Erythrocyte reactive nitrogen species in health and disease

Copyright: © 2018 Saldanha C. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.In general, reactive nitrogen species, like nitric oxide (NO) and peroxynitrite are classified in primary and secondary according its benefic or deleterious effects respectively. Multiple benefic roles through the body has been attributed to nitric oxide (NO), a key signaling molecule, which acts as vasodilator relaxing smooth muscles of arteries; participates in the blood pressure control; hinders the initiation of inflammation NO liberated from endothelium cells or lymphocytes to intravascular corporal fluid enter into erythrocytes through membrane band 3 protein being after captured by deoxyhemoglobin or by oxyhemoglobin. Inside the erythrocytes several chemical reactions occur with generation of S-nitrosoglutathione a NO reservoir molecules and NO derivatives (NOx) molecules namely nitrite, nitrate and peroxynitrite. The erythrocyte acts as sensor of the tissues oxygen partial pressure and consequently change its oxygen levels lead along with NO contents. NO is released through band 3 protein into the blood, at microcirculatory network, in tissues with low oxygen partial pressure. The aim of this mini review was to present the erythrocyte signal transduction pathways associated with NO and S-nitrosoglutathione efflux, metabolic behavior of the NO reservoir molecules and NO reactive species in human erythrocytes under several biological conditions. For examples, the effects of external amphipathic molecules, fibrinogen, and internal manipulation of protein phosphorylation degree and redox status on erythrocyte. NOx, NO and S-nitrosoglutathione efflux levels are herein included. Erythrocyte NO efflux evidenced as biomarkers of inflammatory vascular diseases will be herein highlighted.This work was funded by Fundação para a Ciência e Tecnologia: LISBOA-01-0145-FEDER-007391, project cofunded by FEDER, through POR Lisboa 2020 - Programa Operacional Regional de Lisboa, PORTUGAL 2020.info:eu-repo/semantics/publishedVersio

The ubiquity nature of acetylcholine

© 2014 Saldanha C, et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.This study was supported by grants from the FCT-Fundação para a Ciência e a Tecnologia (EXCL/MAT-NAN/0114/2012).info:eu-repo/semantics/publishedVersio

A designed cyclic analogue of gomesin has potent activity against Staphylococcus aureus biofilms

© The Author(s) 2022. Published by Oxford University Press on behalf of British Society for Antimicrobial Chemotherapy. This is an Open Access article distributed under the terms of the Creative Commons Attribution-NonCommercial License (https:// creativecommons.org/licenses/by-nc/4.0/), which permits non-commercial re-use, distribution, and reproduction in any medium, provided the original work is properly cited. For commercial re-use, please contact [email protected]: Infections caused by bacterial biofilms are very difficult to treat. The use of currently approved antibiotics even at high dosages often fails, making the treatment of these infections very challenging. Novel antimicrobial agents that use distinct mechanisms of action are urgently needed. Objectives: To explore the use of [G1K,K8R]cGm, a designed cyclic analogue of the antimicrobial peptide gomesin, as an alternative approach to treat biofilm infections. Methods: We studied the activity of [G1K,K8R]cGm against biofilms of Staphylococcus aureus, a pathogen associated with several biofilm-related infections. A combination of atomic force and real-time confocal laser scanning microscopies was used to study the mechanism of action of the peptide. Results: The peptide demonstrated potent activity against 24 h-preformed biofilms through a concentration-dependent ability to kill biofilm-embedded cells. Mechanistic studies showed that [G1K,K8R]cGm causes morphological changes on bacterial cells and permeabilizes their membranes across the biofilm with a half-time of 65 min. We also tested an analogue of [G1K,K8R]cGm without disulphide bonds, and a linear unfolded analogue, and found both to be inactive. Conclusions: The results suggest that the 3D structure of [G1K,K8R]cGm and its stabilization by disulphide bonds are essential for its antibacterial and antibiofilm activities. Moreover, our findings support the potential application of this stable cyclic antimicrobial peptide to fight bacterial biofilms.This project received funding from the European Union’s Horizon 2020 research and innovation programme under grant agreement No. 828774. This work was supported by project grants funded by Fundação para a Ciência e a Tecnologia (FCT-MCTES, Portugal; UIDB/04565/2020 and PPBI-POCI-01-0145-FEDER-022122). S.A.D. acknowledges FCT for the fellowship PD/BD/114425/2016. S.T.H. is an Australian Research Council (ARC) Future Fellow (FT150100398) and is supported by the ARC Centre of Excellence for Innovations in Peptide & Protein Science (CE200100012).info:eu-repo/semantics/publishedVersio

Retinal vascular reactivity in type 1 diabetes patients without retinopathy using optical coherence tomography angiography

Copyright © 2020 The Authors. This work is licensed under a Creative Commons Attribution-Non-Commercial-No-Derivatives 4.0 International License.Purpose: We hypothesize that patients with type 1 diabetes (T1D) may have abnormal retinal vascular responses before diabetic retinopathy (DR) is clinically evident. Optical coherence tomography angiography (OCTA) was used to dynamically assess the retinal microvasculature of diabetic patients with no clinically visible retinopathy. Methods: Controlled nonrandomized interventional study. The studied population included 48 eyes of 24 T1D patients and 24 demographically similar healthy volunteers. A commercial OCTA device (AngioVue) was used, and two tests were applied: (1) the hypoxia challenge test (HCT) and (2) the handgrip test to induce a vasodilatory or vasoconstrictive response, respectively. The HCT is a standardized test that creates a mild hypoxic environment equivalent to a flight cabin. The handgrip test (i.e., isometric exercise) induces a sympathetic autonomic response. Changes in the parafoveal superficial and deep capillary plexuses in both tests were compared in each group. Systemic cardiovascular responses were also comparatively evaluated. Results: In the control cohort, the vessel density of the median parafoveal superficial and deep plexuses increased during hypoxia (F1,23 = 15.69, P < 0.001 and F1,23 = 16.26, P < 0.001, respectively). In the T1D group, this physiological response was not observed in either the superficial or the deep retinal plexuses. Isometric exercise elicited a significant decrease in vessel density in both superficial and deep plexuses in the control group (F1,23 = 27.37, P < 0.0001 and F1,23 = 27.90, P < 0.0001, respectively). In the T1D group, this response was noted only in the deep plexus (F1,23 = 11.04, P < 0.01). Conclusions: Our work suggests there is an early impairment of the physiological retinal vascular response in patients with T1D without clinical diabetic retinopathy.info:eu-repo/semantics/publishedVersio

A protocol to evaluate retinal vascular response using optical coherence tomography angiography

Copyright © 2019 Sousa, Leal, Moreira, do Vale, Silva-Herdade, Aguiar, Dionísio, Abegão Pinto, Castanho and Marques-Neves. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY).The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these termsIntroduction: Optical coherence tomography angiography (OCT-A) is a novel diagnostic tool with increasing applications in ophthalmology clinics that provides non-invasive high-resolution imaging of the retinal microvasculature. Our aim is to report in detail an experimental protocol for analyzing both vasodilatory and vasoconstriction retinal vascular responses with the available OCT-A technology. Methods: A commercial OCT-A device was used (AngioVue®, Optovue, CA, United States), and all examinations were performed by an experienced technician using the standard protocol for macular examination. Two standardized tests were applied: (i) the hypoxia challenge test (HCT) and (ii) the handgrip test, in order to induce a vasodilatory and vasoconstriction response, respectively. OCT-A was performed at baseline conditions and during the stress test. Macular parafoveal vessel density of the superficial and deep plexuses was assessed from the en face angiograms. Statistical analysis was performed using STATA v14.1 and p < 0.05 was considered for statistical significance. Results: Twenty-four eyes of 24 healthy subjects (10 male) were studied. Mean age was 31.8 ± 8.2 years (range, 18–57 years). Mean parafoveal vessel density in the superficial plexus increased from 54.7 ± 2.6 in baseline conditions to 56.0 ± 2.0 in hypoxia (p < 0.01). Mean parafoveal vessel density in the deep plexuses also increased, from 60.4 ± 2.2 at baseline to 61.5 ± 2.1 during hypoxia (p < 0.01). The OCT-A during the handgrip test revealed a decrease in vessel density in both superficial (55.5 ± 2.6 to 53.7 ± 2.9, p < 0.001) and deep (60.2 ± 1.8 to 56.7 ± 2.8, p < 0.001) parafoveal plexuses. Discussion: In this work, we detail a simple, non-invasive, safe, and non-costly protocol to assess a central nervous system vascular response (i.e., the retinal circulation) using OCT-A technology. A vasodilatory response and a vasoconstriction response were observed in two physiologic conditions—mild hypoxia and isometric exercise, respectively. This protocol constitutes a new way of studying retinal vascular changes that may be applied in health and disease of multiple medical fields.This study was supported by the Faculty of Medicine of the University of Lisbon, AstraZeneca Foundation – 14th Grant.info:eu-repo/semantics/publishedVersio

Fibrinogen involvement in hemorheology and inflammation

The aim of this chapter is to review the knowledge obtained from; (i) ex vivo and in vitro studies about the effects of human soluble fibrinogen molecules on blood components behavior and their effects on physiological and pathophysiological conditions, and (ii) in vitro studies conducted on experimental animal models. Ex vivo studies which demonstrated the hemorheogical participation of plasma fibrinogen and its involvement in inflammatory vascular disease will be described. In vitro studies of fibrinogen binding to erythrocyte and neutrophils targets and their influence (at normal and high concentrations) on the biorheology properties of erythrocyte (NO metabolism under the external presence of endogenous or exogenous body molecules and by intra erythrocyte manipulation of redox or protein phosphorylation status) and on neutrophils activation will be presented. In vivo studies on the presence and absence of soluble fibrinogen in the bloodstream, and to enable the leukocyte recruitment will be presented. The participation of erythrocyte in leukocyte recruitment in inflammation will also be described.This work was funded by Fundação para a Ciência e Tecnologia: LISBOA-01-0145-FEDER-007391, project cofunded by FEDER, through POR Lisboa 2020 - Programa Operacional Regional de Lisboa, PORTUGAL 2020.info:eu-repo/semantics/publishedVersio

Timolol effects on erythrocyte deformability and nitric oxide metabolism

© 2018 – IOS Press and the authors. All rights reservedTimolol maleate is a compound used in treatment for reducing increased intra-ocular pressure by limiting aqueous humor production. Decreased erythrocyte deformability (ED), increased activity of erythrocyte acetylcholinesterase (AChE), increased values of nitrosoglutathione (GSNO) and nitic oxide (NO) and decreased plasma levels of NO metabolites, were described in primary open angle glaucoma patients. In healthy human red blood cells (RBCs), timolol is an inhibitor of AChE and induces NO efflux and GSNO efflux from that blood component in lower concentration than those obtained in presence of the natural AChE substrate, acetylcholine (ACh). The signal transduction pathway in RBCs described for NO in dependence of AChE-ACh active complex involves Gi protein, protein tyrosine kinase (PTK like Syk and p53/56Lyn), protein tyrosine phosphatase (PTP) and adenylyl cyclase (AC).The aim of this in vitro study was to verify the effect of timolol maleate in ED, NO efflux and NO derivatives molecules (NOx) like nitrite (NO2-), nitrate (NO3-, peroxynitrite (-ONOO) and GSNO under the presence of PTK, PTP, AC and guanylyl cyclase (GC) enzyme proteins inhibitors.Blood samples from healthy donors were each one divided and were performed aliquots in absence (control aliquots) and presence of timolol or timolol plus each inhibitor and Gi protein uncoupling. No significant differences in erythrocyte NO efflux, GSNO, peroxynitrite, nitrite and nitrate concentrations in response to timolol when compared with the untreated blood samples aliquots were obtained.It was observed an increase in erythrocyte deformability at high shear stresses induced by the simultaneous presence of timolol and band 3 protein dephosphorylation by PTK syk inhibitor. No significant differences where verified in peroxynitrite levels in the blood aliquots in presence of timolol plus each enzyme inhibitor and Gi protein uncoupling in relation to the control aliquots. No variation of GSNO concentration occurs under the presence of timolol and AMGT (PTK lyn inhibitor) besides the significant higher values observed with each one of the other inhibitors. Nitrate concentration increases significantly in all aliquots with timolol plus each one of the inhibitors. The same was observe with nitrite levels with exception of the aliquots with timolol plus AMGT or timolol plus Gi protein uncoupling showing no significant values in relation to the control aliquots.Besides the changes in NO derivative molecules and NO efflux from RBCs obtained in this study with blood samples of healthy donors under the effect of timolol plus each inhibitor of the proteins participants in NO signal transduction mechanism, further analogue studies must be promoted with blood samples of patients with glaucoma or any other inflammatory vascular disease.This study was funded by Fundação para a Ciência e Tecnologia: LISBOA-01-0145-FEDER-007391, ˆ project cofunded by FEDER, through POR Lisboa 2020 - Programa Operacional Regional de Lisboa, PORTUGAL 2020.info:eu-repo/semantics/publishedVersio

Modulation of erythrocyte deformability by PKC activity

Copyright © 2008 IOS Press All rights reserved.The interactions between membrane, peripheral and cytoskeleton proteins are responsible for the maintenance of erythrocyte deformability (EEI) and some of these interactions are modulated by PKC activity. Protein band 3 of the erythrocyte membrane is phosphorylated by phosphotyrosine kinases (PTK) and dephosphorylated by phosphotyrosine phosphatase (PTP). It was previously described by us a signal transduction mechanism that describes a possible pathway connecting an erythrocyte external membrane protein, acetylcholinesterase (AChE), with protein band 3. So how does PKC activity modulate EEI when protein band 3 is phosphorylated or dephosphorylated in absence or presence of AChE effectors? To answer this we used phorbol 12-myristate 13-acetate (PMA) as an activator and chelerythrine chloride as inhibitor of PKC and also band 3 modulators of band 3 phosphorylation degree, in presence and absence of AChE effectors in order to measure in whole blood samples EEI. Our results showed that erythrocyte deformability was significantly (i) decreased by inhibition of PKC, in absence and presence of AChE inhibitor velnacrine (ii) increased with PMA in absence and presence of ACh and (iii) decreased in presence of calpeptin in absence and presence of either chelerythrine or PMA. These results establish dependence between cytoskeleton proteins, PKC activity, band 3 phosphorylation degrees and EEI. Better understanding of those proteins interactions on transduction mechanisms might trigger possible targets for drug action that would modulate EEI.info:eu-repo/semantics/publishedVersio

Leukocytes dynamics in microcirculation under shear-thinning blood flow

© 2009 Elsevier Ltd. All rights reserved.We present detailed simulation results of localised hemodynamics for a cluster of rolling leukocytes under shear-thinning blood flow using a lattice Boltzmann model. Leukocytes were modelled as hard spheres moving through a venule of rigid walls. The used hemorheological parameters were obtained from in vivo measurements in blood samples of Wistar rats. Velocities, shear stresses and torques were computed and visualised for each individual cell, for the cluster and for the fluid. We have found that the flow is mainly three-dimensional due to the swirling and the asymmetry of the formed vortices during the recruitment process. The shear stress is maximum on a cap covering the cell and a cone with its base on the endothelial wall at the contact region. The leukocyte is recruited to the wall with the aid of trapping vortices and four stagnant regions surrounding the cell in addition to lateral motion towards the wall. We suggest that these phenomena are highly dependent on the angular velocity of the leukocyte and on the attractive force between the leukocyte and the endothelial wall. For a moving cluster of recruited leukocytes, velocities and shear stresses as well as torques are computed. It was found that the shear stress at the endothelium gets higher as the cluster moves in the main stream enabling early initialisation of the rolling process.This work has been partially supported by the grant SFRH/BPD/20823/2004 of Fundação para a Ciência e a Tecnologia (A. Artoli) and by the project PTDC/MAT/68166/2006. FCT funds from the Centre for Mathematics and its Applications–CEMAT and the Molecular Medicine Institute–Microvascular Biology and Inflammation Unit are highly appreciated.info:eu-repo/semantics/publishedVersio

Fibrinogen signalling in erythrocyte nitric oxide mobilization in presence of PI3-K and adenylyl cyclase inhibitors

Soluble form of fibrinogen (Fib) and the peptide 4N1K are ligands of erythrocyte membrane CD47. Fibrinogen reinforces the ability of erythrocyte to scavenger nitric oxide (NO). Hiperfibrinogenemia increased NO efflux from erythrocyte in dependence of band 3 phosphorylation which is abolished by the presence of 4N1K. Herein we study in vitro the effect of high fibrinogen levels, on the NO efflux from erythrocytes and on its mobilization under influence of phosphoinositide-3 kinase (PI3-K) and adenylyl cyclase (AC) inhibitors in presence of 4N1K. Erythrocyte NO efflux, peroxynitrite, nitrite, nitrate and S-nitrosoglutathione (GSNO) were determined in blood samples in presence of 4N1K, wortmannin (WORT, PI3-K inhibitor) and MDL (AC inhibitor) under high fibrinogen concentrations. 4N1K with WORT and high fibrinogen levels induce, in relation to Fib plus WORT samples no variations on the erythrocyte NO efflux, decreased peroxynitrite, increased of nitrite, nitrate and GSNO concentrations. When 4N1K is present with MDL and high fibrinogen levels show, in relation to fibrinogen plus MDL samples increased erythrocyte NO efflux and nitrite, nitrate and GSNO concentrations. In conclusion, under high Fib levels and 4N1K the erythrocytes show: preservation of NO and impaired peroxynitrite in presence of PI3K inhibition; increased efflux of NO at lower levels of cAMP resulting from adenylyl cyclase inhibition.This study was supported by grants from the FCT— Fundação para a Ciência e a Tecnologia (project reference PTDC/SAU-OSM/73449/2006).info:eu-repo/semantics/publishedVersio