Comparing plasma and skin imprint metabolic profiles in COVID-19 diagnosis and severity assessment.

As SARS-CoV-2 continues to produce new variants, the demand for diagnostics and a better understanding of COVID-19 remain key topics in healthcare. Skin manifestations have been widely reported in cases of COVID-19, but the mechanisms and markers of these symptoms are poorly described. In this cross-sectional study, 101 patients (64 COVID-19 positive patients and 37 controls) were enrolled between April and June 2020, during the first wave of COVID-19, in São Paulo, Brazil. Enrolled patients had skin imprints sampled non-invasively using silica plates; plasma samples were also collected. Samples were used for untargeted lipidomics/metabolomics through high-resolution mass spectrometry. We identified 558 molecular ions, with lipids comprising most of them. We found 245 plasma ions that were significant for COVID-19 diagnosis, compared to 61 from the skin imprints. Plasma samples outperformed skin imprints in distinguishing patients with COVID-19 from controls, with F1-scores of 91.9% and 84.3%, respectively. Skin imprints were excellent for assessing disease severity, exhibiting an F1-score of 93.5% when discriminating between patient hospitalization and home care statuses. Specifically, oleamide and linoleamide were the most discriminative biomarkers for identifying hospitalized patients through skin imprinting, and palmitic amides and N-acylethanolamine 18:0 were also identified as significant biomarkers. These observations underscore the importance of primary fatty acid amides and N-acylethanolamines in immunomodulatory processes and metabolic disorders. These findings confirm the potential utility of skin imprinting as a valuable non-invasive sampling method for COVID-19 screening; a method that may also be applied in the evaluation of other medical conditions. KEY MESSAGES: Skin imprints complement plasma in disease metabolomics. The annotated markers have a role in immunomodulation and metabolic diseases. Skin imprints outperformed plasma samples at assessing disease severity. Skin imprints have potential as non-invasive sampling strategy for COVID-19.

Comparison between two different local hemostatic methods for dental extractions in patients on dual antiplatelet therapy: a within-person, single-blind, randomized study.

BACKGROUND: Dual antiplatelet therapy (DAPT) provides additional risk reduction of ischemic events compared to aspirin monotherapy, at cost of higher bleeding risk. There are few data comparing new techniques for reducing bleeding after dental extractions in these patients. PURPOSE: This study investigated the effectiveness of the HemCon Dental Dressing (HDD) compared to oxidized cellulose gauze. MATERIALS AND METHODS: This randomized study included 60 patients on DAPT who required at least two dental extractions (120 procedures). Each surgical site was randomized to HDD or oxidized regenerated cellulose gauze as the local hemostatic method. Intra-oral bleeding time was measured immediately after the dental extraction and represents our main endpoint for comparison of both hemostatic agents. Prolonged bleeding, platelet reactivity measured by Multiplate Analyser (ADPtest and ASPItest) and tissue healing comparison after 7 days were also investigated. RESULTS: Intra-oral bleeding time was lower in HDD compared with control (2 [2-5] vs. 5 [2-8] minutes, P=0.001). Prolonged postoperative bleeding was observed in 7 cases (11.6%), all of them successfully managed with local sterile gauze pressure. More HDD treated sites presented better healing when compared with control sites [21 (36.8%) vs. 5 (8.8%), P=0.03]. There was poor correlation between platelet reactivity and intra-oral bleeding time. CONCLUSIONS: In patients on DAPT, HDD resulted in a lower intra-oral bleeding time compared to oxidized cellulose gauze after dental extractions. Moreover, HDD also seems to improve healing conditions.

Effects of exercise on platelet reactivity after myocardial infarction: a randomized clinical trial.

Exercise training (ET) can lower platelet reactivity in patients with cardiovascular risk factors. However, the effects of ET on platelet reactivity in higher-risk patients is unknown. The aim of this study was to evaluate the effects of ET on platelet reactivity in patients with recent myocardial infarction (MI). Ninety patients were randomly assigned 1 month post-MI to the intervention (patients submitted to a supervised ET program) or control group. All patients were on dual antiplatelet therapy (DAPT). Platelet reactivity by VerifyNow-P2Y12 (measured by P2Y12 reaction units - PRUs) test was determined at baseline and at the end of 14 ± 2 weeks of follow-up at rest (primary endpoint), and multiplate electrode aggregometry (MEA) adenosine diphosphate (ADP) and aspirin (ASPI) tests were performed immediately before and after the maximal cardiopulmonary exercise test (CPET) at the same time points (secondary endpoints). Sixty-five patients (mean age 58.9 ± 10 years; 73.8% men; 60% ST elevation MI) completed follow-up (control group, n = 31; intervention group, n = 34). At the end of the follow-up, the mean platelet reactivity was 172.8 ± 68.9 PRUs and 166.9 ± 65.1 PRUs for the control and intervention groups, respectively (p = .72). Platelet reactivity was significantly increased after the CPET compared to rest at the beginning and at the end of the 14-week follow-up (among the intervention groups) by the MEA-ADP and MEA-ASPI tests (p < .01 for all analyses). In post-MI patients on DAPT, 14 weeks of supervised ET did not reduce platelet reactivity. Moreover, platelet reactivity was increased after high-intensity exercise (ClinicalTrials.gov: NCT02958657; https://clinicaltrials.gov/ct2/show/NCT02958657).

What is the context? Platelet reactivity is reduced after exercise training in healthy individuals and patients with cardiovascular risk factors, but the effect in higher-risk patients is unknown.High-intensity exercise in untrained individuals increases platelet reactivity. The effect of dual antiplatelet therapy in inhibiting exercise-induced hyperreactivity is poorly understood.What's new?Exercise training did not reduce platelet reactivity in post-myocardial infarction patients.High-intensity exercise increased platelet reactivity in post-myocardial infarction patients on dual antiplatelet therapy.Exercise training did not attenuate the exercise-induced increase in platelet reactivity.What's the impact?The study suggests that strenuous exercise, if indicated, should be applied carefully to patients with high risk of recurrent ischemic events, even if on optimal medical therapy and after being trained.

Effects of DPP4 Inhibitor in Platelet Reactivity and Other Cardiac Risk Markers in Patients with Type 2 Diabetes and Acute Myocardial Infarction.

BACKGROUND: The management of acute myocardial infarction (AMI) presents several challenges in patients with diabetes, among them the higher rate of recurrent thrombotic events, hyperglycemia and risk of subsequent heart failure (HF). The objective of our study was to evaluate effects of DPP-4 inhibitors (DPP-4i) on platelet reactivity (main objective) and cardiac risk markers. METHODS: We performed a single-center double-blind randomized trial. A total of 70 patients with type 2 diabetes (T2DM) with AMI Killip ≤2 on dual-antiplatelet therapy (aspirin plus clopidogrel) were randomized to receive sitagliptin 100 mg or saxagliptin 5 mg daily or matching placebo. Platelet reactivity was assessed at baseline, 4 days (primary endpoint) and 30 days (secondary endpoint) after randomization, using VerifyNow Aspirin™ assay, expressed as aspirin reaction units (ARUs); B-type natriuretic peptide (BNP) in pg/mL was assessed at baseline and 30 days after (secondary endpoint). RESULTS: Mean age was 62.6 ± 8.8 years, 45 (64.3%) male, and 52 (74.3%) of patients presented with ST-segment elevation MI. For primary endpoint, there were no differences in mean platelet reactivity (p = 0.51) between the DPP-4i (8.00 {-65.00; 63.00}) and placebo (-14.00 {-77.00; 52.00}) groups, as well in mean BNP levels (p = 0.14) between DPP-4i (-36.00 {-110.00; 15.00}) and placebo (-13.00 {-50.00; 27.00}). There was no difference between groups in cardiac adverse events. CONCLUSIONS: DPP4 inhibitor did not reduce platelet aggregation among patients with type 2 diabetes hospitalized with AMI. Moreover, the use of DPP-4i did not show an increase in BNP levels or in the incidence of cardiac adverse events. These findings suggests that DPP-4i could be an option for management of T2DM patients with acute MI.

Platelet Reactivity and Coagulation Markers in Patients with COVID-19.

INTRODUTION: COVID-19 is associated with an increased risk of thrombotic events. However, the contribution of platelet reactivity (PR) to the aetiology of the increased thrombotic risk associated with COVID-19 remains unclear. Our aim was to evaluate PR in stable patients diagnosed with COVID-19 and hospitalized with respiratory symptoms (mainly dyspnoea and dry cough), in comparison with a control group comprised of non-hospitalized healthy controls. METHODS: Observational, case control study that included patients with confirmed COVID-19 (COVID-19 group, n = 60) and healthy individuals matched by age and sex (control group, n = 60). Multiplate electrode aggregometry (MEA) tests were used to assess PR with adenosine diphosphate (MEA-ADP, low PR defined as < 53 AUC), arachidonic acid (MEA-ASPI, low PR < 86 AUC) and thrombin receptor-activating peptide 6 (MEA-TRAP, low PR < 97 AUC) in both groups. RESULTS: The rates of low PR with MEA-ADP were 27.5% in the COVID-19 group and 21.7% in the control group (OR = 1.60, p = 0.20); with MEA-ASPI, the rates were, respectively, 37.5% and 22.5% (OR = 3.67, p < 0.001); and with MEA-TRAP, the incidences were 48.5% and 18.8%, respectively (OR = 9.58, p < 0.001). Levels of D-dimer, fibrinogen, and plasminogen activator inhibitor 1 (PAI-1) were higher in the COVID-19 group in comparison with the control group (all p < 0.05). Thromboelastometry was utilized in a subgroup of patients and showed a hypercoagulable state in the COVID-19 group. CONCLUSION: Patients hospitalized with non-severe COVID-19 had lower PR compared to healthy controls, despite having higher levels of D-dimer, fibrinogen, and PAI-1, and hypercoagulability by thromboelastometry. TRIAL REGISTRATION: ClinicalTrials.gov identifier, NCT04447131.

Association between Statin Therapy and Lower Incidence of Hyperglycemia in Patients Hospitalized with Acute Coronary Syndromes. / Associação entre Terapia com Estatinas e Menor Incidência de Hiperglicemia em Pacientes Internados com Síndromes Coronarianas Agudas.

BACKGROUND: Increased risk of new-onset diabetes with statins challenges the long-term safety of this drug class. However, few reports have analyzed this issue during acute coronary syndromes (ACS). OBJECTIVE: To explore the association between early initiation of statin therapy and blood glucose levels in patients admitted with ACS. METHODS: This was a retrospective analysis of patients hospitalized with ACS. Statin-naïve patients were included and divided according to their use or not of statins within the first 24 hours of hospitalization. The primary endpoint was incidence of in-hospital hyperglycemia (defined as peak blood glucose > 200 mg/dL). Multivariable linear and logistic regression models were used to adjust for confounders, and a propensity-score matching model was developed to further compare both groups of interest. A p-value of less than 0.05 was considered statistically significant. RESULTS: A total of 2,357 patients were included, 1,704 of them allocated in the statin group and 653 in the non-statin group. After adjustments, statin use in the first 24 hours was associated with a lower incidence of in-hospital hyperglycemia (adjusted OR=0.61, 95% CI 0.46-0.80; p < 0.001) and lower need for insulin therapy (adjusted OR = 0.56, 95% CI 0.41-0.76; p < 0.001). These associations remained similar in the propensity-score matching models, as well as after several sensitivity analyses, such as after excluding patients who developed cardiogenic shock, severe infection or who died during index-hospitalization. CONCLUSIONS: Among statin-naïve patients admitted with ACS, early statin therapy was independently associated with lower incidence of in-hospital hyperglycemia. (Arq Bras Cardiol. 2021; 116(2):285-294).

FUNDAMENTO: O maior risco de se desenvolver diabetes com o uso de estatinas é um desafio para a segurança do uso dessa classe de medicamentos em longo prazo. No entanto, poucos estudos analisaram essa questão durante síndromes coronarianas agudas (SCA). OBJETIVOS: Investigar a associação entre início precoce da terapia com estatina e níveis de glicemia em pacientes admitidos com SCA. MÉTODOS: Este foi um estudo retrospectivo de pacientes hospitalizados por SCA. Pacientes que nunca haviam usado estatinas foram incluídos e divididos segundo uso ou não de estatina nas primeiras 24 horas de internação. O desfecho primário foi a incidência de hiperglicemia na internação (definida como pico de glicemia > 200mg/dL). Modelos de regressão logística e modelos lineares multivariados foram usados para ajuste quanto a fatores de confusão e um modelo de pareamento por escore de propensão foi desenvolvido para comparações entre os dois grupos de interesses. Um valor de p menor que 0,05 foi considerado estatisticamente significativo. RESULTADOS: Um total de 2357 pacientes foram incluídos, 1704 deles alocados no grupo que receberam estatinas e 653 no grupo que não receberam estatinas nas primeiras 24 horas de internação. Após os ajustes, uso de estatina nas primeiras 24 horas foi associado com uma menor incidência de hiperglicemia durante a internação (OR ajustado = 0,61, IC95% 0,46-0,80; p < 0,001) e menor necessidade de uso de insulina (OR ajustado = 0,56, IC 95% 0,41-0,76; p < 0,001). Essas associações mantiveram-se similares nos modelos de pareamento por escore de propensão, bem como após análises de sensibilidade, como exclusão de pacientes que desenvolveram choque cardiogênico, infecção grave ou pacientes que foram a óbito durante a internação hospitalar. CONCLUSÕES: Entre os pacientes internados com SCA que não receberam estatinas previamente, a terapia precoce com estatina associou-se independentemente com menor incidência de hiperglicemia durante a internação. (Arq Bras Cardiol. 2021; 116(2):285-294).

Associação entre Terapia com Estatinas e Menor Incidência de Hiperglicemia em Pacientes Internados com Síndromes Coronarianas Agudas / Association between Statin Therapy and Lower Incidence of Hyperglycemia in Patients Hospitalized with Acute Coronary Syndromes

Resumo Fundamento O maior risco de se desenvolver diabetes com o uso de estatinas é um desafio para a segurança do uso dessa classe de medicamentos em longo prazo. No entanto, poucos estudos analisaram essa questão durante síndromes coronarianas agudas (SCA). Objetivos Investigar a associação entre início precoce da terapia com estatina e níveis de glicemia em pacientes admitidos com SCA. Métodos Este foi um estudo retrospectivo de pacientes hospitalizados por SCA. Pacientes que nunca haviam usado estatinas foram incluídos e divididos segundo uso ou não de estatina nas primeiras 24 horas de internação. O desfecho primário foi a incidência de hiperglicemia na internação (definida como pico de glicemia > 200mg/dL). Modelos de regressão logística e modelos lineares multivariados foram usados para ajuste quanto a fatores de confusão e um modelo de pareamento por escore de propensão foi desenvolvido para comparações entre os dois grupos de interesses. Um valor de p menor que 0,05 foi considerado estatisticamente significativo. Resultados Um total de 2357 pacientes foram incluídos, 1704 deles alocados no grupo que receberam estatinas e 653 no grupo que não receberam estatinas nas primeiras 24 horas de internação. Após os ajustes, uso de estatina nas primeiras 24 horas foi associado com uma menor incidência de hiperglicemia durante a internação (OR ajustado = 0,61, IC95% 0,46-0,80; p < 0,001) e menor necessidade de uso de insulina (OR ajustado = 0,56, IC 95% 0,41-0,76; p < 0,001). Essas associações mantiveram-se similares nos modelos de pareamento por escore de propensão, bem como após análises de sensibilidade, como exclusão de pacientes que desenvolveram choque cardiogênico, infecção grave ou pacientes que foram a óbito durante a internação hospitalar. Conclusões Entre os pacientes internados com SCA que não receberam estatinas previamente, a terapia precoce com estatina associou-se independentemente com menor incidência de hiperglicemia durante a internação. (Arq Bras Cardiol. 2021; 116(2):285-294)

Abstract Background Increased risk of new-onset diabetes with statins challenges the long-term safety of this drug class. However, few reports have analyzed this issue during acute coronary syndromes (ACS). Objective To explore the association between early initiation of statin therapy and blood glucose levels in patients admitted with ACS. Methods This was a retrospective analysis of patients hospitalized with ACS. Statin-naïve patients were included and divided according to their use or not of statins within the first 24 hours of hospitalization. The primary endpoint was incidence of in-hospital hyperglycemia (defined as peak blood glucose > 200 mg/dL). Multivariable linear and logistic regression models were used to adjust for confounders, and a propensity-score matching model was developed to further compare both groups of interest. A p-value of less than 0.05 was considered statistically significant. Results A total of 2,357 patients were included, 1,704 of them allocated in the statin group and 653 in the non-statin group. After adjustments, statin use in the first 24 hours was associated with a lower incidence of in-hospital hyperglycemia (adjusted OR=0.61, 95% CI 0.46-0.80; p < 0.001) and lower need for insulin therapy (adjusted OR = 0.56, 95% CI 0.41-0.76; p < 0.001). These associations remained similar in the propensity-score matching models, as well as after several sensitivity analyses, such as after excluding patients who developed cardiogenic shock, severe infection or who died during index-hospitalization. Conclusions Among statin-naïve patients admitted with ACS, early statin therapy was independently associated with lower incidence of in-hospital hyperglycemia. (Arq Bras Cardiol. 2021; 116(2):285-294)

Factors associated with actively working in the very long-term following acute coronary syndrome.

OBJECTIVES: Returning to work after an episode of acute coronary syndrome (ACS) is challenging for many patients, and has both personal and social impacts. There are limited data regarding the working status in the very long-term after ACS. METHODS: We retrospectively analyzed 1,632 patients who were working prior to hospitalization for ACS in a quaternary hospital and were followed-up for up to 17 years. Adjusted models were developed to analyze the variables independently associated with actively working at the last contact, and a prognostic predictive index for not working at follow-up was developed. RESULTS: The following variables were significantly and independently associated with actively working at the last contact: age>median (hazard-ratio [HR], 0.76, p<0.001); male sex (HR, 1.52, p<0.001); government health insurance (HR, 1.36, p<0.001); history of angina (HR, 0.69, p<0.001) or myocardial infarction (MI) (HR, 0.76, p=0.005); smoking (HR, 0.81, p=0.015); ST-elevation MI (HR, 0.81, p=0.021); anterior-wall MI (HR, 0.75, p=0.001); non-primary percutaneous coronary intervention (PCI) (HR, 0.77, p=0.002); fibrinolysis (HR, 0.61, p<0.001); cardiogenic shock (HR, 0.60, p=0.023); statin (HR, 3.01, p<0.001), beta-blocker (HR, 1.26, p=0.020), angiotensin-converting enzyme (ACE) inhibitor/angiotensin II receptor blocker (ARB) (HR, 1.37, p=0.001) at hospital discharge; and MI at follow-up (HR, 0.72, p=0.001). The probability of not working at the last contact ranged from 24.2% for patients with no variables, up to 80% for patients with six or more variables. CONCLUSIONS: In patients discharged after ACS, prior and in-hospital clinical variables, as well as the quality of care at discharge, have a great impact on the long-term probability of actively working.

Covid-19 Automated Diagnosis and Risk Assessment through Metabolomics and Machine Learning.

COVID-19 is still placing a heavy health and financial burden worldwide. Impairment in patient screening and risk management plays a fundamental role on how governments and authorities are directing resources, planning reopening, as well as sanitary countermeasures, especially in regions where poverty is a major component in the equation. An efficient diagnostic method must be highly accurate, while having a cost-effective profile. We combined a machine learning-based algorithm with mass spectrometry to create an expeditious platform that discriminate COVID-19 in plasma samples within minutes, while also providing tools for risk assessment, to assist healthcare professionals in patient management and decision-making. A cross-sectional study enrolled 815 patients (442 COVID-19, 350 controls and 23 COVID-19 suspicious) from three Brazilian epicenters from April to July 2020. We were able to elect and identify 19 molecules related to the disease's pathophysiology and several discriminating features to patient's health-related outcomes. The method applied for COVID-19 diagnosis showed specificity >96% and sensitivity >83%, and specificity >80% and sensitivity >85% during risk assessment, both from blinded data. Our method introduced a new approach for COVID-19 screening, providing the indirect detection of infection through metabolites and contextualizing the findings with the disease's pathophysiology. The pairwise analysis of biomarkers brought robustness to the model developed using machine learning algorithms, transforming this screening approach in a tool with great potential for real-world application.

Factors associated with actively working in the very long-term following acute coronary syndrome

OBJECTIVES: Returning to work after an episode of acute coronary syndrome (ACS) is challenging for many patients, and has both personal and social impacts. There are limited data regarding the working status in the very long-term after ACS. METHODS: We retrospectively analyzed 1,632 patients who were working prior to hospitalization for ACS in a quaternary hospital and were followed-up for up to 17 years. Adjusted models were developed to analyze the variables independently associated with actively working at the last contact, and a prognostic predictive index for not working at follow-up was developed. RESULTS: The following variables were significantly and independently associated with actively working at the last contact: age>median (hazard-ratio [HR], 0.76, p<0.001); male sex (HR, 1.52, p<0.001); government health insurance (HR, 1.36, p<0.001); history of angina (HR, 0.69, p<0.001) or myocardial infarction (MI) (HR, 0.76, p=0.005); smoking (HR, 0.81, p=0.015); ST-elevation MI (HR, 0.81, p=0.021); anterior-wall MI (HR, 0.75, p=0.001); non-primary percutaneous coronary intervention (PCI) (HR, 0.77, p=0.002); fibrinolysis (HR, 0.61, p<0.001); cardiogenic shock (HR, 0.60, p=0.023); statin (HR, 3.01, p<0.001), beta-blocker (HR, 1.26, p=0.020), angiotensin-converting enzyme (ACE) inhibitor/angiotensin II receptor blocker (ARB) (HR, 1.37, p=0.001) at hospital discharge; and MI at follow-up (HR, 0.72, p=0.001). The probability of not working at the last contact ranged from 24.2% for patients with no variables, up to 80% for patients with six or more variables. CONCLUSIONS: In patients discharged after ACS, prior and in-hospital clinical variables, as well as the quality of care at discharge, have a great impact on the long-term probability of actively working.

Relation of High Lipoprotein (a) Concentrations to Platelet Reactivity in Individuals with and Without Coronary Artery Disease.

INTRODUCTION: Lipoprotein (a) [Lp(a)] is a risk factor for coronary artery disease (CAD). To the best of our knowledge, this is the first study addressing the relationship between Lp(a) and platelet reactivity in primary and secondary prevention. METHODS: Lp(a) was evaluated in 396 individuals with (82.3%) and without (17.7%) obstructive CAD. The population was divided into two groups according to Lp(a) concentrations with a cutoff value of 50 mg/dL. The primary objective was to evaluate the association between Lp(a) and adenosine diphosphate (ADP)-induced platelet reactivity using the VerifyNow™ P2Y12 assay. Platelet reactivity was also induced by arachidonic acid and collagen-epinephrine (C-EPI) and assessed by Multiplate™, platelet function analyzer™ 100 (PFA-100), and light transmission aggregometry (LTA) assays. Secondary objectives included the assessment of the primary endpoint in individuals with or without CAD. RESULTS: Overall, 294 (74.2%) individuals had Lp(a) < 50 mg/dL [median (IQR) 13.2 (5.8-27.9) mg/dL] and 102 (25.8%) had Lp(a) ≥ 50 mg/dL [82.5 (67.6-114.5) mg/dL], P < 0.001. Univariate analysis in the entire population revealed no differences in ADP-induced platelet reactivity between individuals with Lp(a) ≥ 50 mg/dL (249.4 ± 43.8 PRU) versus Lp(a) < 50 mg/dL (243.1 ± 52.2 PRU), P = 0.277. Similar findings were present in individuals with (P = 0.228) and without (P = 0.669) CAD, and regardless of the agonist used or method of analysis (all P > 0.05). Finally, multivariable analysis did not show a significant association between ADP-induced platelet reactivity and Lp(a) ≥ 50 mg/dL [adjusted OR = 1.00 [(95% CI 0.99-1.01), P = 0.590]. CONCLUSION: In individuals with or without CAD, Lp(a) ≥ 50 mg/dL was not associated with higher platelet reactivity.

Increased bodyweight and inadequate response to aspirin in individuals with coronary artery disease.

Recent reports have suggested that aspirin effect might be influenced by bodyweight, with decreased efficacy in heavier individuals. We investigated the influence of bodyweight on aspirin pharmacodynamics in two independent datasets of patients taking non-enteric coated aspirin 100 mg QD for coronary artery disease (CAD). In the first dataset, 368 patients had their platelet aggregation assessed using VerifyNow Aspirin and measured in Aspirin Reaction Units (ARU). In the second dataset, 70 patients had serum thromboxane B2 (TXB2) dosage assessed by an ELISA assay and measured in pg/mL. Platelet aggregation was independently associated with bodyweight, with 8.41 (95% CI 1.86-14.97; adjusted p-value = 0.012) increase in ARU for every 10 kg. Furthermore, the rate of non-response to aspirin (defined as ARU ≥ 550) was significantly associated with increased bodyweight (adjusted p-value = 0.007), with OR = 1.23 (95% CI 1.06-1.42) for every 10 kg. Similar results were found considering body mass index (in kg/m2), with 15.5 (95% CI 5.0 to 25.9; adjusted p-value = 0.004) increase in ARU for every 10 kg and non-response OR = 1.43 (95% CI 1.13 to 1.81, adjusted p-value = 0.003) for every 5 kg/m2. Moreover, serum TXB2 was higher in patients weighting more than 70 kg (222.6 ± 62.9 versus 194.9 ± 61.9 pg/mL; adjusted p-value = 0.018). In two different datasets of patients with CAD on non-enteric coated aspirin 100 mg QD, increased bodyweight was independently associated with impaired response to aspirin.

Maternal supraphysiological hypercholesterolemia associates with endothelial dysfunction of the placental microvasculature.

Maternal physiological or supraphysiological hypercholesterolemia (MPH, MSPH) occurs during pregnancy. MSPH is associated with foetal endothelial dysfunction and atherosclerosis. However, the potential effects of MSPH on placental microvasculature are unknown. The aim of this study was to determine whether MSPH alters endothelial function in the placental microvasculature both ex vivo in venules and arterioles from the placental villi and in vitro in primary cultures of placental microvascular endothelial cells (hPMEC). Total cholesterol < 280 mg/dL indicated MPH, and total cholesterol ≥280 mg/dL indicated MSPH. The maximal relaxation to histamine, calcitonin gene-related peptide and adenosine was reduced in MSPH venule and arteriole rings. In hPMEC from MSPH placentas, nitric oxide synthase (NOS) activity and L-arginine transport were reduced without changes in arginase activity or the protein levels of endothelial NOS (eNOS), human cationic amino acid 1 (hCAT-1), hCAT-2A/B or arginase II compared with hPMEC from MPH placentas. In addition, it was shown that adenosine acts as a vasodilator of the placental microvasculature and that NOS is active in hPMEC. We conclude that MSPH alters placental microvascular endothelial function via a NOS/L-arginine imbalance. This work also reinforces the concept that placental endothelial cells from the macro- and microvasculature respond differentially to the same pathological condition.

Akt/mTOR Role in Human Foetoplacental Vascular Insulin Resistance in Diseases of Pregnancy.

Insulin resistance is characteristic of pregnancies where the mother shows metabolic alterations, such as preeclampsia (PE) and gestational diabetes mellitus (GDM), or abnormal maternal conditions such as pregestational maternal obesity (PGMO). Insulin signalling includes activation of insulin receptor substrates 1 and 2 (IRS1/2) as well as Src homology 2 domain-containing transforming protein 1, leading to activation of 44 and 42 kDa mitogen-activated protein kinases and protein kinase B/Akt (Akt) signalling cascades in the human foetoplacental vasculature. PE, GDM, and PGMO are abnormal conditions coursing with reduced insulin signalling, but the possibility of the involvement of similar cell signalling mechanisms is not addressed. This review aimed to determine whether reduced insulin signalling in PE, GDM, and PGMO shares a common mechanism in the human foetoplacental vasculature. Insulin resistance in these pathological conditions results from reduced Akt activation mainly due to inhibition of IRS1/2, likely due to the increased activity of the mammalian target of rapamycin (mTOR) resulting from lower activity of adenosine monophosphate kinase. Thus, a defective signalling via Akt/mTOR in response to insulin is a central and common mechanism of insulin resistance in these diseases of pregnancy. In this review, we summarise the cell signalling mechanisms behind the insulin resistance state in PE, GDM, and PGMO focused in the Akt/mTOR signalling pathway in the human foetoplacental endothelium.

Maternal insulin therapy does not restore foetoplacental endothelial dysfunction in gestational diabetes mellitus.

Pregnant women diagnosed with gestational diabetes mellitus subjected to diet (GDMd) that do not reach normal glycaemia are passed to insulin therapy (GDMi). GDMd associates with increased human cationic amino acid transporter 1 (hCAT-1)-mediated transport of L-arginine and nitric oxide synthase (NOS) activity in foetoplacental vasculature, a phenomenon reversed by exogenous insulin. Whether insulin therapy results in reversal of the GDMd effect on the foetoplacental vasculature is unknown. We assayed whether insulin therapy normalizes GDMd-associated foetoplacental endothelial dysfunction. Primary cultures of human umbilical vein endothelial cells (HUVECs) from GDMi pregnancies were used to assay L-arginine transport kinetics, NOS activity, p44/42mapk and protein kinase B/Akt activation, and umbilical vein rings reactivity. HUVECs from GDMi or GDMd show increased hCAT-1 expression and maximal transport capacity, NOS activity, and eNOS, and p44/42mapk, but not Akt activator phosphorylation. Dilation in response to insulin or calcitonin-gene related peptide was impaired in umbilical vein rings from GDMi and GDMd pregnancies. Incubation of HUVECs in vitro with insulin (1 nmol/L) restored hCAT-1 and eNOS expression and activity, and eNOS and p44/42mapk activator phosphorylation. Thus, maternal insulin therapy does not seem to reverse GDMd-associated alterations in human foetoplacental vasculature.

Intracellular and extracellular pH dynamics in the human placenta from diabetes mellitus.

The placenta is a vital organ whose function in diseases of pregnancy is altered, resulting in an abnormal supply of nutrients to the foetus. The lack of placental vasculature homeostasis regulation causes endothelial dysfunction and altered vascular reactivity. The proper distribution of acid- (protons (H(+))) and base-equivalents through the placenta is essential to achieve physiological homeostasis. Several membrane transport mechanisms that control H(+) distribution between the extracellular and intracellular spaces are expressed in the human placenta vascular endothelium and syncytiotrophoblast, including sodium (Na(+))/H(+) exchangers (NHEs). One member of the NHEs family is NHE isoform 1 (NHE1), whose activity results in an alkaline intracellular pH (high intracellular pH (pHi)) and an acidic extracellular pH (pHo). Increased NHE1 expression, maximal transport activity, and turnover are reported in human syncytiotrophoblasts and lymphocytes from patients with diabetes mellitus type I (DMT1), and a positive correlation between NHEs activity and plasma factors, such as that between thrombin and platelet factor 3, has been reported in diabetes mellitus type II (DMT2). However, gestational diabetes mellitus (GDM) could result in a higher sensitivity of the human placenta to acidic pHo. We summarized the findings on pHi and pHo modulation in the human placenta with an emphasis on pregnancies in which the mother diagnosed with diabetes mellitus. A potential role of NHEs, particularly NHE1, is proposed regarding placental dysfunction in DMT1, DMT2, and GDM.

Nitric Oxide is a Central Common Metabolite in Vascular Dysfunction Associated with Diseases of Human Pregnancy.

Preeclampsia (PE), gestational diabetes mellitus (GDM), and maternal supraphysiological hypercholesterolaemia (MSPH) are pregnancy-related conditions that cause metabolic disruptions leading to alterations of the mother, fetus and neonate health. These syndromes result in fetoplacental vascular dysfunction, where nitric oxide (NO) plays a crucial role. PE characterizes by abnormal increase in the placental blood pressure and a negative correlation between NO level and fetal weight, suggesting that increased NO level and oxidative stress could be involved. GDM courses with macrosomia along with altered function of the fetal cardiovascular system and fetoplacental vasculature. Even when NO synthesis in the fetoplacental vasculature is increased, NO bioavailability is reduced due to the higher oxidative stress seen in this disease. In MSPH, there is an early development of atherosclerotic lesions in fetal and newborn arteries, altered function of the fetoplacental vasculature, and higher markers of oxidative stress in fetal blood and placenta, thus, vascular alterations related with NO metabolism occur as a consequence of this syndrome. Potential mechanisms of altered NO synthesis and bioavailability result from transcriptional and post-translational NO synthases (NOS) modulation, including phosphorylation/dephosphorylation cycles, coupling/uncoupling of NOS, tetrahydrobiopterin bioavailability, calcium/calmodulin-NOS and caveolin-1-NOS interaction. Additionally, oxidative stress also plays a role in the reduced NO bioavailability. This review summarizes the available information regarding lower NO bioavailability in these pregnancy pathologies. A common NO-dependent mechanism in PE, GDM and MSPH contributing to fetoplacental endothelial dysfunction is described.

A Hypothesis for the Role of RECK in Angiogenesis.

Angiogenesis is a key process by which new capillary blood vessels are formed, sustaining the supply of oxygen and other nutrients to the body allowing its growth and wound healing, among others. However, angiogenesis also associates with pathological processes, such us tumor growth. Vascular endothelial cells produce different matrix remodeling enzymes such as matrix metalloproteinases and a-disintegrin and metalloproteinases, which have both positive and negative effects on angiogenesis, regulating the cell environment and signaling. However, little is known about the regulation of the activity of these proteases during vascular development. Reversion-inducing cysteine-rich protein with Kazal motifs (RECK) is a membrane-anchored inhibitor of different matrix metalloproteinases and a-disintegrin and metalloproteinases, being a critical regulator of extracellular matrix remodeling and signaling pathway, particularly Notch, which is critical for the maturation of the growing vessels. Reck knockout mice die in utero showing vascular developmental defects and massive hemorrhages. These defects were not observed in knockout mice for secreted-soluble matrix metalloproteinase inhibitors pointing to an exclusive role of RECK in vascular development and maturation since its location at the plasma membrane. Despite the above, the exact role of RECK in this process has not been clarified. This review is focused to summarize the available information on the role of RECK as membrane anchored matrix metalloproteinases and a-disintegrin and metalloproteinases inhibitor, proposing a hypothesis by which RECK play key roles in the physiology and pathophysiology of the angiogenesis processes.

Insulin reverses D-glucose-increased nitric oxide and reactive oxygen species generation in human umbilical vein endothelial cells.

Vascular tone is controlled by the L-arginine/nitric oxide (NO) pathway, and NO bioavailability is strongly affected by hyperglycaemia-induced oxidative stress. Insulin leads to high expression and activity of human cationic amino acid transporter 1 (hCAT-1), NO synthesis and vasodilation; thus, a protective role of insulin on high D-glucose-alterations in endothelial function is likely. Vascular reactivity to U46619 (thromboxane A2 mimetic) and calcitonin gene related peptide (CGRP) was measured in KCl preconstricted human umbilical vein rings (wire myography) incubated in normal (5 mmol/L) or high (25 mmol/L) D-glucose. hCAT-1, endothelial NO synthase (eNOS), 42 and 44 kDa mitogen-activated protein kinases (p42/44mapk), protein kinase B/Akt (Akt) expression and activity were determined by western blotting and qRT-PCR, tetrahydrobiopterin (BH4) level was determined by HPLC, and L-arginine transport (0-1000 µmol/L) was measured in response to 5-25 mmol/L D-glucose (0-36 hours) in passage 2 human umbilical vein endothelial cells (HUVECs). Assays were in the absence or presence of insulin and/or apocynin (nicotinamide adenine dinucleotide phosphate-oxidase [NADPH oxidase] inhibitor), tempol or Mn(III)TMPyP (SOD mimetics). High D-glucose increased hCAT-1 expression and activity, which was biphasic (peaks: 6 and 24 hours of incubation). High D-glucose-increased maximal transport velocity was blocked by insulin and correlated with lower hCAT-1 expression and SLC7A1 gene promoter activity. High D-glucose-increased transport parallels higher reactive oxygen species (ROS) and superoxide anion (O2•-) generation, and increased U46619-contraction and reduced CGRP-dilation of vein rings. Insulin and apocynin attenuate ROS and O2•- generation, and restored vascular reactivity to U46619 and CGRP. Insulin, but not apocynin or tempol reversed high D-glucose-increased NO synthesis; however, tempol and Mn(III)TMPyP reversed the high D-glucose-reduced BH4 level. Insulin and tempol blocked the high D-glucose-increased p42/44mapk phosphorylation. Vascular dysfunction caused by high D-glucose is likely attenuated by insulin through the L-arginine/NO and O2•-/NADPH oxidase pathways. These findings are of interest for better understanding vascular dysfunction in states of foetal insulin resistance and hyperglycaemia.

Reduced L-carnitine transport in aortic endothelial cells from spontaneously hypertensive rats.

Impaired L-carnitine uptake correlates with higher blood pressure in adult men, and L-carnitine restores endothelial function in aortic rings from spontaneously hypertensive rat (SHR). Thus, endothelial dysfunction in hypertension could result from lower L-carnitine transport in this cell type. L-Carnitine transport is mainly mediated by novel organic cation transporters 1 (Octn1, Na(+)-independent) and 2 (Octn2, Na(+)-dependent); however, their kinetic properties and potential consequences in hypertension are unknown. We hypothesize that L-carnitine transport kinetic properties will be altered in aortic endothelium from spontaneously hypertensive rats (SHR). L-Carnitine transport was measured at different extracellular pH (pHo 5.5-8.5) in the absence or presence of sodium in rat aortic endothelial cells (RAECs) from non-hypertensive Wistar-Kyoto (WKY) rats and SHR. Octn1 and Octn2 mRNA relative expression was also determined. Dilation of endothelium-intact or denuded aortic rings in response to calcitonine gene related peptide (CGRP, 0.1-100 nmol/L) was measured (myography) in the absence or presence of L-carnitine. Total L-carnitine transport was lower in cells from SHR compared with WKY rats, an effect due to reduced Na(+)-dependent (Na(+) dep ) compared with Na(+)-independent (Na(+) indep ) transport components. Saturable L-carnitine transport kinetics show maximal velocity (V max), without changes in apparent K m for Na(+) indep transport in SHR compared with WKY rats. Total and Na(+) dep component of transport were increased, but Na(+) indep transport was reduced by extracellular alkalization in WKY rats. However, alkalization reduced total and Na(+) indep transport in cells from SHR. Octn2 mRNA was higher than Octn-1 mRNA expression in cells from both conditions. Dilation of artery rings in response to CGRP was reduced in vessels from SHR compared with WKY rats. CGRP effect was endothelium-dependent and restored by L-carnitine. All together these results suggest that reduced L-carnitine transport (likely via Na(+)-dependent Octn2) could limit this compound's potential beneficial effects in RAECs from SHR.