Imunização de camundongos LDLr-/- com peptídeo mimético da LDL eletronegativa: uma potencial estratégia terapêutica para a ateroscleros / Immunization of LDLr - / - mice with electronegative LDL mimetic peptides: a potential therapeutic strategy for atherosclerosis

Introdução: A aterosclerose é uma doença inflamatória crônica decorrente de alterações na parede das artérias de médio e grande calibre e associadas a diversos fatores de risco, dentre os quais destaca-se as hiperlipidemias, ou seja, o aumento plasmático das lipoproteínas, mas também outras comorbidades, como a Síndrome Metabólica. Entre as lipoproteínas, a lipoproteína de baixa densidade (LDL) é de grande relevância na aterosclerose. Diferentes espécies de LDL modificada (LDLm) são originadas através de lipólise, glicação e proteólise, além da oxidação, variando em densidade e eletronegatividade, sendo melhor denominada LDL eletronegativa [LDL (-)]. Considerando as diferenças conformacionais entre a estrutura da ApoB-100 da LDL nativa e da LDL (-), em um estudo inicial, nosso grupo desenvolveu um anticorpo monoclonal (2C7) a partir da imunização de camundongos Balb/c com a LDL (-) humana. Em uma etapa seguinte foi mapeado o epítopo reconhecido pelo anticorpo monoclonal anti-LDL (-) através de phage display. O peptídeo ligante do anticorpo monoclonal anti-LDL (-) foi nomeado p2C7. Esse peptídeo não representa regiões da sequência linear da ApoB-100 humana, mas microdomínios conformacionais de epítopos da ApoB-100 da LDL (-), tornando-os candidatos para a imunomodulação da aterogênese. Portanto, investigar a imunomodulação induzida pelos peptídeo p2C7 miméticos da LDL (-), por representar um epítopo imunodominante da LDL (-), poderá abrir novas perspectivas terapêuticas futuras para a imunomodulação da aterosclerose. Objetivo: Avaliar a imunomodulação promovida pelo p2C7 in vivo, utilizando camundongos C57BL/6 LDLr -/- e amostras de plasma humano. Adicionalmente, no estágio (BEPE) realizado no Instituto Karolinska (dezembro de 2019 a março de 2021), investigou-se o imunometabolismo como mediador nas doenças cardiovasculares. Na parte II-A, estão descritos os resultados do estudo inicialmente proposto. Na parte II-B, apresenta-se os resultados que foram desenvolvidos posteriormente, com ampliação do escopo do projeto, abordando-se a inflamação vascular envolvida no aneurisma de aorta abdominal através de ferramentas de bioinformática. Na parte II-C, são apresentados os resultados do estudo do envolvimento da enzima indolamina 2,3 dioxigenase (IDO) na esteatohepatite não-alcoólica (NASH) e aterosclerose em camundongos ApoE-/- and ApoE/IDO/double-knockout. Metodologia: Foi avaliada a presença de anticorpos anti-p2C7 em amostras de plasma humano de indivíduos com ou sem síndrome metabólica. Realizamos a determinação de TNF circulante nas mesmas amostras e prosseguimos com regressões lineares associando os parâmetros inflamatórios com os níveis de anticorpos anti-p2C7. Camundongos C57BL/6 LDLr -/- foram imunizados com p2C7 e os adjuvantes Alum ou Montanide ISA 720, analisando-se os títulos de anticorpos contra p2C7 e LDL (-), a produção de citocinas (IL-10, IL-4, IL-2, IL-6, IFNγ, IL-17, TNFα) e células secretoras de anticorpos. Camundongos C57BL/6 LDLr -/- foram tolerizados contra os peptídeos mimotopos, com injeções intravenosas (veia caudal) e desafiados com a imunização contendo LDL (-) + Alum. Avaliou-se os títulos de anticorpos contra p2C7 e LDL (-) e a produção de citocinas (TNF-α, IFNγ, IL-12, IL-6, IL-10 e MCP-1). Os camundongos foram mantidos em dieta hipercolesterolêmica por 3 meses para formação da placa aterosclerótica. Após este período, os camundongos foram eutanasiados, avaliando-se a formação de placa aterosclerótica na artéria abdominal e arco aórtico, assim como a produção de citocinas (TNF-α, IFNγ, IL-12, IL-6, IL-10 e MCP-1). Camundongos C57BL/6 LDLr -/- foram imunizados com OVA-p2C7 e, após dieta hipercolesterolêmica de 3 meses para formação de placa aterosclerótica, foram avaliados os parâmetros inflamatórios e avaliada a captação de 18F-FDG no arco aórtico através de PET/CT. Resultados: A imunização com o p2C7 (livre) não foi capaz de induzir resposta humoral, não se observando títulos detectáveis de anticorpos reativos à p2C7 ou LDL (-) em nenhum camundongo imunizado, assim como não foram detectadas células secretoras de anticorpos específicos para a LDL (-). O grupo imunizado com Alum ou Montanide + p2C7 teve aumento significativo na produção de TNF- quando comparado com os demais grupos. O protocolo de tolerização foi realizado com sucesso, visto que os camundongos tolerizados apresentaram títulos de anticorpos inferiores aos controles para o epítopo utilizado. Apenas os camundongos tolerizados com o p2C7 apresentaram aumento significativo na produção de IL-6, IL-12, IL-10, TNF-α, IFNγ e MCP 1 após dieta hipercolesterolêmica. A imunização ativa com OVA-p2C7 foi capaz de reduzir a produção de TNF induzida pela dieta hipercolesterolêmica, assim como reduzir a captação de 18F-FDG. Conclusão: o epítopo p2C7 é altamente expresso na LDL (-) de pacientes com maior risco cardiovascular. Além disso, a imunização ativa com p2C7 também se mostra uma ferramenta promissora para prevenir e regular a inflamação causada pela LDL (-) no curso da aterosclerose

Introduction: Atherosclerosis is a chronic inflammatory disease resulting from changes in the wall of medium and large-caliber arteries and associated with several risk factors, among which hyperlipidemias stand out, ie, the increase in plasma lipoproteins, but also other comorbidities, such as Metabolic Syndrome. Among the lipoproteins, low-density lipoprotein (LDL) is of great relevance in atherosclerosis. Different isoforms of modified LDL (LDLm) are originated through lipolysis, glycation and proteolysis, in addition to oxidation, varying in density and electronegativity, being better called electronegative LDL [LDL (-)]. Considering the conformational differences between the ApoB-100 structure of native LDL and LDL (-), in an initial study, our group developed a monoclonal antibody (2C7) from the immunization of Balb/c mice with human LDL (-). In a next step, the epitope recognized by the anti-LDL monoclonal antibody (-) was mapped using phage display. The binding peptide of anti-LDL monoclonal antibodies (-) was named p2C7. This peptide does not represent linear sequence regions of human ApoB-100, but conformational microdomains of LDL (-) ApoB-100 epitopes, making them candidates for the immunomodulation of atherogenesis. Therefore, investigating the immunomodulation induced by p2C7 peptide mimetics of LDL (-) as it represents an immunodominant epitope of LDL (-) could open new future therapeutic perspectives for the immunomodulation of atherosclerosis. Objective: To evaluate the immunomodulation promoted by p2C7 in vivo, using C57BL/6 LDLr -/- mice, and human plasma samples. In addition, in the internship (BEPE), held at the Karolinska Institute (December 2019 to March 2021), immunometabolism as a mediator of Cardiovascular Diseases was studied. In part II-A, the results of the initially proposed study are described. In part II-B, the results that were developed later are presented, expanding the scope of the project, approaching the vascular inflammation involved in the abdominal aortic aneurysm through bioinformatics tools. In part II-C, the results of the study of the involvement of the enzyme indoleamine 2,3 dioxygenase (IDO) in non-alcoholic steatohepatitis (NASH) and atherosclerosis in ApoE-/- and ApoE/IDO/double mice are presented -knockout. Methodology: The presence of anti-p2C7 antibodies in human plasma samples with or without Metabolic Syndrome was evaluated. We measured circulating TNF in the same samples and proceeded with linear regressions associating inflammatory parameters with levels of anti-p2C7 antibodies. C57BL/6 LDLr -/- mice were immunized with p2C7 and the adjuvants Alum or Montanide ISA 720, analyzing the antibody titers against p2C7 and LDL (-), the production of cytokines (IL-10, IL-4, IL -2, IL-6, IFNγ, IL-17, TNFα) and antibody-secreting cells. C57BL/6 LDLr -/- mice were tolerized against mimotope peptides with intravenous injections (caudal vein) and challenged with immunization containing LDL (-) + Alum. Antibody titers against p2C7 and LDL (-) and cytokine production (TNF-α, IFNγ, IL-12, IL-6, IL-10 and MCP-1) were evaluated. The mice were kept on a hypercholesterolemic diet for 3 months for atherosclerotic plaque formation. After this period, the mice were euthanized, evaluating the formation of atherosclerotic plaque in the abdominal artery and aortic arch, as well as the production of cytokines (TNF-α, IFNγ, IL-12, IL-6, IL-10 and MCP -1). C57BL/6 LDLr -/- mice were immunized with OVA-p2C7 and, after a 3-month hypercholesterolemic diet for atherosclerotic plaque formation, inflammatory parameters were evaluated and 18F-FDG uptake was evaluated by PET/CT. Results: Immunization with p2C7 (free) was not able to induce a humoral response, with no detectable titers of antibodies reactive to p2C7 or LDL (-) being observed in any immunized mouse, as well as no detectable antibody-secreting cells for the LDL (-). The group immunized with Alum or Montanide + p2C7 had a significant increase in TNF-α production when compared to the other groups. The tolerance protocol was successfully performed, as the tolerized mice had lower antibody titers than controls for the epitope used. Only mice tolerated with p2C7 showed a significant increase in the production of IL-6, IL-12, IL-10, TNF-α, IFNγ and MCP 1 after a hypercholesterolemic diet. Active immunization with OVA-p2C7 was able to reduce TNF production induced by the hypercholesterolemic diet, as well as to reduce 18F-FDG uptake. Conclusion: the p2C7 epitope is highly expressed in LDL (-) of patients with higher cardiovascular risk. Furthermore, active immunization with p2C7 is also a promising tool to prevent and regulate inflammation caused by LDL (-) in the course of atherosclerosis

Ligustrazine suppresses platelet aggregation through inhibiting the activities of calcium sensors

Abstract Ligustrazine is widely used for the treatment of cardiovascular diseases in traditional Chinese medication. It has been reported that Ligustrazine decreases the concentration of intracellular calcium ions (Ca2+); however, the underlying mechanism remains unknown. In the present study, the effect of Ligustrazine on adenosine diphosphate (ADP)-induced platelet aggregation was evaluated using a turbidimetric approach. The changes in concentration of intracellular Ca2+ stimulated by ADP was measured using fluo-4, a fluorescent Ca2+ indicator dye. The mRNA expression of stromal interaction molecule l (STIM1) and Orai1, calcium sensor, was determined using real-time PCR. In addition, the protein expression of STIM1, Orai1, and serum/glucocorticoid-regulated protein kinase 1 (SGK1) was determined using Western blot analysis. The data demonstrated that Ligustrazine significantly suppressed platelet aggregation in a dose-dependent manner and reduced the concentration of intracellular Ca2+ triggered by ADP. Our data showed that Ligustrazine treatment inhibited the expression of STIM1 and Orai1 induced by ADP at both mRNA and protein levels, and suppressed the protein expression of SGK1. Taken together, our data indicated that Ligustrazine suppressed platelet aggregation by partly inhibiting the activities of calcium sensors, thereby suggesting that Ligustrazine may be a promising candidate for the treatment of platelet aggregation.

Proteomics: a tool to investigate of composition and function of HDL in hyperlipidemia / Proteômica: uma ferramenta para a investigação da composição e função da HDL em hiperlipidemia

The inverse relationship between HDL-C (high-density lipoprotein cholesterol) and cardiovascular disease is well established. However, it is consensus that the cholesterol content present in HDL does not capture its complexity, and other metrics need to be explored. HDL is a heterogeneous, protein-enriched particle with functions going beyond lipid metabolism. In this way, its protein content seems to be attractive to investigate its behavior in the face of pathologies. Many of the proteins with important function in HDL are in low abundance (<1% of total proteins), which makes their detection challenging. Quantitative proteomics allows detecting proteins with high precision and robustness in complex matrix. However, quantitative proteomics is still poorly explored in the context of HDL. In this sense, in the second chapter of this thesis, the analytical performance of two quantitative methodologies was carefully investigated. These methods achieved adequate linearity and high precision using labeled peptides in a pool HDL, in addition to comparable ability to differentiate proteins from HDL subclasses of healthy subjects. Another bottleneck that waits for a solution in proteomics is the lack of standardization in data processing and analysis after mass spectrometry acquisition. In addition, interest in the cardioprotective properties of omega-3 is growing, but little is known about its effects on the HDL proteome. Thus, in the third chapter of this thesis, we compared five protein quantification strategies using Skyline and MaxDIA software platforms in order to investigate the HDL proteome from mice submitted to a high-fat diet supplemented or not with omega-3. MaxDIA with label-free quantification (MaxLFQ) achieved high precision to show that polyunsaturated fatty acids remodel the HDL proteome to a less inflammatory profile. Therefore, the two studies presented in this thesis begin to open new paths for a deeper and more reliable understanding of HDL, both at the level of protein quantification by mass spectrometry and after data acquisition

A inversa relação entre HDL-C (do inglês, high-density lipoprotein cholesterol) e doenças cardiovasculares é bem estabelecida. No entanto, é consenso que o conteúdo de colesterol presente na HDL não captura sua complexidade, e outras métricas precisam ser exploradas. A HDL é uma partícula heterogênea, enriquecida em proteínas, com funções que vão além do metabolismo de lipídeos. Dessa forma, seu conteúdo proteico parece ser mais atrativo para exprimir seu comportamento frente às patologias. Muitas das proteínas com função importante estão em baixa abundância (<1% do total de proteínas), o que torna a detecção desafiadora. Métodos quantitativos de proteômica permitem detectar proteínas com alta precisão e robustez em matrizes complexas. No entanto, a proteômica quantitativa ainda é pouco explorada no contexto da HDL. Nesse sentido, no segundo capítulo dessa tese, a performance analítica de dois métodos quantitativos foi criteriosamente investigada, os quais alcançaram adequada linearidade e alta precisão usando peptídeos marcados em um pool de HDL, além de comparável habilidade em diferenciar as proteínas das subclasses da HDL de indivíduos saudáveis. Outro gargalo que aguarda por solução em proteômica é a falta de padronização no processamento e análise de dados após a aquisição por espectrometria de massas. Além disso, é crescente o interesse das propriedades cardioprotetivas do ômega-3, porém pouco se conhece sobre seus efeitos no proteoma da HDL. Então, no terceiro capítulo dessa tese, comparamos cinco estratégias de quantificação de proteínas utilizando os softwares Skyline e MaxDIA com o intuito de comparar o proteoma da HDL de camundongos submetidos a uma dieta hiperlipídica suplementados ou não com ômega-3. MaxDIA com quantificação label-free (MaxLFQ) apresentou alta precisão para mostrar que o ômega-3 remodela o proteoma da HDL para um perfil menos inflamatório. Portanto, os dois estudos apresentados nessa tesa começam a abrir novos caminhos para o entendimento mais profundo e confiável da HDL tanto por meio da quantificação das proteínas por espectrometria de massas quanto após à aquisição dos dados

Clinacanthus nutans: Its potential against diabetic vascular diseases

Diabetes is an independent risk factor for the development of cardiovascular disease, with approximately 80% of cardiovascular mortality and morbidity linked to vascular complications such as atherosclerosis. It has been estimated that up to one-third of patients with diabetes mellitus use some form of complementary and/or alternative medicine. One plant that has received attention from diabetic patients for its perceived antidiabetic properties is Clinacanthus nutans, a member of the Acanthaceae family that is known as snake grass. Ethnomedical applications of this herb have been identified for the treatment of certain conditions, including fever, diabetes, skin rashes, and insect bites. This review aims to assess the potential of C. nutans to be used in the prevention and/or treatment of diabetic vasculopathy. Evidence for antidiabetic, anti-inflammatory, and dyslipidemic properties of C. nutans, as shown from experimental studies, is presented and discussed. Diabetes, inflammation, and hyperlipidemia are known to play significant roles in the initiation and severity of diabetic cardiovascular disease; thus, targeting these factors might be beneficial for preventing and/or treating diabetic vasculopathy.

Gαq-RGS2 loop activator modulates the activity of vario us agonists on isolated heart and aorta of normal rats

The Gαq-RGS2 loop activator, 1-(5-chloro-2-hydroxyphenyl)-3-(4-(trifluoromethyl)-phenyl)-1H-1,2,4-triazol-5(4H)-one has demonstrated Gαq signaling inhibitor activity. Therefore, we aimed to study the effect of Gαq-RGS2 loop activator on isolated heart and aorta of normal rats. Heart and aorta were isolated from the sacrificed rats (n=6) and mounted on the langendroff's and organ bath assembly, respectively. The effect of various receptor-dependent (acetylcholine, angiotensin II and adrenaline) and independent (calcium chloride and sodium nitroprusside) agonists in absence and presence of Gαq-RGS2 loop activator on left ventricular systolic pressure (LVSP) and the contractile responseswere evaluated in isolated heart and aorta, respectively. Gαq-RGS2 loop activator (100 µM) significantly attenuated the adrenaline (p<0.001,) and angiotensin II (p<0.001) induced increase in LVSP in isolated heart and contractile response of adrenaline (p<0.01) and angiotensin II (p<0.01) in the aorta. However, effect calcium chloride did not significantly alter by Gαq-RGS2 loop activator. The effect of acetylcholinewas significantly (p<0.01, p<0.05) increased by Gαq-RGS2 loop activator in isolated heart and aorta. The effect of sodium nitroprusside significantly (p<0.01) potentiated by Gαq-RGS2 loop activator (100 µM) in isolated heart while it did not significantly alters in the aorta. Ultimately, the Gαq-RGS2 loop activator modulated the action of receptor-dependent agonists in isolated heart and aorta

Evaluation of Glucosamine Effect Against Heart and Brain Damage Induced by Y-radiation or Aluminium Chloride in Female Rats

Abstract Glucosamine is known as anti-inflammatory, antioxidant and as neuroprotective as well as using to treat many of diseases. This work aimed to investigate the remedial effect of glucosamine (20mg/kg b.wt) against the damage induced by a single dose of γ-radiation (8Gy) or aluminium chloride (AlCl3) (100mg/kg b.wt) in the heart and brain tissues of female rats. Serum aspartate aminotransferase (AST), cholesterol, triglycerides (TGs), LDH and creatine kinase (CPK) were measured. Moreover, gene expression of amyloid protein precursor (APP) and seladin-1 were estimated in the brain tissue. Also, acetylcholinesterase activity (AChE) and p-tau protein expression were estimated in brain homogenate. Metallothioneine (MT) was estimated in the heart and brain tissues. Heart and brain histopathological examination was performed. Irradiation significantly decreased serum AST, CPK and LDH, as well as MT levels in heart and brain tissues. Also, gene expression of seladin-1 decreased. On the other hand, irradiation significantly increased serum TGs level and brain AchE activity, tau protein, and β-amyloid percursor (APP). AlCl3 administration (21 days) induced disturbance in most of the estimated parameters, especially AST, TGs, and MT. Glucosamine treatment with irradiation or AlCl3 improved most of the measured parameters. In addition, histopathological examination confirmed the biochemical results. In conclusion: Glucosamine could be used to improve the heart and brain damages induced by γ-radiation exposure or AlCl3.

Efectos cardiovasculares en usuarios de cocaína / Cardiovascular effects in cocaine users

Resumen La cocaína es una sustancia psicoactiva ampliamente utilizada de forma recreativa. El uso indiscriminado de esta sustancia genera múltiples consultas a los servicios de emergencias, principalmente debido a sus efectos cardiovasculares. Los mecanismos fisiopatológicos actúan principalmente en sistema nervioso simpático, cardiomiocito, plaquetas, canales de sodio y potasio, entre otros. El objetivo principal de esta revisión es demostrar los efectos cardiovasculares asociados al uso de la cocaína.

Abstract Cocaine is a psychoactive substance widely used recreationally. The indiscriminate use of this substance generates multiple consultations to the emergency services, mainly due to its cardiovascular effects. The physio pathological mechanisms act mainly in sympathetic nervous system, cardiomyocyte, platelets, sodium and potassium channels, among others. The main objective of this review is to demonstrate the cardiovascular effects associated with the use of cocaine.

The relation between echocardiographic epicardial fat thickness and cha2ds2-vasc score in patients with sinus rhythm

Abstract Objective: To evaluate the predictive value of epicardial fat thickness (EFT) in CHA2DS2-VASc (congestive heart failure, hypertension, age ≥75 years, diabetes mellitus, previous stroke or transient ischemic attack, vascular disease, age 65-74 years, sex category) score risk groups. Methods: A total of 158 consecutive patients (75 females, 83 males, mean age 70.8±6.3 years) admitted routinely for cardiologic control were divided into two groups according to their CHA2DS2-VASc scores (scores 0 and 1 were regarded as low risk, and score ≥2 as high risk). One hundred twenty-five of 158 patients had a high-risk score. Results: Mean EFT was significantly higher in the high-risk group than in the low-risk group (4.34±0.62 vs. 5.37±1.0; P<0.001). EFT was positively correlated with CHA2DS2-VASc score (r=0.577, P<0.001). According to receiver operating characteristics (ROC) analysis, EFT value of 4.4 mm was found to be predictive of high risk in CHA2DS2-VASc score with 80% of sensitivity and 79% of specificity (C-statistic = 0.875, P<0.001, 95% confidence interval [CI] = 0.76-0.90). And according to multivariate logistic regression analysis, EFT was an independent predictor of high thromboembolic risk in terms of CHA2DS2-VASc score. Conclusion: Our findings suggest that echocardiographic EFT measurement could provide additional information on assessing cardiovascular risks, such as thromboembolic events, and individuals with increased EFT should receive more attention to reduce unfavorable cardiovascular risk factors and the development of future cardiovascular events.

Estudo de riscos cardiovasculares em adolescentes (Erica): resultados principais e perspectivas / Perspectivas study of cardiovascular risks in adolescents (Erica): main results and perspectives

As doenças cardiovasculares são a principal causa de morte no Brasil. Os fatores de risco modificáveis dessas doenças podem ser observados desde a infância e sua persistência está associada ao diagnóstico precoce de morbidades. Neste contexto, a adolescência é considerada um período chave para estratégias de prevenção. Com objetivo de avaliar a prevalência de fatores de risco cardiovascular e seus fatores as-sociados foi planejado o Estudo de riscos cardiovasculares em adolescentes (ERICA), um inquérito multicêntrico de base escolar com abrangência nacional que envolveu mais de 70 mil adolescentes, entre 12 e 17 anos, de cidades brasileiras com mais de 100 mil habitantes. Os principais resultados do estudo apontaram prevalências preo-cupantes de sobrepeso/obesidade (25,5%, IC 95%: 24,4%-26,6%), pressão arterial elevada (9,6%, IC 95%: 8,9%-10,3%) e HDL-c baixo (47,3%, IC 95%: 45,2%-49,3%). A prevalência de síndrome metabólica foi de 2,6% (IC 95%: 2,3%-2,9%). Além disso, mais da metade dos adolescentes reportaram um estilo de vida sedentário. As estimativas regionais apontaram a região sul como a que tem maiores prevalências de fatores de risco. O ERICA foi um estudo pioneiro no Brasil e os adolescentes que participaram do estudo no Rio de Janeiro, Porto Alegre, Brasília e Fortaleza serão acompanhados em uma nova etapa. Os primeiros resultados do ERICA podem servir de referência para futuras pesquisas sobre riscos cardiovasculares entre adolescentes brasileiros, bem como subsidiar políticas públicas de saúde.

Cardiovascular disease is the leading cause of mortality in Brazil. Modifiable risk factors for this group of diseases can be observed since childhood, and their persisten-ce is associated with the early diagnosis of morbidities. In this context, adolescence is considered a key period for prevention strategies. In order to evaluate the prevalence of cardiovascular risk factors and their associated factors, the Study of Cardiovascular Risks in Adolescents ("ERICA") was planned. This is a multicenter school-based nationwide survey involving more than 70,000 adolescents aged between 12 and 17 years from Brazilian cities with more than 100 thousand inhabitants. The main results of the study indicated concerning prevalence of overweight/obesity (25.5%, 95% CI: 24.4%-26.6%), high blood pressure (9.6%, 95% CI: 8.9%-10.3%) and low HDL-c (47.3%, 95% CI: 45.2%-49.3%). The prevalence of metabolic syndrome was 2.6% (95% CI: 2.3%-2.9%). In addition, more than half of adolescents reported a sedentary lifestyle. Regional estimates pointed to the south as the region with the highest prevalence of risk factors. ERICA was a pioneering study in Brazil and the adolescents who participated in the study in Rio de Janeiro, Porto Alegre, Brasília and Fortaleza will be followed in a new stage. The first results of ERICA can both serve as a reference for future research on cardiovascular risks among Brazilian adolescents and support public health policies.

Kinin B2-Receptor in human iPSC differentiation into cardiomyocytes / Receptor B2 de cininas na diferenciação de iPSC humanas em cardiomiócitos

Cardiovascular diseases are responsible for almost one third of all global deaths yearly, and therefore are largely studied. Cardiomyocytes derived from human induced pluripotent stem cells (hiPSC-CM) have emerged as an exciting technology for cardiac disease modelling and personalised therapy. Nevertheless, issues concerning functional and molecular maturation are still faced. In addition to this, differentiation protocols generally yield a heterogeneous mixed population comprised of nodal, atrial and ventricular-like subtypes, being unsuitable for therapeutic purposes. Bradykinin (BK) is a vasoactive peptide which exerts important physiological roles in the cardiovascular system, having been previously described as important for cellular, keratinocyte and skeletal muscle differentiation. This project performed in cooperation with PluriCell Biotech, a startup specialized in the production and differentiation of hiPSC-CM, has sought (1) characterizing gene and protein expression of molecular markers of maturation and of subtype specification throughout of differentiation; (2) Assessing the electrical functionality of hiPSC-CM through the characterization of subtype-specific action potentials (APs) and (3) Investigating whether the progress of hiPSCCM maturation is regulated by BK through kinin-B2 receptors (B2R). Our results have validated the model that proposes a developmental-dependent switch between skeletal (ssTnI) and cardiac (cTnI) isoforms of troponin I as differentiation progresses, at least to some extent. Furthermore, prolonged time in culture has resulted in higher levels of expression of the ventricular marker MLC2v and in increased rates of ventricular-like action APs. Electrophysiological analysis of hiPSC-CM reveals a mixed population with AP morphologies correspondent to nodal, atrial and ventricular subtypes, all showing pronounced automaticity as well as other features of immature cardiomyocytes, such as low amplitude and depolarization velocity. Such findings are coherent with those from other groups who have attempted to differentiate mature native-like cardiac cells from pluripotent stem cells sources, without fully succeeding. After showing that differentiating hiPSC-CM express a functional and responsive B2R, the receptor was subjected to chronic activation with 10µM BK and 1µM BK or inhibition with 5µM Firazyr+BK. Even though B2R modulation has not interfered negatively with differentiation yields nor cell morphology, analysis of gene andprotein expression of ssTnI or cTnI and of the ventricular marker MLC2v, have revealed no significant results in comparison to untreated controls. This suggests that BK does not interfere on hiPSC-CM maturation nor subtype specification, although we cannot rule out that it could be leading to other unexplored effects. We recommend a closer look into which intracellular signalling pathways become active upon B2R stimulation in hiPSC-CM, in order to narrow down cellular processes for further investigation

Doenças cardiovasculares são responsáveis por quase um terço de todas as mortes globais anualmente, e por isto o sistema cardiovascular é amplamente estudado. Cardiomiócitos derivados a partir de células-tronco pluripotentes induzidas humanas (hiPSCCM) emergiram como uma promissora tecnologia para modelagem de doenças cardíacas e terapia personalizada. No entanto, desafios acerca de sua maturação funcional e molecular ainda são enfrentados. Além disso, protocolos de diferenciação geralmente levam à obtenção de populações heterogêneas contendo células com fenótipos similares aos de cardiomiócitos nodais, atriais e ventriculares sendo, portanto, inapropriadas para fins terapêuticos. A bradicinina (BK) é um peptídio vasoativo que exerce importantes papeis fisiológicos no sistema cardiovascular, além de ter sido previamente descrita como importante para a diferenciação neuronal, de queratinócitos e de músculo esquelético. Este projeto foi realizado em colaboração com a empresa PluriCell Biotech, uma startup especializada na produção e diferenciação de hiPSC-CM, e buscou (1) caracterizar a expressão gênica e proteíca de marcadores moleculares de maturação e de especificação de subtipos cardíacos durante a diferenciação; (2) avaliar a funcionalidade elétrica de hiPSC-CM por meio da caracterização de seus potenciais de ação (PAs) e (3) Investigar se o progresso da diferenciação de hiPSCCM é regulado por bradicinina por meio do receptor B2 (B2R). Nossos resultados validaram o modelo que propõe um switch na expressão das isoformas funcionais de troponina I esquelética (ssTnI) e cardíaca (cTnI), durante o desenvolvimento e diferenciação celular, pelo menos parcialmente. Além disso, tempo prolongado em cultura resultou em maiores níveis de expressão do marcador ventricular MLC2v, assim como maiores frequências de PAs com morfologias similares a de cardiomiócitos ventriculares. Análise eletrofisiológica de hiPSCCM revelam a existência de uma população mista contendo PAs correspondentes aos subtipos nodais, atriais e ventriculares, assim como pronunciada automaticidade e outros atributos típicos de cardiomiócitos imaturos, como baixa amplitude e devagar velocidade de despolarização. Estes resultados são coerentes com os de outros grupos que ainda não foram totalmente bem-sucedidos em diferenciar células cardíacas maduras similares acardiomiócitos nativos a partir de células-troncos pluripotentes. Após mostrar que as hiPSCCM expressam receptores B2 funcionais e responsivos, submetemos o receptor a uma ativação crônica com BK 10µM e BK 1µM ou inibição crônica com Firazyr 5µM + BK. Apesar da modulação do B2R não ter interferido de forma negativa no rendimento da diferenciação ou na morfologia celular, análise de expressão gênica e proteica de ssTnI e cTnI e do marcador ventricular MLC2v não revelou resultados significativos em comparação aos controles não-tratados. Isto sugere que a BK não interfere na maturação e especificação de subtipos cardíacos em hiPSC-CM, apesar de não podermos ignorar o fato de que ela poderia estar desencadeando outros efeitos inexplorados. Nós recomendamos um estudo mais aprofundado acerca de quais vias de sinalização se tornam ativas após estimulação do receptor B2 em hiPSC-CM, com o objetivo de afunilar quais processos celulares poderiam ser investigados em uma próxima etapa deste estudo

Diabetes Mellitus: a raiz do problema / Diabetes mellitus: the root of the problem

A doença cardiovascular (DCV) de origem aterosclerótica é a principal causa da morbidade e mortalidade em pacientes com diabetes mellitus (DM). Tanto os fatores de risco cardiovascular associados à resistência à insulina (RI) no contexto da síndrome da adiposidade visceral (SAV) quanto a hiperglicemia crônica contribuem para o risco da DCV na DM. A hiperinsulinemia compensatória que se estabelece na RI estimula os fatores de transcrição SREBP1c e SREBP1 em que se ativam os genes lipogênicos, levando à grande produção hepática de triglicérides. A hipertrigliceridemia é o gatilho para as demais alterações lipídicas que contribuem para o perfil pró-aterogênico na RI, caracterizando-se pelo predomínio de LDL pequenas e densas e redução do colesterol HDL. A hiperinsulinemia, também, está intimamente ligada à hipertensão arterial, pois aumenta o tônus simpático e a reabsorção renal de sódio. A RI é considerada o melhor fator preditivo para a ocorrência de DM tipo 2 (DM2), sendo necessário um defeito concomitante na secreção de insulina para que a hiperglicemia se estabeleça. Os efeitos deletérios da hiperglicemia devem-se à ativação de vias bioquímicas que resultam em inflamação e estresse oxidativo celular. A dislipidemia e a hipertensão arterial secundárias à RI, assim como a hiperglicemia, são importantes moduladores do risco cardiovascular na SAV e na DM2 e devem ser intensiva e conjuntamente abordados no tratamento e prevenção da DCV.

Cardiovascular disease (CVD) of atherosclerotic origin is the main cause of morbidity and mortality in patients with diabetes mellitus (DM). Both the cardiovascular risk factors associated with insulin resistance (IR) in the context of visceral adiposity syndrome (VAS) and chronic hyperglycemia contribute to the risk of CVD in DM. Compensatory hyperinsulinemia established in IR stimulates the transcription factors SREBP1c and SREBP1a, which activate lipogenic genes, leading to high hepatic production of triglycerides. Hypertriglyceridemia triggers other lipid changes that contribute to the pro-atherogenic profile in IR, which is characterized by the predominance of small and dense LDL and reduction of HDL-cholesterol. Hyperinsulinemia is also closely linked to arterial hypertension, as it increases sympathetic tone and renal sodium reabsorption. IR is considered the best predictive factor for the occurrence of type 2 DM (DM2), and a concomitant defect in insulin secretion is required for hyperglycemia to be established. The harmful effects of hyperglycemia are due to activation of biochemical pathways that result in inflammation and cellular oxidative stress. Dyslipidemia and hypertension secondary to IR, as well as hyperglycemia, are important modulators of cardiovascular risk in VAS and DM2 and should be intensively and jointly addressed in the management and prevention of CVD.

Aberrant Origin of Vertebral Artery and its Clinical Implications

Abstract Aberrant origin of vertebral artery is rare. The anatomical features and clinical significance of this lesion remain to be clarified. A comprehensive collection of the pertinent literature resulted in a cohort of 1286 cases involving 955 patients and 331 cadavers. There were more left than right and more unilateral than bilateral aberrant vertebral arteries. Patients with aberrant origin of vertebral artery were often asymptomatic and in only 5.5% of the patients their symptoms were probably related to the aberrant origin of vertebral artery. The acquired cardiovascular lesions were present in 9.5% of the patients, 20.9% of which were vertebral artery-associated lesions. Eight (0.8%) patients had a vertebral artery dissection. Logistic regression analysis showed significant regressions between bovine trunk and left vertebral artery (P=0.000), between the dual origins of vertebral artery and cerebral infarct/thrombus (P=0.041), between associated alternative congenital vascular variants and cervical/aortic dissection/atherosclerosis (P=0.008). Multiple logistic regression demonstrated that side of the aberrant origin of vertebral artery (left vertebral artery) (P=0.014), arch branch pattern (direct arch origin) (P=0.019), presence of the common trunk (P=0.019), associated acquired vascular disorder (P=0.034) and the patients who warranted management (P=0.000) were significant risk predictors for neurological sequelea. The patients with neurological symptoms and those for neck and chest operations/ interventions should be carefully screened for the possibility of an aberrant origin of vertebral artery. The results from the cadaver metrology study are very helpful in the design of the aortic stent. The arch branch pattern has to be taken into consideration before any maneuver in the local region so as to avoid unexpected events in relation to aberrant vertebral artery.

Autofagia en el sistema cardiovascular: pasado, presente y futuro / Cardiovascular autophagy: past, present and future

La enfermedad cardiovascular se mantiene como la principal causa de morbimortalidad a nivel mundial a pesar de los avances científicos y tecnológicos recientes, por esto existe la necesidad de búsqueda de nuevas dianas terapéuticas. La autofagia es un mecanismo de degradación de proteínas y organelos disfuncionales que ocurre en vacuolas especializadas de doble membrana denominadas autofagosomas y que requiere la participación de los lisosomas. Este proceso permite el auto abastecimiento celular de energía a través del reciclaje de diversos substratos energéticos. Se activa en respuesta a diversas formas de estrés, principalmente debido a la ausencia de nutrientes y su presencia ha sido caracterizada en todos los tipos celulares que componen el sistema cardiovascular. Existe una ventana de actividad de autofagia óptima la que se relaciona con la mantención de la homeostasis cardiovascular y su desregulación participa en la patogénesis de diversas patologías cardiovasculares. En este artículo se revisa el curso temporal que llevó el descubrimiento de la autofagia, la contribución al área del Dr. Ohsumi, reciente Premio Nobel de Medicina, los principales conceptos, mecanismos celulares y moleculares de la formación del auto-fagosoma, nodos de regulación y sintetizamos su participación en la homeostasis del corazón y en la patogénesis de las enfermedades cardiovasculares y sus perspectivas futuras.

Cardiovascular disease continues to be the leading cause of morbi-mortality worldwide despite the recent scientific and technological advances. Therefore, more research is needed to discover novel therapeutic targets. Autophagy mediates the removal of dysfunctional proteins and organelles. This process takes place in double-membrane vesicles, named autophagosomes, which later fuse with lysosomes. The mechanism allows self-renewal energy repletion through diverse energy substrate recycling. Diverse forms of cellular stress, mainly nutrient deprivation, activate this process. Autophagy has been widely characterized within the cells of the cardiovascular system. There is a window of optimal autophagy activity implicated in maintaining cardiovascular homeostasis and its dysregulation participates in the pathogenesis of different cardiovascular diseases. In this article, we review the time course of auto-phagy discovery, the Nobel Prize winner Dr. Ohsumi contribution, main concepts, mechanisms involved in autophagosome formation and its regulatory no-des. Additionally, we summarized the role of auto-phagy in cardiovascular homeostasis and pathogenesis and future perspectives.

Microparticles as Potential Biomarkers of Cardiovascular Disease / Micropartículas como Possíveis Biomarcadores da Doença Cardiovascular

Primary prevention of cardiovascular disease is a choice of great relevance because of its impact on health. Some biomarkers, such as microparticles derived from different cell populations, have been considered useful in the assessment of cardiovascular disease. Microparticles are released by the membrane structures of different cell types upon activation or apoptosis, and are present in the plasma of healthy individuals (in levels considered physiological) and in patients with different pathologies. Many studies have suggested an association between microparticles and different pathological conditions, mainly the relationship with the development of cardiovascular diseases. Moreover, the effects of different lipid-lowering therapies have been described in regard to measurement of microparticles. The studies are still controversial regarding the levels of microparticles that can be considered pathological. In addition, the methodologies used still vary, suggesting the need for standardization of the different protocols applied, aiming at using microparticles as biomarkers in clinical practice.

A prevenção primária da doença cardiovascular constitui uma opção de grande relevância pelos seus impactos na saúde. Alguns biomarcadores têm sido considerados úteis na avaliação da doença cardiovascular, dentre eles micropartículas originadas de diferentes populações de células. Micropartículas são estruturas liberadas pela membrana de diferentes tipos celulares após ativação ou apoptose, presentes tanto no plasma de indivíduos saudáveis (níveis considerados fisiológicos) quanto em portadores de diferentes doenças. Muitos estudos têm sugerido uma associação entre micropartículas e diferentes condições patológicas, destacando-se a relação com o desenvolvimento das doenças cardiovasculares. Além disso, têm sido descritos os efeitos de diferentes terapias hipolipemiantes na mensuração de micropartículas. Os estudos ainda são controversos quanto aos níveis de micropartículas que possam ser considerados patológicos, e os métodos utilizados ainda são variados, o que sugere a necessidade da padronização dos diferentes protocolos utilizados, visando à utilização de micropartículas como biomarcadores úteis na prática clínica.

Efeito da administração In Bolus da heparina sódica no remodelamento de partículas lepoprotéicas associadas ao transporte reverso do colesterol / In bolus administration the effect of heparin sodium in remodeling lepoprotéicas particles associated with reverse cholesterol transport

Introdução: as doenças cardiovasculares acometem milhares de pessoas no mundo. Destas, a doença arterosclerótica está entre as de maior morbimortalidade. Para a avaliação da necessidade de intervenções hemodinâmicas e/ou revascularização miocárdica, há a necessidade da realização do cateterismo (CATE), procedimento de imagem indicado para evidenciar pontos de obstrução e determinar a melhor estratégia cirúrgica. Para a realização do CATE utiliza-se heparina sódica (5000 UI) in bolus. Atualmente, sabe-se que a heparina interfere no remodelamento de partículas lipoproteicas por liberação da lipoproteína lipase (LPL) e da lipase hepática (LH), essa ação pode alterar o transporte reverso do colesterol (TRC), em função de modificações no metabolismo das lipoproteínas. Métodos: foram selecionados por conveniência 20 pacientes, 10 do sexo masculino e 10 do sexo feminino, ambos os sexos, entre 45 e 73 anos, admitidos no Hospital Ana Neri, submetidos à cineangiocoronariografia (CATE)...

Introduction: cardiovascular diseases affect thousands of people worldwide. Of these, the atherosclerotic disease is one of the most morbidity and mortality. To evaluate the need for hemodynamic interventions and / or CABG, the catheterization (CATE) is performed, an imaging procedure to evidence obstruction and to determine the best surgical strategy. To perform CATE, is necessary to use in bolus sodium heparin (5000 IU). Currently, it is known that heparin interferes with the remodeling of the lipoprotein particles by releasing lipoprotein lipase (LPL) and hepatic lipase (HL), this action may alter the reverse cholesterol transport (TRC), by changes in lipoprotein metabolism. Methods: were selected by convenience 20 patients, 10 male and 10 female, both gender, between 45 and 73 years old, admitted to the Hospital Ana Neri, who underwent coronary angiography (CATE)...

Caracterización de los pacientes en una consulta de hipertensión arterial de Dosquebradas: Risaralda-Colombia: 2008-2012 / Patients characterization in one high blood pressure consultation from Dosquebradas: Risaralda-Colombia: 2008-2012

El objetivo fue describir las características de los pacientes evaluados en la consulta de hipertensión arterial del Hospital Santa Mónica de Dosquebradas, Risaralda, Colombia, atendidos en el período 2008-2012. Estudio observacional descriptivo realizado en el Hospital Santa Mónica de Dosquebradas, Risaralda, Colombia. Los pacientes corresponden a la población atendida en el programa de detección de hipertensión arterial con sospecha de la enfermedad que fueron ingresados a la consulta de hipertensión arterial del Hospital. Se evaluaron variables demográficas y clínicas, incluyendo presión arterial sistólica y diastólica, entre otras. De un total de 8900 pacientes con evaluación de la presión arterial en el período, se atendieron en promedio 1780 por año (±934), el promedio por mes durante el período fue de 148 pacientes (intervalo 93-286). De los pacientes 73,3% correspondió al sexo femenino y 26,7% al masculino. La edad promedio de los mismos fue de 62,91 años (±13,09). Del total de pacientes, 92,57% (IC95% 92,02-93,12) presentaban valores de prehipertensión/hipertensión arterial, siendo mayor en las mujeres de > igual 70 años (93,7%) que en las mujeres < 70 años (92,1%) (OR=1,269; IC95% 1,026-1,569). No se observaron diferencias significativas por edad en los pacientes del sexo masculino. El presente estudio permitió caracterizar una población de casi 9000 pacientes que acudieron a la consulta de hipertensión arterial del hospital de referencia del municipio durante 5 años, del cual no existen estudios previos y que muestra una considerable proporción de población con alteraciones de la presión arterial, especialmente en mujeres de más de 70 años.

The objetive was to describe the characteristics of patients evaluated at the high blood pressure consultation of the Hospital Santa Monica, Dosquebradas, Risaralda, Colombia, attended between 2008-2012. Observational, descriptive study realized in the Hospital Santa Monica,Dosquebradas, Risaralda, Colombia. The patients comes from the population attended at the hypertension screening program with disease who were admitted to the Hospital with high blood pressure diagnosis. We evaluated demographic and clinical variables, including systolic and diastolic blood pressure, among others. Results: from a total of 8900 patients with blood pressure assessment in the period, were attended on average 1780 per year(±934), the average per month during the period was 148 patients (range 93-286). Of the patients 73.3% were female and 26.7% male. The average age of them was 62.91 years (±13.09). Of the patients, 92.57% (95%CI 92.02-93.12) had values of prehypertension/hypertension, being higher in women < igual 70 years (93.7%) that in woman < 70 years (92.1%) (OR=1.269;95% CI 1.026-1.569). There were no significant differences by age in male patients. This study allowed us to characterize a population of nearly 9000 patients who were attended at the reference hospital of municipality for five years, of which there are no previous studies that show a considerable proportion of the population with blood pressure changes, especially in women over 70 years.