Longitudinal SARS-CoV-2 seroprevalence in Portugal and antibody maintenance 12 months after infection.

During the COVID-19 pandemic, Portugal has experienced three distinct SARS-CoV-2 infection waves. We previously documented the prevalence of SARS-CoV-2 immunity, measured by specific antibodies, in September 2020, 6 months after the initial moderate wave. Here, we show the seroprevalence changes 6 months later, up to the second week of March 2021, shortly following the third wave, which was one of the most severe in the world, and 2 months following the start of the vaccination campaign. A longitudinal epidemiological study was conducted, with a stratified quota sample of the Portuguese population. Serological testing was performed, including ELISA determination of antibody class and titers. The proportion of seropositives, which was 2.2% in September 2020, rose sharply to 17.3% (95% CI: 15.8-18.8%) in March 2021. Importantly, circulating IgG and IgA antibody levels were very stable 6 months after the initial determination and up to a year after initial infection, indicating long-lasting infection immunity against SARS-CoV-2. Moreover, vaccinated people had higher IgG levels from 3 weeks post-vaccination when compared with previously infected people at the same time post-infection.

Association between miR-146a and Tumor Necrosis Factor Alpha (TNF-α) in Stable Coronary Artery Disease.

Background and Objectives: Tumor necrosis factor alpha (TNF-α) is proatherogenic and associated with the risk of acute ischemic events, although the mechanisms that regulate TNF-α expression in stable coronary artery disease (SCAD) are not fully understood. We investigated whether metabolic, inflammatory, and epigenetic (microRNA (miRNA)) markers are associated with TNF-α expression in SCAD. Materials and Methods: Patients with SCAD were prospectively recruited and their metabolic and inflammatory profiles were assessed. TNF-α levels were assessed using an enzyme-linked immunosorbent assay. The relative expression of six circulating miRNAs associated with the regulation of inflammation and/or atherosclerosis was determined. Results: Of the 24 included patients with the mean age of 65 (9) years, 88% were male, and 54% were diabetic. The TNF-α levels were (median (interquartile range)) 1.0 (0.7-1.1) pg/mL. The percentage of glycosylated hemoglobin (r = 0.418, p = 0.042), serum triglyceride levels (r = 0.429, p = 0.037), and C-reactive protein levels (r = 0.407, p = 0.048) were positively correlated with TNF-α levels. Of the candidate miRNAs, miR-146a expression levels were negatively correlated with TNF-α levels (as indicated by r = 0.500, p = 0.035 for correlation between delta cycle threshold (ΔCt) miR-146a and TNF-α levels). In multivariate analysis, serum triglyceride levels and miR-146a expression levels were independently associated with TNF-α levels. miR-146 expression levels were not associated with metabolic or other inflammatory parameters and were negatively correlated with the number of coronary vessels with obstructive disease (as indicated by r = 0.556, p = 0.017 for correlation between ΔCt miR-146a and number of diseased vessels). Conclusions: miR-146a expression levels were negatively correlated with TNF-α levels in patients with SCAD, irrespective of other metabolic or inflammatory markers, and with the severity of coronary artery disease. The results add to the knowledge on the role of miR-146a in TNF-α-based inflammation in SCAD and support future research on the potential therapeutic use of miR-146a in such a clinical scenario.

Cigarette Smoking, miR-27b Downregulation, and Peripheral Artery Disease: Insights into the Mechanisms of Smoking Toxicity.

Cigarette smoking is a risk factor for the development of peripheral artery disease (PAD), although the proatherosclerotic mediators of cigarette smoking are not entirely known. We explored whether circulating microRNAs (miRNAs) are dysregulated in cigarette smokers and associated with the presence of PAD. Ninety-four participants were recruited, including 58 individuals without and 36 with PAD, 51 never smokers, 28 prior smokers, and 15 active smokers. The relative expression of six circulating miRNAs with distinct biological roles (miR-21, miR-27b, miR-29a, miR-126, miR-146, and miR-218) was assessed. Cigarette smoking was associated with the presence of PAD in multivariate analysis. Active smokers, but not prior smokers, presented miR-27b downregulation and higher leukocyte, neutrophil, and lymphocyte counts; miR-27b expression levels were independently associated with active smoking. Considering the metabolic and/or inflammatory abnormalities induced by cigarette smoking, miR-27b was independently associated with the presence of PAD and downregulated in patients with more extensive PAD. In conclusion, the atheroprotective miR-27b was downregulated in active smokers, but not in prior smokers, and miR-27b expression was independently associated with the presence of PAD. These unreported data suggest that the proatherogenic properties of cigarette smoking are mediated by a downregulation of miR-27b, which may be attenuated by smoking cessation.

Circulating miRNAs Are Associated with the Systemic Extent of Atherosclerosis: Novel Observations for miR-27b and miR-146.

The mechanisms that regulate the systemic extent of atherosclerosis are not fully understood. We investigated whether the expression of circulating miRNAs is associated with the extent of stable atherosclerosis to a single territory or multiple territories (polyvascular) and with the severity of atherosclerosis in each territory. Ninety-four participants were prospectively recruited and divided into five age- and sex-matched groups: presenting no atherosclerosis, isolated coronary atherosclerosis, coronary and lower extremity atherosclerosis, coronary and carotid atherosclerosis, and atherosclerosis of the coronary, lower extremity, and carotid territories. The expression of six circulating miRNAs with distinct biological roles was assessed. The expression of miR-27b and miR-146 differed across groups (p < 0.05), showing a decrease in the presence of atherosclerosis, particularly in the three territories. miR-27b and miR-146 expression decreased in association with a higher severity of coronary, lower extremity, and carotid atherosclerosis. Polyvascular atherosclerosis involving the three territories was independently associated with a decreased miR-27b and miR-146 expression. Both miRNAs presented an area under the curve of ≥0.75 for predicting polyvascular atherosclerosis involving the three territories. To conclude, miR-27b and miR-146 were associated with the presence of severe polyvascular atherosclerosis and with the atherosclerosis severity in each territory. Both are potential biomarkers of severe systemic atherosclerosis.

The Proinflammatory Soluble CD40 Ligand Is Associated with the Systemic Extent of Stable Atherosclerosis.

Background and objectives: Polyvascular atherosclerosis is frequent and associated with a high cardiovascular risk, although the mechanisms regulating the atherosclerosis extent to single or multiple arterial territories are still poorly understood. Inflammation regulates atherogenesis and soluble CD40 ligand (sCD40L) is an inflammatory mediator associated with the presence of single-territorial atherosclerosis. We assessed whether the sCD40L expression is associated with the atherosclerosis extent to single or multiple arterial territories and with the atherosclerosis severity in different territories. Materials and Methods: We prospectively enrolled 94 participants with no atherosclerosis (controls, n = 26); isolated coronary atherosclerosis (group 1, n = 20); coronary and lower extremity (LE) atherosclerosis (group 2, n = 18); coronary and carotid atherosclerosis (group 3, n = 12); and coronary, LE, and carotid atherosclerosis (group 4, n = 18). Serum sCD40L levels were quantified. Results: The sCD40L levels (ng/mL, mean (standard deviation)) were 4.0 (1.5), 5.6 (2.6), 7.2 (4.2), 5.9 (3.7), and 5.1 (2.4) in controls and groups 1 to 4, respectively (ANOVA p = 0.012). In nonrevascularized patients, the sCD40L levels were significantly higher in group 2 than in group 1 and were correlated with the number of LE diseased segments. Prior LE bypass surgery was associated with lower sCD40L levels. Coexistence of coronary and LE atherosclerosis was independently associated with the sCD40L levels. Conclusions: The sCD40L levels were increased in stable atherosclerosis, particularly in polyvascular coronary and LE atherosclerosis. The number of LE diseased segments and prior LE revascularization were associated with sCD40L expression. To our knowledge, these are novel data, which provide insights into the mechanisms underlying multi-territorial atherosclerosis expression. sCD40L may be a promising noninvasive tool for refining the stratification of the systemic atherosclerotic burden.

Soluble CD40 ligand expression in stable atherosclerosis: A systematic review and meta-analysis.

BACKGROUND AND AIMS: The role of inflammation in atherosclerosis development and expression in different arterial territories is unclear. Soluble CD40 ligand (sCD40L) mediates inflammation and atherogenesis. Through a systematic review and meta-analysis, we assessed whether sCD40L was dysregulated in stable atherosclerosis, irrespective of the diseased arterial territory, and whether this dysregulation differed according to the specific territory. METHODS: Systematic literature searches were performed in MEDLINE, Cochrane Library, Web of Science, and Embase for studies reporting circulating sCD40L levels in individuals with and without stable atherosclerosis. sCD40L levels were compared using random-effects meta-analysis, weighted by the inverse variance method (study protocol: PROSPERO CRD42020181392). RESULTS: Fifty-four studies (59 estimates) including 7705 patients and 7841 controls were analyzed. sCD40L levels were found to be increased in patients with atherosclerosis, irrespective of the territory (standardized mean difference [SMD] 0.43, 95% CI 0.29-0.57; 59 estimates; χ2 heterogeneity p < 0.001; I2 = 92%). SMD was greatest in carotid atherosclerosis (SMD 0.58, 95% CI 0.30-0.86; 17 estimates), followed by coronary (SMD 0.43, 95% CI 0.24-0.62; 33 estimates), lower extremity (SMD 0.26, 95% CI -0.02-0.54; 7 estimates), and renal atherosclerosis (SMD -0.07, 95% CI -2.77-2.64; 2 estimates) (χ2 heterogeneity p < 0.001; I2 ≥ 80% for all). Subgroup analysis revealed that sCD40L levels were increased in clinical, but not subclinical, atherosclerosis. CONCLUSIONS: sCD40L levels were increased in stable atherosclerosis, particularly in the carotid and coronary territories. These novel data support sCD40L as a marker of systemic atherosclerosis, possibly with differential roles in specific territories.

Inflammation is associated with the presence and severity of chronic coronary syndrome through soluble CD40 ligand.

INTRODUCTION: Inflammation contributes to the initiation and progression of atherosclerosis, although the underlying inflammatory pathways are not entirely known. Specifically, the role of the proinflammatory soluble CD40 ligand (sCD40L) on the expression of chronic coronary syndrome (CCS) is not completely understood. We evaluated whether sCD40L expression is associated with the presence of CCS and with the clinical and anatomical severity of CCS. METHODS: We prospectively recruited 94 participants, assigned to two groups matched by age and sex, without coronary artery disease (n=26) and with CCS (n=68). Clinical, laboratory and anatomical data were prospectively collected, and serum levels of sCD40L were measured. RESULTS: In patients with CCS, classic cardiovascular risk factors were more prevalent, and the sCD40L levels, leukocyte and neutrophil counts, and neutrophil/lymphocyte ratio, but not the C-reactive protein levels, were significantly higher than those in controls. sCD40L was independently associated with the presence of obstructive coronary artery disease in multivariate analysis. Regarding CCS severity, sCD40L levels showed a significant stepwise increase with increasing angina severity (ANOVA P=0.001). In addition, sCD40L was independently associated with the anatomical severity of coronary artery disease, as assessed by the Gensini score. Among patients with CCS, those with previous coronary artery bypass grafting (n=23) had lower sCD40L levels than patients waiting for revascularization (n=45) [4.3 (2.1) ng/mL vs. 6.8 (3.5) ng/mL, P=0.001]. CONCLUSIONS: The expression of the proinflammatory sCD40L was associated with the presence of CCS and reflected the clinical and anatomical severity of CCS. In addition, we describe for the first time the association between prior CABG and reduced sCD40L levels in patients with CCS.

Myocardial infarction before and after the age of 45: Possible role of platelet receptor polymorphisms.

INTRODUCTION: We examined the potential role of polymorphisms of the platelet genes GP1BA (rs2243093, rs6065 and VNTR), ITGB3 (rs5918), ITGA2 (rs938043469) and P2RY12 (rs2046934, rs6801273 and rs6798347) as risk factors for myocardial infarction (MI). METHODS: The study population was divided into three groups: controls (n=235), MI at age ≤45 years (MI ≤45, n=44), and MI at age >45 years (MI >45, n=78). The control group was further divided into two subgroups (control ≤45 and >45), and subgroups including only men were also considered for statistical analysis. Polymorphisms were detected by polymerase chain reaction and restriction fragment length polymorphism analysis. RESULTS: Regarding non-genetic risk factors, the control group differed statistically from the MI ≤45 group (p<00.5) in terms of smoking, hypertension, diabetes and obesity, and from the MI >45 group (p<0.05) in terms of hypertension, diabetes, obesity, family history of thrombosis and high cholesterol. For the studied ITGA2 polymorphism, a statistical difference was found when MI >45 was compared with the control group, with a higher risk of MI in the TT genotype (OR 2.852; 95% CI: 1.092-7.451; p=0.032). In the GP1BA rs6065 polymorphism, a statistically significant difference was found between control ≤45 only men and MI ≤45 only men, with a higher risk in the CT genotype (OR 5.568; 95% CI: 1.421-21.822; p=0.016), despite the low numbers included. The other polymorphisms studied did not show any statistically significant correlations. CONCLUSION: There is a statistically significant association between the TT genotype of the ITGA2 rs938043469 polymorphism and increased risk for MI >45.

Circulating microRNA profiles in different arterial territories of stable atherosclerotic disease: a systematic review.

AIMS: Atherosclerosis is associated with altered circulating microRNA profiles. It is yet unclear whether the expression of these potential biomarkers differs according to the location of atherosclerosis. We assessed whether atherosclerosis of different arterial territories, except the coronary, is associated with specific circulating microRNA profiles. METHODS: A systematic search in PubMed, Web of Science, Embase, and Cochrane Library was carried out using a retrieval strategy including MESH and non-MSH terms. Eligible studies have compared circulating microRNA profiles between individuals with and without stable atherosclerotic disease of large or medium size arteries. The review protocol was registered in PROSPERO database (reference CRD42017073846). RESULTS: Eighteen studies were selected for qualitative synthesis: ten focused on carotid, six on lower limbs, and two on renal arteries atherosclerosis, none reporting on other locations. A common microRNA profile to different atherosclerotic disease locations was identified, including deregulation of miR-21, miR-30, miR-126, and miR-221-3p. Specific microRNA profiles for each territory were also identified, with consistency across studies, such as deregulation of miR-21 and miR-29 in carotid atherosclerosis, and let 7e, miR-27b, miR-130a, and miR-210 in lower limbs atherosclerosis. The robustness of the results was very high for let 7e, miR-29, miR-30, considering both the adjustment of microRNA expression for baseline variables and the replication of results in different studies (miR-29 in carotid, let 7e in lower limbs, and miR-30 in carotid and lower limbs atherosclerosis). Globally, the deregulated microRNAs are associated with control of angiogenesis, endothelial cell function, inflammation, cholesterol metabolism, oxidative stress and extracellular matrix composition. CONCLUSIONS: A common microRNA profile to different atherosclerotic disease locations and specific microRNA profiles for each territory were identified. These findings may provide insights into pathophysiology and be useful for selecting potential biomarkers for clinical practice. To the best of our knowledge, no systematic data on this subject has been reported.

Erythrocyte nitric oxide in glaucoma patients - ex vivo study.

BACKGROUND: Glaucoma is an optic neuropathy associated with vascular dysregulation and increased intra-ocular pressure (IOP). Timolol is used as treatment for reducing IOP, by limiting aqueous humour production. Increased NOS expression as well as decreased levels of nitric oxide (NO) metabolites, and high activity of erythrocyte acetylcholinesterase (AChE) were observed in primary open angle glaucoma patients. OBJECTIVE: This ex vivo study aims to evaluate timolol effect in NO efflux and its derivatives in glaucoma patient's erythrocytes. METHODS: Venous blood from 15 glaucoma patients was collected. Erythrocyte suspensions were incubated with the AChE modulators acetylcholine (ACh) and timolol at 10 µM. Erythrocyte NO efflux and S-nitrosoglutathione (GSNO) concentration were measured. RESULTS: No significant differences were obtained in erythrocyte NO efflux and GSNO concentration in response to ACh or timolol when compared with the untreated erythrocytes of glaucoma patients. When comparing the same incubation conditions for erythrocyte suspensions between glaucoma patients and healthy subjects, those from glaucoma patients showed higher NO efflux in presence and absence of timolol, and higher values of GSNO in the presence of timolol. CONCLUSIONS: We demonstrated that erythrocytes from glaucoma patients have similar availability to release NO both in absence and presence of timolol, and have higher GSNO values in presence of timolol.

An ex vivo study of nitric oxide efflux from human erythrocytes in both genders.

INTRODUCTION: Acetylcholinesterase (AChE) is located on outer surface of erythrocyte membrane. Gender-related differences in erythrocyte AChE enzyme activity had been verified in young adults. It is also known that binding of acetylcholine (ACh) with AChE on erythrocyte membrane initiates a signal transduction mechanism that stimulates nitric oxide (NO) efflux. AIMS: This ex vivo study was done to compare the amount of NO efflux obtained from erythrocytes of healthy donors in males and females. METHODS: We included 66 gender age-matched healthy donors (40-60 years old). We performed quantification of erythrocyte NO efflux from erythrocytes and of the membrane AChE enzyme activity. RESULTS: There are no significant differences in NO efflux from erythrocytes between men and women. Regarding AChE enzyme activity values, in this range of age, no differences between genders were obtained. However, the values of AChE enzyme activity in the third quartile of NO efflux values were significantly higher (p < 0.05) in women than in men. CONCLUSIONS: The efflux of NO from erythrocyte of healthy humans did not change with gender. For the same range of values of NO efflux from erythrocytes, in both gender, it was verified higher values of AChE enzyme activity in women.

Effect of oxidized LDL on erythrocyte nitric oxide metabolism.

AIMS: Oxidized low density lipoprotein (ox-LDL) has been reported as an inhibitor of nitric oxide (NO)-mediated dilatation in microcirculation. Oxidized LDL effect on NO metabolism of erythrocytes is not known. Therefore, this study aims to evaluate the effect of ox-LDL on erythrocytes NO metabolism. METHODS: The effect of different concentrations of human purified ox-LDL (25, 50 and 100 µg/mL) on NO metabolism was evaluated on blood of healthy subjects. RESULTS: An inhibitory effect of higher concentrations of ox-LDL on erythrocyte NO efflux levels was verified. Concentrations of NO efflux from erythrocytes were lower as consequence of treatments with 50 µg/mL ox-LDL treatment (1.6±0.27 nM) and 100 µg/mL ox-LDL treatment (1.3±0.22 nM) than control (1.9±0.28 nM). Opposite, ox-LDL incubation has a positive effect on GSNO content of erythrocytes. That effect is proportional to concentrations of ox-LDL treatments (10.8±1.4 nM for 25 µg/mL, 12.9±1.5 nM for 50 µg/mL and 12.1±1.9 nM for 100 µg/mL) and is significant relative to control (8.56±0.76 µM) and ACh (8.9±0.52 µM) aliquots. CONCLUSIONS: Presence of oxidized LDL in erythrocyte NO metabolism induces a decrease of NO efflux amount and an increase on intra-erythrocyte GSNO concentrations. These results suggest a role of ox-LDL in mobilization of NO between NO derivatives molecules in dependence of oxidized LDL concentration. An anti - reactive nitrogene role can be attributed to ox-LDL for its contribution in the erythrocyte scavenged ability for nitric oxide.

Soluble CD40 ligand profiles in patients with septic shock.

AIM: Soluble CD40 ligand (sCD40L) has been considered as a marker of thrombosis and inflammation in several diseases, including sepsis. Recent studies challenge this view and point to a role of sCD40L in vascular and endothelial function. An indication of that association in sepsis has not been obtained so far. Therefore, herein we evaluated association between sCD40L and markers of hemorheology and inflammation on context of septic shock. METHODS: Time-changes of sCD40L levels over 72 hours of Intensive Care Unit (ICU) internment were assessed in 22 patients with septic shock and compared with 36 healthy volunteers. Association of sCD40L levels with erythrocyte deformability and aggregation (as markers of hemorheology), plasma concentrations of haemoglobin (Hb, as markers of endothelial function) and white blood cells (WBC) count (as marker of low-grade inflammation) were assessed in patients with septic shock. RESULTS: At ICU admission, sCD40L concentrations in patients with septic shock were lower (p = 0.024) than levels of healthy volunteers. However, sCD40L did not change over 72 hours of internment (F = 2.1, p = 0.137). Soluble CD40L levels in patients with septic shock at ICU admission correlate with concentrations of Hb (r = 0.61, p = 0.00) and WBC count (r = 0.63, p = 0.00), but not to erythrocyte deformability (r≥0.157, p≤0.235) and aggregation (r≥-0.109, p≤0.192). CONCLUSIONS: These results seem to highlight a possible association of sCD40L to endothelial function and inflammation in septic shock context.

Identification of erythrocyte biomarkers in amyotrophic lateral sclerosis.

INTRODUCTION: Amyotrophic Lateral Sclerosis (ALS) is a fatal neurodegenerative disease of the motor system. It has been hypothesised that red blood cells (RBCs) may be involved in the disease process by the release of damaging molecules. OBJECTIVE: The aim of this ex vivo study is to compare RBCs biochemical and hemorheological parameters between ALS patients and healthy donors to identify novel biomarkers of the ALS disease. METHODS: We included 82 ALS patients and 40 gender age-matched healthy donors. We performed quantification of erythrocyte aggregation and deformability, nitric oxide (NO) efflux from RBCs, acetylcholinesterase (AChE) enzyme activity and intraerythrocytic concentration of nitrite, nitrate and S-nitrosogluthatione (GSNO). RESULTS: Erythrocyte deformability and AChE activity were increased in patients with ALS in comparison to healthy donors. NO efflux from RBCs and concentration of intraerythrocytic nitrite were lower in ALS patients. In patients, we found that for higher NO range of values the respiratory function is worse and that for higher AChE range of values the RBCs nitrite content increase. CONCLUSION: The results of the present study indicate that NO efflux from RBCs and RBCs AChE should be further explored as potential biomarkers for ALS.

Stratification of ST-elevation myocardial infarction patients based on soluble CD40L longitudinal changes.

Involvement of soluble CD40 ligand (sCD40L) in thrombosis and inflammation on the context of coronary artery disease is currently being revised. In that perspective, we had studied the association of sCD40L with markers of platelet activation and markers of endothelial and vascular function. On that cohort, a stratification of patients with acute myocardial infarction (AMI) 1 month after percutaneous coronary intervention (PCI) was observed based on concentrations of sCD40L. The study intended to identify the groups of AMI patients with different profiles of sCD40L concentrations and verify how medication, clinical evolution, biochemical data, and markers of regulation of endothelial function at genetic (endothelial nitric oxide synthase polymorphisms) and post-transcriptional levels (circulating microRNAs) affect sCD40L serum levels. Lower quartiles of sCD40L (<2.3 ng/mL) were associated with higher concentrations of N-terminal pro-brain natriuretic peptide (NT-proBNP), high frequency of G894T polymorphism, and altered expression of a set of microRNAs assumed to be involved in the regulation of endothelial and cardiac function and myocardium hypertrophy, relative to patients in sCD40L upper quartiles. A characteristic sCD40L variation pattern in STEMI patients was identified. Low levels of sCD40L 1 month after PCI distinguish STEMI patients with worse prognosis, a compromised cardiac healing, and a persistent endothelial dysfunction, as given by the association between sCD40L, NT-proBNP, G894T polymorphism, and specific profile of miRNA expression. These results suggest sCD40L could have a prognostic value in STEMI patients.

Changes of soluble CD40 ligand in the progression of acute myocardial infarction associate to endothelial nitric oxide synthase polymorphisms and vascular endothelial growth factor but not to platelet CD62P expression.

Reported in vitro data implicated soluble CD40 ligand (sCD40L) in endothelial dysfunction and angiogenesis. However, whether sCD40L could exert that influence in endothelial dysfunction and angiogenesis after injury in acute myocardial infarction (AMI) patients remains unclear. In the present study, we evaluated the association of sCD40L with markers of platelet activation, endothelial, and vascular function during a recovery period early after AMI. To achieve this goal, the time changes of soluble, platelet-bound, and microparticle-bound CD40L levels over 1 month were assessed in AMI patients and correlated with endothelial nitric oxide synthase (eNOS) polymorphisms, vascular endothelial growth factor (VEGF) concentrations, and platelet expression of P-selectin (CD62P). The association of soluble form, platelet-bound, and microparticle-bound CD40L with CD62P expression on platelets, a marker of platelet activation, was also assessed to evaluate the role of CD40L in the thrombosis, whereas the association with eNOS and VEGF was to evaluate the role of CD40L in vascular dysfunction. This work shows for the first time that time changes of sCD40L over 1 month after myocardial infarct onset were associated with G894T eNOS polymorphism and with the VEGF concentrations, but not to the platelet CD62P expression. These results indicate that, in terms of AMI pathophysiology, the sCD40L cannot be consider just as being involved in thrombosis and inflammation but also as having a relevant role in vascular and endothelial dysfunction.

Acetylcholinesterase conformational states influence nitric oxide mobilization in the erythrocyte.

In the human erythrocyte, band 3 protein mediates nitric oxide (NO) translocation and its effects are strongly related to phosphorylated/dephosphorylated intracellular states. The metabolism of NO could change in the presence of acetylcholinesterase (AChE). Therefore, the present study was designed to assess the effect of conformational changes in AChE (via N-19 and C-16 antibodies) and enzymatic inhibition/activation of protein kinase C (PKC) in erythrocyte NO mobilization in vitro. Our results show that by inhibiting PKC with cheletrine, impaired erythrocyte NO efflux and s-nitrosoglutathione (GSNO) levels were verified, while PKC's activation by Phorbol 12-myristate 13-acetate had the opposite effect. Those results demonstrate the influence of 4.1R complex and band 3 protein level of phosphorylation on NO efflux and GSNO concentration mediated by PKC inhibition/activation. In addition, the present study shows evidence that conformational changes in AChE promoted by incubation with N-19 and C-16 antibodies alter the enzyme's functional connection to acetylcholine (ACh) (AChE-ACh complex) in an irreversible manner, resulting in impaired GSNO concentration and NO efflux from the erythrocyte. Novel insight into NO metabolism in the erythrocyte is brought with the presented findings allowing new possibilities of modulating NO delivery, possibly involving PKC and AChE conformational alterations in combination.

S-nitrosoglutathione efflux in the erythrocyte.

Glutathione is an abundant molecule inside erythrocyte, originating S-nitrosoglutathione (GSNO) by reacting with nitric oxide (NO). GSNO has been regarded as a store and transporter of NO, with significant interest as a potential therapeutic agent, acting as an NO donor.NO metabolism inside the erythrocyte generates several derivatives, which can be altered by external and internal stimuli such as acetylcholine (ACh), a natural substrate of acetylcholinesterase (AChE). In spite of the knowledge gained in the last decades concerning NO efflux in erythrocytes little is known regarding erythrocyte GSNO efflux, which has also a significant role in microcirculation. Hence, the objective of this research was to evaluate the efflux of GSNO, concomitant with the efflux of NO, after stimulation with AChE effectors. To achieve these goals, the in vitro effect of AChE modulators - ACh and timolol - in erythrocyte NO and GSNO were studied. Timolol is an erythrocyte AChE inhibitor. Venous blood samples were collected from 18 healthy Caucasian men. For each blood sample, erythrocyte suspensions were obtained and incubated in the absence (controls) and presence of ACh and timolol maleate (10 µM final concentration of each modulator). Both timolol and ACh induced significant GSNO efflux in the erythrocyte when compared to the control; however the efflux was lower in the presence of timolol compared to ACh. Although erythrocyte NO efflux in presence of timolol is similar to the control, the efflux decreased when compared to the ACh treatment. The presence of timolol induces significant decrease of intra-erythrocyte GSNO levels, relative to control and ACh treatment. In conclusion, when erythrocytes were stimulated with ACh or timolol, GSNO efflux occurred associated with NO efflux. These new results bring new insight into the metabolism of erythrocyte NO and new possible therapeutic applications for GSNO.

Prognostic value of VEGF in patients submitted to percutaneous coronary intervention.

We examined the longitudinal changes of VEGF levels after percutaneous coronary intervention for predicting major adverse cardiac events (MACE) in coronary artery disease (CAD) patients. VEGF was measured in 94 CAD patients' serum before revascularization, 1-month and 1-year after. Independently of clinical presentation, patients had lower VEGF concentration than a cohort of healthy subjects (median, IQ: 15.9, 9.0-264 pg/mL versus 419, 212-758 pg/mL; P < 0.001) at baseline. VEGF increased to 1-month (median, IQ: 276, 167-498 pg/mL; P < 0.001) and remained steady to 1-year (median, IQ: 320, 173-497 pg/mL; P < 0.001) approaching control levels. Drug eluting stent apposition and previous medication intake produced a less steep VEGF evolution after intervention (P < 0.05). Baseline VEGF concentration <40.8 pg/mL conveyed increased risk for MACE in a 5-year follow-up. Results reflect a positive role of VEGF in recovery and support its importance in CAD prognosis.