Carlota Saldanha

Multidisciplinary utilization of dimethyl sulfoxide: pharmacological, cellular, and molecular aspects

DMSO is an amphipathic molecule with a highly polar domain and two apolar methyl groups, making it soluble in both aqueous and organic media. It is one of the most common solvents for the in vivo administration of several water-insoluble substances. Despite being frequently used as a solvent in biological studies and as a vehicle for drug therapy, the side-effects of DMSO (undesirable for these purposes) are apparent from its utilization in the laboratory (both in vivo and in vitro) and in clinical settings. DMSO is a hydrogen-bound disrupter, cell-differentiating agent, hydroxyl radical scavenger, intercellular electrical uncoupler, intracellular low-density lipoprotein-derived cholesterol mobilizing agent, cryoprotectant, solubilizing agent used in sample preparation for electron microscopy, antidote to the extravasation of vesicant anticancer agents, and topical analgesic. Additionally, it is used in the treatment of brain edema, amyloidosis, interstitial cystitis, and schizophrenia. Several systemic side-effects from the use of DMSO have been reported, namely nausea, vomiting, diarrhea, hemolysis, rashes, renal failure, hypertension, bradycardia, heart block, pulmonary edema, cardiac arrest, and bronchospasm. Looking at the multitude of effects of DMSO brought to light by these studies, it is easily understood how many researchers working with DMSO (or studying one of its specific effects) might not be fully aware of the experiences of other groups who are working with it but in a different context.

Evaluation of Lipopolysaccharide Aggregation by Light Scattering Spectroscopy

Lipopolysaccharides (LPS) are cell wall components of Gram‐negative bacteria. These molecules behave as bacterial endotoxins and their release into the bloodstream is a determinant of the development of a wide range of pathologies. These amphipathic molecules can self‐aggregate into supramolecular structures with different shapes and sizes. The formation of these structures occurs when the LPS concentration is higher than the apparent critical micelle concentration (CMCa). Light scattering spectroscopy (both static and dynamic) was used to directly characterize the aggregation process of LPS from Escherichia coli serotype 026:B6. The results point to a CMCa value of 14 μg mL−1 and the existence of premicelle LPS oligomers below this concentration. Both structures were characterized in terms of molecular weight (5.5×106 and 16×106 g mol−1 below and above the CMCa, respectively), interaction with the aqueous environment, gyration radius (56 and 105 nm), hydrodynamic radius, (60 and 95 nm) and geometry of the supramolecular structures (nearly spherical). Our data indicates that future in vitro experiments should be carried out both below and above the CMCa. The search for drugs that interact with the aggregates, and thus change the CMCa and condition LPS interactions in the bloodstream, could be a new way to prevent certain bacterial‐endotoxin‐related pathologies.

An overview about erythrocyte membrane

In the sixties and seventies, erythrocytes or red blood cells (RBCs) were extensively studied. Much has been learnt particularly concerning their metabolism and gas transporter function.In the past decade, the use of new approaches and methodologies, such as proteomic analysis, has contributed for a renewed interest on the erythrocyte. Recent studies have provided us with a more detailed and comprehensive picture on the composition and organization of its cellular membrane that will be the main subject of this minireview. Unexpectedly, it has been recognized that this cell expresses several adhesion molecules on its surface, like other cellular types such blood circulating cells or endothelial cells. Taking into consideration the cellular functions of the erythrocyte, the clarification of the role of those adhesion molecules may in the future open new horizons for the biological significance of this cellular player.

Modulation of erythrocyte deformability by PKC activity

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.

Expression and Subcellular Localization of a Novel Nuclear Acetylcholinesterase Protein

Acetylcholine is found in the nervous system and also in other cell types (endothelium, lymphocytes, and epithelial and blood cells), which are globally termed the non-neuronal cholinergic system. In this study we investigated the expression and subcellular localization of acetylcholinesterase (AChE) in endothelial cells. Our results show the expression of the 70-kDa AChE in both cytoplasmic and nuclear compartments. We also describe, for the first time, a nuclear and cytoskeleton-bound AChE isoform with ∼55 kDa detected in endothelial cells. This novel isoform is decreased in response to vascular endothelial growth factor via the proteosomes pathway, and it is down-regulated in human leukemic T-cells as compared with normal T-cells, suggesting that the decreased expression of the 55-kDa AChE protein may contribute to an angiogenic response and associate with tumorigenesis. Importantly, we show that its nuclear expression is not endothelial cell-specific but also evidenced in non-neuronal and neuronal cells. Concerning neuronal cells, we can distinguish an exclusively nuclear expression in postnatal neurons in contrast to a cytoplasmic and nuclear expression in embryonic neurons, suggesting that the cell compartmentalization of this new AChE isoform is changed during the development of nervous system. Overall, our studies suggest that the 55-kDa AChE may be involved in different biological processes such as neural development, tumor progression, and angiogenesis.

Hemorheological parameters are related to subclinical atherosclerosis in systemic lupus erythematosus and rheumatoid arthritis patients

OBJECTIVES Rheological characteristics of blood are strongly linked to atherothrombosis in the general population, but its contribution to atherosclerosis in systemic lupus erythematosus (SLE) and rheumatoid arthritis (RA) is currently unclear. This work examines the relationship between blood rheology, traditional cardiovascular (CV) risk factors, inflammation and subclinical atherosclerosis in SLE and RA. METHODS Whole blood viscosity (WBV), plasma viscosity (PV), erythrocyte deformability (ED), aggregation (EA) and erythrocyte NO production were measured in 197 patients (96 SLE and 101 RA) and compared to 97 controls, all females without previous CV events. Clinical information was obtained and fasting lipids and acute phase reactants were measured. The relationship between hemorheological parameters, CV risk factors and inflammation was assessed in patients and the impact of these variables on carotid intima-media thickness (cIMT) was evaluated in univariate followed by multivariate regression analyses. RESULTS WBV and ED are significantly lower in patients, while EA is elevated as compared with controls. Hemorheological disturbances correlate with CV risk factors and markers of inflammation and are more profound in patients with metabolic syndrome. Multivariable analysis showed that menopause (OR 34.72, 95%CI 4.44-271.77), obesity (OR 4.09, 95%CI 1.00-16.68) and WBV (OR 3.98; 95%CI 1.23-12.83) are positively associated whereas current corticosteroid dose (OR 0.87; 95%CI 0.78-0.98), and erythrocyte NO production (OR 0.16; 95%CI 0.05-0.52) are negatively associated with cIMT. CONCLUSION Disturbed hemorheological parameters in SLE and RA women are related to the presence of CV risk factors and inflammation. WBV and erythrocyte NO are independently associated with the early stages of atherosclerosis.

Integrin-associated protein (CD47) is a putative mediator for soluble fibrinogen interaction with human red blood cells membrane

Fibrinogen is a multifunctional plasma protein that plays a crucial role in several biological processes. Elevated fibrinogen induces erythrocyte hyperaggregation, suggesting an interaction between this protein and red blood cells (RBCs). Several studies support the concept that fibrinogen interacts with RBC membrane and this binding, due to specific and non-specific mechanisms, may be a trigger to RBC hyperaggregation in inflammation. The main goals of our work were to prove that human RBCs are able to specifically bind soluble fibrinogen, and identify membrane molecular targets that could be involved in this process. RBCs were first isolated from blood of healthy individuals and then separated in different age fractions by discontinuous Percoll gradients. After isolation RBC samples were incubated with human soluble fibrinogen and/or with a blocking antibody against CD47 followed by fluorescence confocal microscopy, flow cytometry acquisitions and zeta potential measurements. Our data show that soluble fibrinogen interacts with the human RBC membrane in an age-dependent manner, with younger RBCs interacting more with soluble fibrinogen than the older cells. Importantly, this interaction is abrogated in the presence of a specific antibody against CD47. Our results support a specific and age-dependent interaction of soluble fibrinogen with human RBC membrane; additionally we present CD47 as a putative mediator in this process. This interaction may contribute to RBC hyperaggregation in inflammation.

Non-neuronal cholinergic system and signal transduction pathways mediated by band 3 in red blood cells

BACKGROUND Non-neuronal acetylcholine (ACh) and acetylcholinesterase (AChE) have been recognized in the past. Vascular ACh has been associated by us with the regulation of microcirculatory flow by modulating nitric oxide (NO) intracellular mobilization, metabolism (NOx) and release from erythrocytes, as well as the glycolytic flux. Velnacrine maleate is a well-known AChE inhibitor which plays a competitive role by decreasing NO-mediated erythrocyte responses. A plausible hypothesis to explain the mechanisms underlying those events hinges on the NO translocation among nitrosylated molecules and phosphorylated/dephosphorylated states of band 3 protein, processed by major tyrosine-kinases (PTK: p72syk, p53/56lyn and p59/61hck) and phosphotyrosine-phosphatases (PTP). METHODS To assess this hypothesis under the influence of AChE effectors (acetylcholine/velnacrine), blood samples from healthy donors were harvested and Western blot analysis was subsequently used to determine the degree of band 3 phosphorylation, in the presence and absence of PTK/PTP inhibitors. NO and nitrites/nitrates were quantified using an amperometric method and the Griess Reaction, respectively, in erythrocyte suspensions. Measurements of erythrocyte metabolites (2,3-bisphosphoglycerate; glyceraldehyde 3-phosphate dehydrogenase; glucose-6-phosphodehydrogenase; lactate), hemoglobin and cyclic nucleotides were conducted afterwards. RESULTS Increased levels of phosphorylated-band 3 obtained upon p72syk inhibition suggest p59/61hck and p53/56lyn as secondary involved kinases. As to NO/NOx quantification, in the presence of PTKi we reported higher levels with velnacrine-AChE, as opposed to acetylcholine-AChE. Calpeptin, a PTP inhibitor which triggers full band 3-phosphorylation, led to the opposite NO mobilization, being reinforced by ACh. Oxy-hemoglobin, glyceraldehyde 3-phosphate dehydrogenase and glucose-6-phosphodehydrogenase were found to decrease with ACh, whereas P50, lactate and both cGMP/cAMP happened to increase. CONCLUSION Changes on human erythrocyte NOx mobilization and metabolic fluxes occur under influence of non-neuronal ACh/AChE, in turn dependent on the degree of band 3-phosphorylation. Since these vascular events may potentially change under pathological conditions, coadjuvant drugs could become accessible in the setting of microcirculation disease.

Red blood cell membrane integrity in primary open angle glaucoma: ex vivo and in vitro studies

Purpose There is increasing evidence that abnormal perfusion of the optic nerve head is an important factor involved in the pathophysiology of glaucoma. Transport and distribution of oxygen to the tissues takes place through the erythrocyte membrane. Red blood cell (RBC) acetylcholinesterase (AChE) is a marker of RBC membrane integrity. The aim of this study was to find out whether RBC membrane integrity is preserved in primary open angle glaucoma (POAG), and whether it is modified by the use of topical timolol maleate and pilocarpine.Method RBC AChE activity was determined ex vivo by Kaplans spectrophotometric method in 19 POAG patients undergoing topical treatment for glaucoma with timolol, pilocarpine or a combination of the two drugs, and compared with that in 20 controls. To assess the effect of antiglaucomatous therapy in our findings, we carried out an in vitro study in 26 non-glaucomatous patients in which we measured RBC AChE activity after incubation of blood with either timolol maleate, pilocarpine or a combination of the two drugs, using the same spectrophotometric method.Results There was a significant increase in RBC AChE enzyme activity in POAG patients compared with the control group (p < 0.002). However, timolol and pilocarpine, individually or in combination, have the opposite effect, significantly decreasing RBC AChE activity (p < 0.05).Conclusion This change in RBC AChE enzyme activity could suggest alterations in RBC membrane integrity in primary open angle glaucoma. Whether or not this finding has implications regarding the microcirculation at the optic nerve head needs to be investigated further.

Erythrocyte deformability - A partner of the inflammatory response.

We aim to establish an in vivo animal model of acute inflammation using PAF (platelet activating factor) as inflammatory agent and to study the erythrocyte deformability changes induced by the inflammatory response. Counting the number of rolling and adherent neutrophils to endothelium after 2, 4 and 6h of intrascrotal injection of PAF we showed the induction of an inflammatory state. Blood samples are collected in order to measure the erythrocyte deformability and to quantify NO efflux from the red blood cells (RBCs). The results show an increased number of rolling and adherent neutrophils after 2h and 4h of inflammation as well as decreased values of erythrocyte deformability in the same time-points. This result is in line with the need of a low blood viscosity to the recruitment process that will improve leukocyte migration towards the endothelial wall. NO efflux from RBCs is also affected by the inflammatory response at the first hours of inflammation. This animal model demonstrates in vivo the association between an acute inflammatory response and the rheological properties of the blood, namely the RBCs deformability. For those reasons we consider this as an adequate model to study acute inflammatory responses as well as hemorheological parameters.