The microRNA-7-mediated reduction in EPAC-1 contributes to vascular endothelial permeability and eNOS uncoupling in murine experimental retinopathy

To investigate the consequences of oxidative stress and hypoxia on EPAC-1 expression during retinopathy. Oxygen-induced retinopathy was induced in mice and EPAC-1 expression investigated by immunofluorescence. In silico analyses were used to identify a link between EPAC-1 expression and microRNA-7-5p in endothelial cells and confirmed by western blot analyses on cells expressing microRNA-7-5p. In vitro, endothelial cells were either incubated at 2% oxygen or transfected with microRNA-7-5p, and the effects of these treatments on EPAC-1 expression, endothelial hyperpermeability and NO production were assessed. In the Ins2Akita mouse model, levels of EPAC-1 expression as well as microRNA-7-5p were assessed by qPCR. Endothelial nitric oxide synthase was assessed by immunoblotting in the Ins2Akita model. Hypoxia induces the expression of microRNA-7-5p that translationally inhibits the expression of EPAC-1 in endothelial cells, resulting in hyperpermeability and the loss of eNOS activity. Activation of EPAC-1 by the cAMP analogue 8-pCPT-2'-O-Me-cAMP reduced the sensitivity of EPAC-1 to oxidative stress and restored the endothelial permeability to baseline levels. Additionally, 8-pCPT-2'-O-Me-cAMP rescued eNOS activity and NO production. In mouse models of retinopathy, i.e., oxygen-induced retinopathy and the spontaneous diabetic heterozygous Ins2(Akita) mice, EPAC-1 levels are decreased which is associated with an increase in microRNA-7-5p expression and reduced eNOS activity. In retinopathy, EPAC-1 expression is decreased in a microRNA-7-mediated manner, contributing to endothelial dysfunction. Pharmacological activation of remnant EPAC-1 rescues endothelial function. Collectively, these data indicate that EPAC-1 resembles an efficacious and druggable target molecule for the amelioration of (diabetic) retinopathy

Crambescin C1 Acts as A Possible Substrate of iNOS and eNOS Increasing Nitric Oxide Production and Inducing In Vivo Hypotensive Effect

Crambescins are guanidine alkaloids from the sponge Crambe crambe. Crambescin C1 (CC) induces metallothionein genes and nitric oxide (NO) is one of the triggers. We studied and compared the in vitro, in vivo, and in silico effects of some crambescine A and C analogs. HepG2 gene expression was analyzed using microarrays. Vasodilation was studied in rat aortic rings. In vivo hypotensive effect was directly measured in anesthetized rats. The targets of crambescines were studied in silico. CC and homo-crambescine C1 (HCC), but not crambescine A1 (CA), induced metallothioneins transcripts. CC increased NO production in HepG2 cells. In isolated rat aortic rings, CC and HCC induced an endothelium-dependent relaxation related to eNOS activation and an endothelium-independent relaxation related to iNOS activation, hence both compounds increase NO and reduce vascular tone. In silico analysis also points to eNOS and iNOS as targets of Crambescin C1 and source of NO increment. CC effect is mediated through crambescin binding to the active site of eNOS and iNOS. CC docking studies in iNOS and eNOS active site revealed hydrogen bonding of the hydroxylated chain with residues Glu377 and Glu361, involved in the substrate recognition, and explains its higher binding affinity than CA. The later interaction and the extra polar contacts with its pyrimidine moiety, absent in the endogenous substrate, explain its role as exogenous substrate of NOSs and NO production. Our results suggest that CC serve as a basis to develop new useful drugs when bioavailability of NO is perturbed.Fil: Rubiolo, Juan Andrés. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas; Argentina. Ministerio de Ciencia, Tecnologia E Innovacion Productiva (santa Fe). - Gobierno de la Provincia de Santa Fe. Ministerio de Ciencia, Tecnologia E Innovacion Productiva (santa Fe).; Argentina. Universidad de Santiago de Compostela; España. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario; ArgentinaFil: Lence, Emilio. Universidad de Santiago de Compostela; EspañaFil: González Bello, Concepción. Universidad de Santiago de Compostela; EspañaFil: Roel, María. Universidad de Santiago de Compostela; EspañaFil: Gil Longo, José. Universidad de Santiago de Compostela; EspañaFil: Campos Toimil, Manuel. Universidad de Santiago de Compostela; EspañaFil: Ternon, Eva. Université Nice Sophia Antipolis. Laboratoire Jean-alexandre Dieudonné.; FranciaFil: Thomas, Olivier P.. National University of Ireland Galway; IrlandaFil: González Cantalapiedra, Antonio. Universidad de Santiago de Compostela; EspañaFil: López Alonso, Henar. Universidad de Santiago de Compostela; EspañaFil: Vieytes, Mercedes R.. Universidad de Santiago de Compostela; EspañaFil: Botana, Luis M.. Universidad de Santiago de Compostela; Españ

The Treatment With the SGLT2 Inhibitor Empagliflozin Modifies the Hepatic Metabolome of Male Zucker Diabetic Fatty Rats Towards a Protective Profile

[Abstract] The EMPA-REG OUTCOME (Empagliflozin, Cardiovascular Outcome Event Trial in patients with Type 2 Diabetes Mellitus (T2DM)) trial evidenced the potential of sodium-glucose cotransporter 2 (SGLT2) inhibitors for the treatment of patients with diabetes and cardiovascular disease. Recent evidences have shown the benefits of the SGLT2 inhibitor empagliflozin on improving liver steatosis and fibrosis in patients with T2DM. Metabolomic studies have been shown to be very useful to improve the understanding of liver pathophysiology during the development and progression of metabolic hepatic diseases, and because the effects of empagliflozin and of other SGLT2 inhibitors on the complete metabolic profile of the liver has never been analysed before, we decided to study the impact on the liver of male Zucker diabetic fatty (ZDF) rats of a treatment for 6 weeks with empagliflozin using an untargeted metabolomics approach, with the purpose to help to clarify the benefits of the use of empagliflozin at hepatic level. We found that empagliflozin is able to change the hepatic lipidome towards a protective profile, through an increase of monounsaturated and polyunsaturated glycerides, phosphatidylcholines, phosphatidylethanolamines, lysophosphatidylinositols and lysophosphatidylcholines. Empagliflozin also induces a decrease in the levels of the markers of inflammation IL-6, chemerin and chemerin receptor in the liver. Our results provide new evidences regarding the molecular pathways through which empagliflozin could exert hepatoprotector beneficial effects in T2DM.This work was supported by Boehringer Ingelheim Pharma GmbH and Co., by the National Institute of Health “Fondo de Investigaciones Sanitarias del Instituto de Salud Carlos III” Madrid, Spain (PI15/00681, PI17/00409, PI18/00821, PI20/00902, RETICS Programme RD16/0012/0014 and CIBER de Enfermedades Cardiovasculares (CIBERCV)); European Regional Development Fund (FEDER) and European Union framework MSCA-RISE-H2020 Programme (Project number 734899). AH-A was funded by predoctoral research grants from Xunta de Galicia and FPU Program of the Spanish Ministry of Science, Innovation and Universities (Spain); MF-S was funded by the predoctoral research grants “Programa Científico do Centro de Investigación en Medicina Molecular e Enfermidades Crónicas (CiMUS) (Spain) and Xunta de Galicia; and AV-L was funded by the predoctoral research grant from the PFIS Program of the Spanish Ministry of Science and Instituto de Salud Carlos III (Spain

Disease Progression in MRL/lpr Lupus-Prone Mice Is Reduced by NCS 613, a Specific Cyclic Nucleotide Phosphodiesterase Type 4 (PDE4) Inhibitor

Systemic lupus erythematosus is a polymorphic and multigenic inflammatory autoimmune disease. Cyclic AMP (cAMP) modulates inflammation and the inhibition of cyclic nucleotide phosphodiesterase type 4 (PDE4), which specifically hydrolyzes cAMP, inhibits TNFα secretion. This study was aimed at investigating the evolution of PDE activity and expression levels during the course of the disease in MRL/lpr lupus-prone mice, and to evaluate in these mice the biological and clinical effects of treatments with pentoxifylline, denbufylline and NCS 613 PDE inhibitors. This study reveals that compared to CBA/J control mice, kidney PDE4 activity of MRL/lpr mice increases with the disease progression. Furthermore, it showed that the most potent and selective PDE4 inhibitor NCS 613 is also the most effective molecule in decreasing proteinuria and increasing survival rate of MRL/lpr mice. NCS 613 is a potent inhibitor, which is more selective for the PDE4C subtype (IC50 = 1.4 nM) than the other subtypes (PDE4A, IC50 = 44 nM; PDE4B, IC50 = 48 nM; and PDE4D, IC50 = 14 nM). Interestingly, its affinity for the High Affinity Rolipram Binding Site is relatively low (Ki = 148 nM) in comparison to rolipram (Ki = 3 nM). Finally, as also observed using MRL/lpr peripheral blood lymphocytes (PBLs), NCS 613 inhibits basal and LPS-induced TNFα secretion from PBLs of lupus patients, suggesting a therapeutic potential of NCS 613 in systemic lupus. This study reveals that PDE4 represent a potential therapeutic target in lupus disease

Concerted Regulation of cGMP and cAMP Phosphodiesterases in Early Cardiac Hypertrophy Induced by Angiotensin II

Left ventricular hypertrophy leads to heart failure and represents a high risk leading to premature death. Cyclic nucleotides (cAMP and cGMP) play a major role in heart contractility and cyclic nucleotide phosphodiesterases (PDEs) are involved in different stages of advanced cardiac diseases. We have investigated their contributions in the very initial stages of left ventricular hypertrophy development. Wistar male rats were treated over two weeks by chronic infusion of angiotensin II using osmotic mini-pumps. Left cardiac ventricles were used as total homogenates for analysis. PDE1 to PDE5 specific activities and protein and mRNA expressions were explored

Probiotics as Beneficial Dietary Supplements to Prevent and Treat Cardiovascular Diseases: Uncovering Their Impact on Oxidative Stress

The gut microbiota, the ecosystem formed by a wide symbiotic community of nonpathogenic microorganisms that are present in the distal part of the human gut, plays a prominent role in the normal physiology of the organism. The gut microbiota’s imbalance, gut dysbiosis, is directly related to the origin of various processes of acute or chronic dysfunction in the host. Therefore, the ability to intervene in the gut microbiota is now emerging as a possible tactic for therapeutic intervention in various diseases. From this perspective, evidence is growing that a functional dietary intervention with probiotics, which maintain or restore beneficial bacteria of the digestive tract, represents a promising therapeutic strategy for interventions in cardiovascular diseases and also reduces the risk of their occurrence. In the present work, we review the importance of maintaining the balance of the intestinal microbiota to prevent or combat such processes as arterial hypertension or endothelial dysfunction, which underlie many cardiovascular disorders. We also review how the consumption of probiotics can improve autonomic control of cardiovascular function and provide beneficial effects in patients with heart failure. Among the known effects of probiotics is their ability to decrease the generation of reactive oxygen species and, therefore, reduce oxidative stress. Therefore, in this review, we specifically focus on this antioxidant capacity and its relationship with the beneficial cardiovascular effects described for probiotics