of IUGR and PE secondary to oxidative stress.

The subthreshold micropulse laser treatment of the retina restores the oxidant/antioxidant balance and counteracts programmed forms of cell death in the mice eyes.

PURPOSE: Subthreshold micropulse laser (SMPL) has been increasingly used for the treatment of different retinal and choroidal macular disorders. However, the exact mechanisms of action have not yet been clearly defined. Therefore, we aimed to examine the role of SMPL treatment in the modulation of oxidant/antioxidant systems, apoptosis and autophagy in the mice eyes. METHODS: A specific laser contact lens for retina was positioned on the cornea of 40 mice (20 young and 20 old) in order to focus the laser on the eye fundus for SMPL treatment. Within 6 months, 20 animals received one treatment only, whereas the others were treated three times. Eye specimens underwent histological analysis and were used for thiobarbituric acid reactive substances (TBARS) and glutathione (GSH) quantification, as well as for the superoxide dismutase 1 (SOD1) and the selenoprotein thioredoxin reductase 1 (TrxR1) expression evaluation. Western blot was performed for nitric oxide synthase (NOS) subtypes detection and to examine changes in apoptotic/autophagy proteins expression. RESULTS: SMPL treatment reduced TBARS and increased GSH and SOD1 in the mice eyes. It also reduced cytochrome c, caspase 3 expression and activity and cleaved caspase 9, and increased Beclin 1, p62 and LC3ß. The effects were more relevant in the elderly animals. CONCLUSION: Our results showed that SMPL therapy restored the oxidant/antioxidant balance within retinal layers and modulated programmed forms of cell death. Further studies may confirm these data and could evaluate their relevance in clinical practice.