Browsing by Author "Boric, Mauricio P."
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- ItemBoldine Prevents Renal Alterations in Diabetic Rats(2013) Hernandez-Salinas, Romina; Vielma, Alejandra Z.; Arismendi, Marlene N.; Boric, Mauricio P.; Saez, Juan C.; Velarde, VictoriaDiabetic nephropathy alters both structure and function of the kidney. These alterations are associated with increased levels of reactive oxygen species, matrix proteins, and proinflammatory molecules. Inflammation decreases gap junctional communication and increases hemichannel activity leading to increased membrane permeability and altering tissue homeostasis. Since current treatments for diabetic nephropathy do not prevent renal damage, we postulated an alternative treatment with boldine, an alkaloid obtained from boldo with antioxidant, anti-inflammatory, and hypoglycemic effects. Streptozotocin-induced diabetic and control rats were treated or not treated with boldine (50 mg/Kg/day) for ten weeks. In addition, mesangial cells were cultured under control conditions or in high glucose concentration plus proinflammatory cytokines, with or without boldine (100 mu mol/L). Boldine treatment in diabetic animals prevented the increase in glycemia, blood pressure, renal thiobarbituric acid reactive substances and the urinary protein/creatinine ratio. Boldine also reduced alterations in matrix proteins and markers of renal damage. In mesangial cells, boldine prevented the increase in oxidative stress, the decrease in gap junctional communication, and the increase in cell permeability due to connexin hemichannel activity induced by high glucose and proinflammatory cytokines but did not block gap junction channels. Thus boldine prevented both renal and cellular alterations and could be useful for preventing tissue damage in diabetic subjects.
- ItemCoordinated Endothelial Nitric Oxide Synthase Activation by Translocation and Phosphorylation Determines Flow-Induced Nitric Oxide Production in Resistance Vessels(2013) Figueroa, Xavier F.; Gonzalez, Daniel R.; Puebla, Mariela; Acevedo, Juan P.; Rojas-Libano, Daniel; Duran, Walter N.; Boric, Mauricio P.Background/Aims: Endothelial nitric oxide synthase (eNOS) is associated with caveolin-1 (Cav-1) in plasma membrane. We tested the hypothesis that eNOS activation by shear stress in resistance vessels depends on synchronized phosphorylation, dissociation from Cav-1 and translocation of the membrane-bound enzyme to Golgi and cytosol. Methods: In isolated, perfused rat arterial mesenteric beds, we evaluated the effect of changes in flow rate (2-10 ml/min) on nitric oxide (NO) production, eNOS phosphorylation at serine 1177, eNOS subcellular distribution and co-immunoprecipitation with Cav-1, in the presence or absence of extracellular Ca2+. Results: Increases in flow induced a biphasic rise in NO production: a rapid transient phase (3-5-min) that peaked during the first 15 s, followed by a sustained phase, which lasted until the end of stimulation. Concomitantly, flow caused a rapid translocation of eNOS from the microsomal compartment to the cytosol and Golgi, paralleled by an increase in eNOS phosphorylation and a reduction in eNOS-Cav-1 association. Transient NO production, eNOS translocation and dissociation from Cav-1 depended on extracellular Ca2+, while sustained NO production was abolished by the PI3K-Akt blocker wortmannin. Conclusions: In intact resistance vessels, changes in flow induce NO production by transient Ca2+-dependent eNOS translocation from membrane to intracellular compartments and sustained Ca2+-independent PI3K-Akt-mediated phosphorylation. (C) 2013 S. Karger AG, Basel
- ItemDevelopment of a Novel Perfusable Solution for ex vivo Preservation: Towards Photosynthetic Oxygenation for Organ Transplantation(Frontiers Media SA, 2021) Veloso Gimenez, Valentina; Escamilla, Rosalba; Necuñir, David; Corrales Orovio, Rocio; Riveros, Sergio; Marino, Carlo; Ehrenfeld, Carolina; Guzman, Christian Dani; Boric, Mauricio P.; Rebolledo, Rolando; Egaña, José TomasOxygen is the key molecule for aerobic metabolism, but no animal cells can produce it, creating an extreme dependency on external supply. In contrast, microalgae are photosynthetic microorganisms, therefore, they are able to produce oxygen as plant cells do. As hypoxia is one of the main issues in organ transplantation, especially during preservation, the main goal of this work was to develop the first generation of perfusable photosynthetic solutions, exploring its feasibility for ex vivo organ preservation. Here, the microalgae Chlamydomonas reinhardtii was incorporated in a standard preservation solution, and key aspects such as alterations in cell size, oxygen production and survival were studied. Osmolarity and rheological features of the photosynthetic solution were comparable to human blood. In terms of functionality, the photosynthetic solution proved to be not harmful and to provide sufficient oxygen to support the metabolic requirement of zebrafish larvae and rat kidney slices. Thereafter, isolated porcine kidneys were perfused, and microalgae reached all renal vasculature, without inducing damage. After perfusion and flushing, no signs of tissue damage were detected, and recovered microalgae survived the process. Altogether, this work proposes the use of photosynthetic microorganisms as vascular oxygen factories to generate and deliver oxygen in isolated organs, representing a novel and promising strategy for organ preservation.
- ItemDifferential role of S-nitrosylation and the NO-cGMP-PKG pathway in cardiac contractility(2008) Gonzalez, Daniel R.; Fernandez, Ignacio C.; Ordenes, Pablo P.; Treuer, Adriana V.; Eller, Gisela; Boric, Mauricio P.The role of nitric oxide (NO) in cardiac contractility is complex and controversial. Several NO donors have been reported to cause positive or negative inotropism. NO can bind to guanylate cyclase, increasing cGMP production and activating PKG. NO may also directly S-nitrosylate cysteine residues of specific proteins. We used the isolated rat heart preparation to test the hypothesis that the differential inotropic effects depend on the degree of NO production and the signaling recruited. SNAP (S-nitroso-N-acetylpenicillamine), a NO donor, increased contractility at 0.1, 1 and 10 M. This effect was independent of phospholamban phosphorylation, was not affected by PKA inhibition with H-89 (N-[2((p-bromocinnamyl)amino)ethyl]-5-isoquinolinesulfonamide), but it was abolished by the radical scavenger Tempol (4-hydroxy-[2,2,4,4]-tetramethyl-piperidine-1-oxyl). However, at 100 mu M SNAP reduced contractility, effect reversed to positive inotropism by guanylyl cyclase blockade with ODQ (1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one), and abolished by PKG inhibition with KT5823, but not affected by Tempol. SNAP increased tissue cGMP at 100 M, but not at lower concentrations. Consistently, a cGMP analog also reduced cardiac contractility. Finally, SNAP at 1 mu M increased the level of S-nitrosylation of various cardiac proteins, including the ryanodine receptor. This study demonstrates the biphasic role for NO in cardiac contractility in a given preparation; furthermore, the differential effect is clearly ascribed to the signaling pathways involved. We conclude that although NO is highly diffusible, its output determines the fate of the messenger: low NO concentrations activate redox processes (S-nitrosylation), increasing con-tractility;while the cGMP-PKG pathway is activated at high NO concentrations, reducing contractility. (c) 2007 Elsevier Inc. All rights reserved.
- ItemEndothelial nitric oxide synthase regulates microvascular hyperpermeability in vivo(2006) Hatakeyama, Takuya; Pappas, Peter J.; Hobson, Robert W., II; Boric, Mauricio P.; Sessa, William C.; Duran, Walter N.Nitric oxide (NO) is an important regulator of blood flow, but its role in permeability is still challenged. We tested in vivo the hypotheses that: (a) endothelial nitric oxide synthase (eNOS) is not essential for regulation of baseline permeability; (b) eNOS is essential for hyperpermeability responses in inflammation; and (c) molecular inhibition of eNOS with caveolin-1 scaffolding domain (AP-Cav) reduces eNOS-regulated hyperpermeability. We used eNOS-deficient (eNOS-/-) mice and their wild-type control as experimental animals, platelet-activating factor (PAF) at 10(-7) m as the test pro-inflammatory agent, and integrated optical intensity (IOI) as an index of microvascular permeability. PAF increased permeability in wild-type cremaster muscle from a baseline of 2.4 +/- 2.2 to a peak net value of 84.4 +/- 2.7 units, while the corresponding values in cremaster muscle of eNOS-/- mice were 1.0 +/- 0.3 and 15.6 +/- 7.7 units (P < 0.05). Similarly, PAF increased IOI in the mesentery of wild-type mice but much less in the mesentery of eNOS-/- mice. PAF increased IOI to comparable values in the mesenteries of wild-type mice and those lacking the gene for inducible NOS (iNOS). Administration of AP-Cav blocked the microvascular hyperpermeability responses to 10(-7) m PAF. We conclude that: (1) baseline permeability does not depend on eNOS; (2) eNOS and NO are integral elements of the signalling pathway for the hyperpermeability response to PAF; (3) iNOS does not affect either baseline permeability or hyperpermeability responses to PAF; and (4) caveolin-1 inhibits eNOS regulation of microvascular permeability in vivo. Our results establish eNOS as an important regulator of microvascular permeability in inflammation.
- ItemGalectin-8 induces endothelial hyperpermeability through the eNOS pathway involving S-nitrosylation-mediated adherens junction disassembly(2019) Zamorano, Patricia; Koning, Tania; Oyanadel, Claudia; Mardones, Gonzalo A.; Ehrenfeld, Pamela; Boric, Mauricio P.; Gonzalez, Alfonso; Soza, Andrea; Sanchez, Fabiola A.The permeability of endothelial cells is regulated by the stability of the adherens junctions, which is highly sensitive to kinase-mediated phosphorylation and endothelial nitric oxide synthase (eNOS)-mediated S-nitrosylation of its protein components. Solid tumors can produce a variety of factors that stimulate these signaling pathways leading to endothelial cell hyperpermeability. This generates stromal conditions that facilitate tumoral growth and dissemination. Galectin-8 (Gal-8) is overexpressed in several carcinomas and has a variety of cellular effects that can contribute to tumor pathogenicity, including angiogenesis. Here we explored whether Gal-8 has also a role in endothelial permeability. We show that recombinant Gal-8 activates eNOS, induces S-nitrosylation of p120-catenin (p120) and dissociation of adherens junction, leading to hyperpermeability of the human endothelial cell line EAhy926. This pathway involves focal-adhesion kinase (FAK) activation downstream of eNOS as a requirement for eNOS-mediated p120 S-nitrosylation. This suggests a reciprocal, yet little understood, regulation of phosphorylation and S-nitrosylation events acting upon adherens junction permeability. In addition, glutathione S-transferase (GST)-Gal-8 pull-down experiments and function-blocking beta 1-integrin antibodies point to beta 1-integrins as cell surface components involved in Gal-8-induced hyperpermeability. Endogenous Gal-8 secreted from the breast cancer cell line MCF-7 has similar hyperpermeability and signaling effects. Furthermore, the mouse cremaster model system showed that Gal-8 also activates eNOS, induces S-nitrosylation of adherens junction components and is an effective hyperpermeability agent in vivo. These results add endothelial permeability regulation by S-nitrosylation as a new function of Gal-8 that can potentially contribute to the pathogenicity of tumors overexpressing this lectin.
- ItemMicroalgae share key features with human erythrocytes and can safely circulate through the vascular system in mice(2023) Ehrenfeld, Carolina; Veloso-Giménez, Valentina; Corrales-Orovio, Rocío; Rebolledo, Rolando; Boric, Mauricio P.; Egaña, José TomásAs animal cells cannot produce oxygen, erythrocytes are responsible for gas interchange, being able to capture and deliver oxygen upon tissue request. Interestingly, several other cells in nature produce oxygen by photosynthesis, raising the question of whether they could circulate within the vascular networks, acting as an alternative source for oxygen delivery. To address this long-term goal, here some physical and mechanical features of the photosynthetic microalga Chlamydomona reinhardtii were studied and compared with erythrocytes, revealing that both exhibit similar size and rheological properties. Moreover, key biocompatibility aspects of the microalgae were evaluated in vitro and in vivo, showing that C. reinhardtii can be co-cultured with endothelial cells, without affecting each other’s morphology and viability. Moreover, short-term systemic perfusion of the microalgae showed a thoroughly intravascular distribution in mice. Finally, the systemic injection of high numbers of microalgae did not trigger deleterious responses in living mice. Altogether, this work provides key scientific insights to support the notion that photosynthetic oxygenation could be achieved by circulating microalgae, representing another important step towards human photosynthesis.
- ItemOxygenation by Intravascular Photosynthesis Reduces Kidney Damage During ex Vivo Preservation(2024) Veloso-Gimenez, Valentina; Cardenas-Calderon, Camila; Castillo, Valentina; Carvajal, Felipe; Gallardo-Aguero, Daniela; Gonzalez-Itier, Sergio; Corrales-Orovio, Rocio; Becerra, Daniela; Miranda, Miguel; Rebolledo, Rolando; San Martin, Sebastian; Boric, Mauricio P.; Egana, Jose TomasSeveral clinical issues are associated with reduced oxygen delivery to tissues due to impaired vascular perfusion; moreover, organs procured for transplantation are subjected to severe hypoxia during preservation. Consequently, alternative tissue oxygenation is an active field in biomedical research where several innovative approaches have been recently proposed. Among these, intravascular photosynthesis represents a promising approach as it relies on the intrinsic capacity of certain microorganisms to produce oxygen upon illumination. In this context, this work aims at the development of photosynthetic perfusable solutions that could be applied to preserve organs for transplantation purposes. Our findings demonstrate that a biocompatible physiological solution containing the photosynthetic microalgae Chlamydomonas reinhardtii can fulfill the metabolic oxygen demand of rat kidney slices in vitro. Furthermore, intravascular administration of this solution does not induce tissue damage in the rat kidneys. Moreover, kidney slices obtained from these algae-perfused organs exhibited significantly improved preservation after 24 h of incubation in hypoxia while exposed to light, resulting in reduced tissue damage and enhanced metabolic status. Overall, the results presented here contribute to the development of alternative strategies for tissue oxygenation, supporting the use of perfusable photosynthetic solutions for organ preservation in transplantation.
- ItemS-Nitrosation of β-Catenin and p120 Catenin A Novel Regulatory Mechanism in Endothelial Hyperpermeability(2012) Marin, Natalie; Zamorano, Patricia; Carrasco, Rodrigo; Mujica, Patricio; Gonzalez, Francisco G.; Quezada, Claudia; Meininger, Cynthia J.; Boric, Mauricio P.; Duran, Walter N.; Sanchez, Fabiola A.Rationale: Endothelial adherens junction proteins constitute an important element in the control of microvascular permeability. Platelet-activating factor (PAF) increases permeability to macromolecules via translocation of endothelial nitric oxide synthase (eNOS) to cytosol and stimulation of eNOS-derived nitric oxide signaling cascade. The mechanisms by which nitric oxide signaling regulates permeability at adherens junctions are still incompletely understood.
- ItemTNF-α-activated eNOS signaling increases leukocyte adhesion through the S- nitrosylation pathway(2021) Aguilar, Gaynor; Cordova, Francisco; Koning, Tania; Sarmiento, Jose; Boric, Mauricio P.; Birukov, Konstantin; Cancino, Jorge; Varas-Godoy, Manuel; Soza, Andrea; Alves, Natascha G.; Mujica, Patricio E.; Duran, Walter N.; Ehrenfeld, Pamela; Sanchez, Fabiola A.Nitric oxide ( NO) is a key factor in inflammation. Endothelial nitric oxide synthase (eNOS), whose activity increases after stimulation with proinflammatory cytokines, produces NO in endothelium. NO activates two pathways: 1) soluble guanylate cyclase-protein kinase G and 2) S-nitrosylation (NO-induced modification of free-thiol cysteines in proteins). S-nitrosylation affects phosphorylation, localization, and protein interactions. NO is classically described as a negative regulator of leukocyte adhesion to endothelial cells. However, agonists activating NO production induce a fast leukocyte adhesion, which suggests that NO might positively regulate leukocyte adhesion. We tested the hypothesis that eNOS-induced NO promotes leukocyte adhesion through the S-nitrosylation pathway. We stimulated leukocyte adhesion to endothelium in vitro and in vivo using tumor necrosis factor-alpha (TNF-alpha) as proinflammatory agonist. ICAM-1 changes were evaluated by immunofluorescence, subcellular fractionation, immunoprecipitation, and fluorescence recovery after photobleaching (FRAP). Protein kinase C sigma (PKC sigma) activity and S-nitrosylation were evaluated by Western blot analysis and biotin switch method, respectively. TNF-alpha, at short times of stimulation, activated the eNOS S-nitrosylation pathway and caused leukocyte adhesion to endothelial cells in vivo and in vitro. TNF-alpha-induced NO led to changes in ICAM-1 at the cell surface, which are characteristic of clustering. TNF-alpha-induced NO also produced S-nitrosylation and phosphorylation of PKCf, association of PKCf with ICAM-1, and ICAM-1 phosphorylation. The inhibition of PKCf blocked leukocyte adhesion induced by TNF-alpha. Mass spectrometry analysis of purified PKCf identified cysteine 503 as the only S-nitrosylated residue in the kinase domain of the protein. Our results reveal a new eNOS S-nitrosylation-dependent mechanism that induces leukocyte adhesion and suggests that S-nitrosylation of PKCf may be an important regulatory step in early leukocyte adhesion in inflammation.