Direct/indirect protective effects elicited by anti-vascular endothelial growth factor drugs on retinal pigment epithelium cells (ARPE-19 cell line)

The Age-Related Macular Degeneration (AMD) is the leading cause of irreversible loss of vision. In the mechanisms of action of the anti-VEGF agents the involvement of nitric oxide (NO), the mitochondria function and apoptosis have not been examined. In the present study, in order to analyze the cross-talk between retinal pigment epithelium (RPE; ARPE-19) cells and endothelial cells, we conducted co-culture experiments between RPE and human umbilical vascular endothelial cells (HUVEC). RPE and HUVEC/RPE co-culture, were exposed to Ranibizumab/Aflibercept in the absence/presence of NO synthase (NOS) inhibitor, phosphatidylinositol 3′-kinase (PI3K), extracellular signal regulated kinases 1/2 (ERK1/2) and p38 mitogen-activated protein kinase (p38 MAPK) blockers. Specific kits were used for cell viability, NO, ROS detection, apoptosis and mitochondrial membrane potential measurement. Western blot was performed for apoptosis markers, NOS isoforms, and other kinases detection. Cell migration was measured by wound healing assay and cell proliferation was analyzed by using xCELLigence. In RPE alone or in co-culture with HUVEC, Aflibercept/Ranibizumab increased NO release in a dose and time-dependent way, in physiologic conditions. Opposite results were obtained in RPE pretreated with hydrogen peroxide. Both anti-VEGF drugs were able to prevent the fall of cell viability and of mitochondrial membrane potential. Those effects were reduced by inhibitors of NOS, PI3K, ERK1/2 and p38MAPK. Both drugs were able to increase cell viability and migration. Finally, Aflibercept/Ranibizumab counteracted the changes of apoptosis markers, NOS expression/activation, protein kinase B (PKB or Akt) and ERK1/2 activation caused by peroxidation. This study has shown for the first time new mechanisms involving NO and mitochondria, in the actions of Aflibercept/Ranibizumab in RPE

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

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

Genistein and 17β-Estradiol Protect Hepatocytes from Fatty Degeneration by Mechanisms Involving Mitochondria, Inflammasome and Kinases Activation

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Monomeric adiponectin modulates nitric oxide release and calcium movements in porcine aortic endothelial cells in normal/high glucose conditions.

AIMS: Perivascular adipose tissue can be involved in the process of cardiovascular pathology through the release of adipokines, namely adiponectins. Monomeric adiponectin has been shown to increase coronary blood flow in anesthetized pigs through increased nitric oxide (NO) release and the involvement of adiponectin receptor 1 (AdipoR1). The present study was therefore planned to examine the effects of monomeric adiponectin on NO release and Ca2+ transients in porcine aortic endothelial cells (PAEs) in normal/high glucose conditions and the related mechanisms. MAIN METHODS: PAEs were treated with monomeric adiponectin alone or in the presence of intracellular kinases blocker, AdipoR1 and Ca2+-ATPase pump inhibitors. The role of Na+/Ca2+ exchanger was examined in experiments performed in zero Na+ medium. NO release and intracellular Ca2+ were measured through specific probes. KEY FINDINGS: In PAE cultured in normal glucose conditions, monomeric adiponectin elevated NO production and [Ca2+]c. Similar effects were observed in high glucose conditions, although the response was lower and not transient. The Ca2+ mobilized by monomeric adiponectin originated from an intracellular pool thapsigargin- and ATP-sensitive and from the extracellular space. Moreover, the effects of monomeric adiponectin were prevented by kinase blockers and AdipoR1 inhibitor. Finally, in normal glucose condition, a role for Na+/Ca2+ exchanger and Ca2+-ATPase pump in restoring Ca2+ was found. SIGNIFICANCE: Our results add new information about the control of endothelial function elicited by monomeric adiponectin, which would be achieved by modulation of NO release and Ca2+ transients. A signalling related to Akt, ERK1/2 and p38MAPK downstream AdipoR1 would be involved

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

Modulation of Oxidative Stress by 17 β-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 17\u3b2-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 17\u3b2-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 \u3b1-smooth-muscle actin expression. RESULTS: In Huh7.5, 17\u3b2-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 17\u3b2-estradiol and genistein. CONCLUSION: In Huh7.5 and LX-2, 17\u3b2-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