Erythrocyte Nitric Oxide

Nitric oxide (NO) is a vasoactive molecule that, by stimulated and functional vascular endothelial cells, is released to the lumen of the vessel and into the surrounding smooth muscle cells. Once in the lumen, NO is captured by red blood cells and scavenged inside through hemoglobin and derived as NO metabolites. The delivery ability of erythrocytes allowing the NO efflux also occurs. Manipulation of NO levels inside the erythrocyte through different external (acetylcholine, acetylcholinesterase inhibitors, fibrinogen and CD47 4N1K peptide) and internal (redox and protein phosphorylation levels) stimuli will be described. The values of NO efflux from the erythrocytes and its association with the data quantified in the hemorheology properties and in clinical parameters obtained from patients with vascular diseases will also be present. The in vivo animal experimental studies highlighting the ability of NO efflux (delivered) from the erythrocytes where is scavenged and its influence in inflammatory and hemorheological responses will be addressed. So, the aim of this chapter is to present the knowledge obtained about the NO signal transduction mechanism in erythrocytes and the association between erythrocyte availability in NO with clinical biomarkers obtained in inflammatory vascular diseases. A final question is raised—namely, could NO be considered a hemorheological parameter

Microcirculation and inflammation in a numerical simulation approach

Inflammation is the response of the organism to eradicate the agent of lesion or infection in order to achieve hemostasis. This response requires the migration of specific leukocyte populations from the blood circulation towards the inflamed area. Leukocyte recruitment constitutes a complex cellular process by which leukocytes are first recruited to the endothelial vascular wall of post-capillary venules across which they further extravasate into the interstitial tissue. Recruitment is mediated via cell-cell interactions between the leukocyte and the endothelium and occurs through a multi-step cascade: tethering, rolling, slow rolling, arrest, crawling, adhesion and transmigration. However, whether or not the leukocytes adhere to the endothelium depends not only on the chemical forces generated by adhesion molecules on leukocytes and endothelial cells, but also on the physical forces that act on those cells. It has been suggested that fluid shear stress resulting from blood flow also regulates leukocyte activity which makes the fluid dynamic environment of the circulation to be considered an important aspect for leukocyte recruitment and migration during the inflammatory response. Most of the studies on the inflammatory response and in particular on leukocyte recruitment are based on animal models and involve, among others, the quantification of inflammatory mediators and cellular players, and/or the analysis of the leukocyte-endothelial cell interactions by intravital microscopy. However, the contribution of hemodynamics for leukocyte recruitment has been seldom addressed in those studies. This is mostly due to the fact that the study of hemodynamics in in vivo animal models is not straightforward and moreover, that several hemodynamic parameters cannot be experimentally determined due to technical constraints. In this work, we reasoned that these limitations could be circumvented by the development and use of numerical simulations to describe leukocyte recruitment. Many of the processes, which take place in living organisms, can be expressed as mathematical equations. This applies to leukocyte recruitment, for which scarce numerical models existed before the beginning of this work. Importantly, these mathematical simulations were performed without considering simultaneously all the players in the process, namely the vessel, the blood flow and the leukocytes. Moreover, most of these studies were two dimensional, assumed blood as a Newtonian fluid with constant viscosity and did not take into account in vivo experimental data. Taken this, our major goal with this work was to understand the contribution of hemodynamics to leukocyte recruitment in inflammation. For such purpose, we aimed here at developing numerical simulations that more adequately reproduced this process. For such, we set up animal models of inflammation to obtain the experimental data required for the development of those numerical simulations. Finally, we used these models to investigate the role of hemodynamics in leukocyte recruitment in inflammation. First, we considered the simpler case of a numerical simulation that assumed leukocytes to be rigid spheres and blood, a non-Newtonian fluid. For such, we initially developed an animal model of inflammation in Wistar rats using a lipopolysaccharide (LPS) as an inflammatory agent. Blood samples were collected for determination of TNF-α levels to ensure the triggering of the inflammatory process. Importantly, the number of rolling and adherent leukocytes in post-capillary venules was monitored using an intravital microscopy approach. As expected, our results showed that there is an increase in TNF-α concentrations after 15 minutes of LPS administration and a significant increase in the number of rolling and adherent leukocytes. The recorded intravital microcopy images, along with other recorded parameters, were then used, in collaboration with a group of mathematicians, to develop a numerical model capable of describing leukocyte recruitment in the microcirculation. To evaluate the contribution of hemodynamics, the localized velocity fields and shear stresses on the surface of leukocytes and near the vessel wall contact points have been computed in two discrete situations, namely as a single leukocyte or when a cluster of them are recruited towards the vessel wall. In the first situation, our numerical results showed the presence of one region of maximum shear stress on the surface of the leuko- cyte close to the endothelial wall and of two regions of minimum shear stress on the op- posite side of the cell. The different areas of shear stress observed in the surface of the leukocyte may be important in directing it towards the endothelial wall during an inflammatory response. The identification of a region of maximum shear stress is consistent with the molecular mechanisms that govern leukocyte rolling because it may actually cor- respond to the area that supports the interaction with the endothelium. On the other hand, the relatively lower shear stress regions may correlate with leukocyte surface areas where binding to the endothelium is not occurring at the moment, thus enabling the roll- ing of the cell along the endothelium. It was also observed that the shear stress at the endothelium gets higher as a cluster of leukocytes moves in the main stream. This sug- gests that the presence of a cluster of leukocytes may potentiate leukocyte rolling, as the increase in the shear stress promoted by the recruited leukocytes may support the migra- tion and recruitment of additional cells. Despite closely simulating leukocyte recruitment, our initial numerical simulation consid- ered the simple case of leukocytes as rigid spheres. However, while circulating leukocytes maintain an approximately spherical shape, rolling leukocytes are known to deform. In order to account for the leukocyte deformability changes that occur during its recruit- ment in inflammation, we needed to assess the deformability profile of the leukocytes under flow and therefore, to “directly” observe them regardless of the other blood cells. For such, intravital microscopy was performed in the mouse cremaster of a transgenic mice strain (Lys-EGFP-ki) in which fluorescent neutrophils can be individually tracked. By using PAF as an inflammatory agent, the analysis of the leukocyte-endothelial cell interac- tions showed a continuous increase in the number of rolling and adherent neutrophils up to 4 hours after the introduction of the inflammatory stimuli, thus confirming the devel- opment of an inflammatory response. As the properties of the red blood cells modulate blood flow properties, erythrocyte deformability was also addressed in this model. A con- tinuous decrease of this parameter was observed throughout time. The decrease in the erythrocyte deformability will most probably lead to an increase in the blood viscosity and to the decrease of the blood flow velocity. These conditions should facilitate the mi- gration of leukocytes from the mainstream to the endothelial wall and promote leukocyte slow rolling and adhesion during the inflammatory response. Importantly, in the intravital microcopy images obtained with this latter model, we clearly observed the deformation of neutrophils along the endothelial wall during rolling, as well as the formation of tethers. As such, in these images, leukocyte trajectories were tracked and their velocities and diameters were measured and further applied to the numerical simulations. Using a recent validated mathematical model describing the coupled defor- mation-flow of an individual leukocyte and the respective experimental results, numerical simulations of the recruitment of an individual leukocyte and of two leukocytes under different velocities were performed, considering a constant blood viscosity. The mathe- matical models obtained showed that under conditions of increased velocity the cell movement is accelerated along the endothelial layer, favouring the dissociation of leuko- cyte-endothelium interactions at designated attraction points. These observations lead us to propose that, in order to attain an efficient inflammatory response, the blood flow ve- locity needs so as to decrease to facilitate slow rolling and subsequent adhesion. These results are corroborated by the decrease in the erythrocyte deformability observed in our animal model, which will ultimately have an impact on the blood flow velocity. Our results further showed that in the vicinity of an adherent leukocyte there is an early slight decel- eration of the rolling leukocyte when compared with the case of an individual leukocyte. As such, these observations strongly suggest that the presence of an adherent cell in the vicinity should decrease the velocity of another leukocyte that is being recruited, thus promoting its slow rolling, and contributing to its capture by the endothelial cells. Altogether, our experimental data and numerical simulations support our working hy- pothesis that the hemodynamic properties of the flow and of the cells in circulation should play an essential role in the margination and rolling of the leukocytes to the endo- thelial wall, which in turn will impact the success of the inflammatory response. In partic- ular, our results strongly suggest that changes in hemodynamic conditions, such as de- creased flow velocities and the increase of the shear stress, will contribute to target leu- kocytes to the endothelial wall. Given our results, we propose that any change in the he- modynamic properties will certainly influence the outcome of the inflammatory response. As such, the adherence of the leukocytes to the endothelium should depend not only on the relative magnitude of the chemical forces generated by the interaction of adhesion molecules between leukocytes and endothelial cells, but also on the physical forces that act on the leukocytes. In this respect, our results suggest that alterations in the blood flow, for example in the flow velocity, will occur during an inflammatory process, thus potentiating the recruitment of more leukocytes towards the inflamed area and contrib- uting to a successful inflammatory response. Overall, the numerical simulations allowed us to better understand the contribution of the hemodynamic properties of the flow to the progression of an inflammatory response and to deepen our knowledge on leukocyte recruitment in inflammation. Importantly, our work provided numerical tools that can be used for the subsequent study and modulation of the hemodynamic parameters involved in an inflammatory response. In particular, these numerical simulations will surely enable us, in the near future, to determine or es- timate a large set of parameters which are unlikely to be recoverable by in vivo experi- ments. Moreover, our methods will allow us to analyze how the parameters evolve over time. Altogether our results further reinforce the notion that the improvement and de- velopment of animal models and numerical tools will certainly provide the medical and biological community with useful tools to study leukocyte recruitment in inflammation. By closely reproducing the microcirculation and the inflammatory process, these tools will be critical for a better comprehension of the inflammatory process and of the mecha- nisms underlying a multitude of inflammatory pathological conditions

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

Erythrocyte as a therapeutic target

© All rights are reserved by Carlota SaldanhaErythrocytes are powerful components of blood flow designed to scavenger or deliver nitic oxide (NO) and oxygen to all cells in the body and transport carbon dioxide from them to the lungs. Blood components started to be quantified and erythrocyte blood shapes used as diagnostic and prognostic tools in clinical practice. Erythrocytes have hemorheological, hemostatic and pro or anti-inflammatory properties enlarging their physiological implications in health and disease. As blood component the erythrocytes establish interaction with others white blood cells, platelets, plasma lipoproteins and vascular endothelial cells. The aim of this mini review is highlight the signaling pathway of nitric oxide in which some steps explain the efficacy of some therapeutic drugs already used and could point new targets for further application in inflammatory vascular diseasesinfo:eu-repo/semantics/publishedVersio

Erythrocyte acetylcholinesterase is a signaling receptor

© All rights are reserved by Carlota SaldanhaThe acetylcholinesterase (AChE), is an enzyme located in the erythrocyte membrane that motivate continually questions, about its physiological function. The aim of this mini review is highlight the receptor behavior of AChE in human red blood cells for chemical signals associated with nitric oxide mobilization and efflux from erythrocytes. Data from the experimental models used are presented. Consequently, key participative proteins in the steps of the signal transduction pathways, in dependence of AChE receptor, are suggested to be further considered as potential therapeutic targets.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

Functional food components, intestinal permeability and inflammatory markers in patients with inflammatory bowel disease

Copyright: © 2021 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https://creativecommons.org/licenses/by/4.0/).Inflammatory bowel diseases (IBD) are characterized by a chronic inflammatory process that affects the intestinal barrier structure. Recent evidence suggests that some food components can influence the integrity of the intestinal barrier and thus its permeability. We aimed at assessing the effect of food components on the intestinal permeability (IP) and on inflammatory markers in individuals with IBD by a single-blind randomized clinical study. Of the 53 individuals included, 47% (n = 25) had been diagnosed with IBD. The participants were divided into 4 groups. IBD patients were allocated to intervention group (n = 14) vs. no intervention group (n = 11), and the same happened with 28 control participants without disease (n = 14 in intervention group vs. n = 14 without intervention). Symptomatology, nutritional status, biochemical parameters (specifically serum zonulin (ZO) to measure IP) were evaluated on all individuals on an eight week period following a diet plan with/without potentially beneficial foods for the IP. At the beginning of the study, there were no significant differences in ZO values between individuals with and without IBD (p > 0.05). The effect of specific food components was inconclusive; however, a trend in the reduction of inflammatory parameters and on the prevalence of gastrointestinal symptomatology was observed. More controlled intervention studies with diet plans, including food components potentially beneficial for the integrity of the intestinal barrier, are of the utmost importance.info:eu-repo/semantics/publishedVersio

Fibrinogen effects of erythrocyte nitric oxide metabolism in presence of timolol

©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 creditedTimolol inhibits erythrocyte membrane acetylcholinesterase (AChE) enzyme activity originates less active enzyme complex forms unable to change the normal values of the nitric oxide (NO) efflux from erythrocytes. The active complex resulting from acetylcholine (ACh) binding to AChE induces higher values of NO efflux. Increased values of NO efflux were also observed in presence of high fibrinogen (Fib) concentration with return to normal levels of NO efflux when ACh is added. Association between NO efflux and erythrocyte deformability values has been evidenced. The objective of this study was to evaluate the effects of high fibrinogen concentration on the erythrocyte NO metabolism and deformability in presence of timolol without and plus adenylyl cyclase or plus guanylyl cyclase inhibitors. Blood samples from healthy donors were divided in aliquots without (control) or with Fib or Fib plus timolol in absence or presence of adenylyl cyclase inhibitor (MDL) or guanylyl cyclase inhibitor (Ly). Erythrocyte deformability (ED), NO efflux, S-nitrosoglutathione (GSNO), nitrite and nitrate values were evaluated by referenced methods. Fib increased the values of NO efflux under the influence of timolol or MDL. Increased values of NO efflux, GSNO, nitrites and nitrates where obtained in presence of Fib plus timolol plus MDL in relation to control to Fib or Fib plus MDL aliquots. Increased nitrites and nitrates values where obtained in presence of Fib plus timolol plus Ly in relation to control, to Fib and Fib plus Ly. Only at high shear stress increased ED values was observed in Fib plus timolol in absence and presence of MDL. In conclusion fibrinogen plus timolol increased ED at high shear stress without or with MDL. Fibrinogen increased NO efflux from erythrocytes and GSNO, nitrite and nitrate levels in presence of timolol plus guanylyl cyclase inhibitor.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

Effects of diabetes on microcirculation and leukostasis in retinal and non-ocular tissues : implications for diabetic retinopathy

© 2020 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).Changes in retinal microcirculation are associated with the development of diabetic retinopathy (DR). However, it is unclear whether such changes also develop in capillary beds of other non-retinal tissues. Here, we investigated microcirculatory changes involving velocity of rolling neutrophils, adherence of neutrophils, and leukostasis during development of retinal vascular lesions in diabetes in other non-retinal tissues. Intravital microscopy was performed on post-capillary venules of cremaster muscle and ear lobe of mice with severe or moderate diabetes and compared to those of non-diabetic mice. Additionally, number and velocity of rolling leukocytes, number of adherent leukocytes, and areas of leukostasis were quantified, and retinal capillary networks were examined for acellular capillaries (AC) and pericyte loss (PL), two prominent vascular lesions characteristic of DR. The number of adherent neutrophils and areas of leukostasis in the cremaster and ear lobe post-capillary venules of diabetic mice was increased compared to those of non-diabetic mice. Similarly, a significant increase in the number of rolling neutrophils and decrease in their rolling velocities compared to those of non-diabetic control mice were observed and severity of diabetes exacerbated these changes. Understanding diabetes-induced microcirculatory changes in cremaster and ear lobe may provide insight into retinal vascular lesion development in DR.info:eu-repo/semantics/publishedVersio

Application of a nitric oxide sensor in biomedicine

© 2014 by the authors; licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution license (http://creativecommons.org/licenses/by/3.0/).In the present study, we describe the biochemical properties and effects of nitric oxide (NO) in intact and dysfunctional arterial and venous endothelium. Application of the NO electrochemical sensor in vivo and in vitro in erythrocytes of healthy subjects and patients with vascular disease are reviewed. The electrochemical NO sensor device applied to human umbilical venous endothelial cells (HUVECs) and the description of others NO types of sensors are also mentioned.This study was supported by grants from the FCT—Fundação para a Ciência e a Tecnologia (project reference EXCL/Mat-NAN/0114/2012).info:eu-repo/semantics/publishedVersio