CD36 (SR-B2) as a Target to Treat Lipid Overload-Induced Cardiac Dysfunction

The heart faces the challenge of adjusting the rate of fatty acid uptake to match myocardial demand for energy provision at any given moment, avoiding both too low uptake rates, which could elicit an energy deficit, and too high uptake rates, which pose the risk of excess lipid accumulation and lipotoxicity. The transmembrane glycoprotein cluster of differentiation 36 (CD36), a scavenger receptor (B2), serves many functions in lipid metabolism and signaling. In the heart, CD36 is the main sarcolemmal lipid transporter involved in the rate-limiting kinetic step in cardiac lipid utilization. The cellular fatty acid uptake rate is determined by the presence of CD36 at the cell surface, which is regulated by subcellular vesicular recycling from endosomes to the sarcolemma. CD36 has been implicated in dysregulated fatty acid and lipid metabolism in pathophysiological conditions, particularly high-fat diet-induced insulin resistance and diabetic cardiomyopathy. Thus, in conditions of chronic lipid overload, high levels of CD36 are moved to the sarcolemma, setting the heart on a route towards increased lipid uptake, excessive lipid accumulation, insulin resistance, and eventually contractile dysfunction. Insight into the subcellular trafficking machinery of CD36 will provide novel targets to treat the lipid-overloaded heart. A screen for CD36-dedicated trafficking proteins found that vacuolar-type H⁺-ATPase and specific vesicle-associated membrane proteins, among others, were uniquely involved in CD36 recycling. Preliminary data suggest that these proteins may offer clues on how to manipulate myocardial lipid uptake, and thus could be promising targets for metabolic intervention therapy to treat the failing heart.

Papel do receptor B2 de cininas na terapia da neurodegeneração dopaminérgica em modelo animal / Targeting Kinin-B2 receptors for the treatment of dopaminergic neurodegeneration in an animal mode

A Doença de Parkinson (DP) é um distúrbio neurodegenerativo, caracterizada em parte pela perda de neurônios dopaminérgicos da via nigroestriatal, originada na substância negra com projeções para o estriado, causando vários déficits motores. Atualmente, o tratamento mais utilizado é a administração de L-DOPA, um análogo da dopamina. Porém, essa droga apresenta eficácia limitada e induz diversos efeitos colaterais. A exploração dos efeitos neuroprotetores, proliferativos e neuroregenerativos da bradicinina (BK) em modelo animal de DP pode conduzir à substituição celular do tecido lesionado pela 6-hidroxidopamina (6-OHDA). De fato, a BK e seus receptores possuem um grande espectro de ações fisiológicas, estando classicamente envolvida no controle da homeostase cardiovascular e inflamação, além de exercer efeitos protetores em fisiopatologias do sistema nervoso, como em modelos de acidente vascular cerebral. Vários tipos celulares têm suas vias de sinalização associadas à ativação do receptor B2 de cininas (B2BKR). Trabalhos anteriores de nosso grupo mostraram que a BK está envolvida na diferenciação neural de células progenitoras neurais por um loop autócrino que resulta em ativação do B2BKR. Os resultados apresentados neste trabalho mostram a eficácia do tratamento com BK, um agonista de B2BKR, em animais submetidos à lesão da via nigro-estriatal induzida por 6-OHDA. Além disso, há uma recuperação comportamental e histológica desses animais quando tratados com Captopril®, um potencializador dos efeitos farmacológicos da BK, e com [Phe8Ψ(CH-NH)Arg9]-Bradicinina, agonista estável do receptor B2BKR. Assim, concluímos que a ativação de B2BKR pela BK desencadeiaum processo de neuroregeneração dopaminérgica de animais submetidos à lesão por 6-OHDA. Trabalhos recentes mostram que o receptor B2BKR desempenha um importante papel neuroprotetor em modelo animal da Doença de Alzheimer, o que corrobora nossos achados. Juntos, esses resultados contribuem para o estabelecimento da ação neuroprotetora e neurorregenerativa da BK no modelo de animal de neurodegeneração dopaminérgica, tornando-a uma excelente candidata para aplicação em terapias de reparo neuronal

Parkinson's disease (PD) is a neurodegenerative disorder partially characterized by the loss of dopaminergic neurons from the nigrostriatal pathway, originated in the substantia nigra with projections to the striatum, which causes several motor deficits. Currently, the most commonly used drug for PD treatment is levodopa. However, it has limited efficacy and induces several side effects. Elucidation of the neuroprotective, proliferative and neuroregenerative effects of bradykinin (BK) in animal models of PD can culminate in cellular replacement of the tissue damaged by 6-hydroxydopamine (6-OHDA). In fact, BK and its receptor have several physiological effects, being classically involved in the control of cardiovascular homeostasis and inflammation. Besides, BK exerts protective effects on nervous system pathophysiology, as observed in stroke models. Several cell types have their signaling pathways associated with the B2 kinin receptor (B2BKR) activation. Previous work from our group showed that BK is involved in differentiation of neural progenitor cells by an autocrine loop that results in activation of B2BKR. The results presented in this thesis show the efficacy of treatment with BK, through B2BKR activation, in animals submitted to nigrostriatal pathway injury induced by 6-OH dopamine. Furthermore, behavioral and histological recoveries of these animals were observed when treated with Captopril®, a potentiator of BK pharmacological effects, and with [Phe8Ψ (CH-NH) Arg9] -BK, a stable agonist of the B2BKR receptor. Thus, we conclude that BK activation of B2BKR triggers neuroregenerative processes in animals submitted to 6- OHDA injury. Recent studies showed that the B2BKR receptor plays an important neuroprotective role in an animal model of Alzheimer's disease, which corroboratesour findings. Together, these results contribute to the establishment of the neuroprotective and neuroregenerative actions of BK - an excellent candidate for neural repair therapies

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

Expression and distribution of kinin B2 receptor in medulla and spinal cord tissues of Rats treated with capsaicin / Expresión y distribución del receptor B2 de cininas en la médula y tejido medular espinal de ratones tratados con capsaicina

Kinins are vasoactive peptides that promote pain and inflammation, yet centrally believed to participate to cardiovascular defensive reflexes produced by noxious stimuli. These peptides signal through the activation of two transmembrane G-protein-coupled receptors named B1 and B2 receptors (B1R and B2R). The B2R is constitutive in healthy tissues and animals. The aim of the study was to measure the gene and protein expression of B2R kinin receptors in central and peripheral tissues isolated from control rats and rats were pre-treated with capsaicin on the second day of life (50 mg/kg, s.c.) or two weeks prior to sacrifice (125 mg/kg over three days, s.c.). The same treatment with saline was made in control animals. Levels of mRNA for B2R were measured by quantitative RT-PCR and Qualitative while receptor binding sites were measured on tissue sections with the radioligands 125I-HPP-Hoe 140 (B2R). B2R was expressed in all studied tissues (hypothalamus, paratrigeminal nucleus, nucleus of solitary tract, spinal cord, aorta and liver) and treatment capsaicin neonates when compared to controls, did not affect its level of expression. Capsaicin had no significant effect on the expression of B2R in some tissues on binding sites. The synthesis of B2R kinin receptor is not associated with sensory C-fibre and tissues showed no significant difference indicating that B2R was regulated by distinct mechanisms.

Las cininas son péptidos vaso activos que participan de mecanismos de dolor e inflamación, con la promoción de reflejos cardiovasculares defensivos producidos por estímulos nocivos. Estos péptidos señalizan por medio de la activación de dos receptores transmembrana acoplados a proteína G llamados receptores B1 y B2 (B1R y B2R). El B2R es constitutivo en tejidos saludables y en animales. El objetivo de este estudio consistió en tomar las medidas de la expresión del gen y la proteína del receptor B2R de cinina en tejidos centrales y periféricos aislados de ratones control y ratones pre tratados con capsaicina en el segundo día de vida (50 mg/kg, s.c.) o con dos semanas antes del sacrificio (125 mg/kg por tres días, s.c.). El mismo tratamiento fue hecho con solución salina en los animales control. Fueron medidos niveles de mRNA para B2R por RT-PCR en forma cuantitativa y cualitativa, en cuanto los sitios de unión del receptor fueron medidos en secciones de tejido con radioligante 125I-HPP-Hoe 140 (B2R). B2R fue expresado en todos los tejidos estudiados (hipotálamo, núcleo paratrigeminal, núcleo del tracto solitario, médula espinal, aorta e hígado). El tratamiento con capsaicina en ratones neonatos comparados con controles no presentaron afectación del nivel de expresión. Por otro lado, la capsaicina no tuvo efecto significante en la expresión de los sitios de unión de B2R en los tejidos. La síntesis del receptor B2R de cininas no está asociado con fibras C sensoriales así como los tejidos no mostraron diferencias significativas indicando que B2R es regulado por mecanismos distintos.

Expressions of GABARB2 in brain regions of rats with high-level and low-level aggressive behavior / 中华行为医学与脑科学杂志

ObjectiveTo compare the mRNA and protein expression levels ofγ-aminobutyric acid receptor B2 ( GABABR2 ) in brain regions of male rats with high level aggressive behaviors and low level aggressive behaviors respectively,and provide clues for exploring mechanism of GABA in aggressive behaviors.MethodsWistar male rats were randomly divided into two groups:the normal group and the aggressive behavior group.Then social isolation and resident intruder stresses were used to establish high level and low level aggressive behavior in the aggressive behavior group.The mRNA and protein level of GABABR2 in parietal cortex,prefrontal cortex,hypothalamus and hippocampus of the three groups rats ( n=10 in each group) were detected using RT-PCR and Western blot respectively.ResultsThe GABABRB22 RT-PCR/Western blot relative integrated optical density of parietal cortex,prefrontal cortex,hypothalamus and hippocampus in the normal group rats respectively were.Those of the above four brain regions in high aggressive behavior group rats respectively were ( 0.507 ± 0.049/0.626 ±0.038 ),(0.609 ± 0.049/0.652 ± 0.010 ),( 0.359 ± 0.030/0.731 ± 0.044 ) and ( 0.296 ± 0.054/0.452 ±0.079) were significantly lower (P＜0.05) compared with the normal group rats.In the low aggressive behavior group rats,the GABABRB2 RT-PCR/Western blot relative integrated optical density of parietal cortex and hippocampus increased statistically(P＜ 0.05 ),while those of prefrontal cortex and hypothalamus decreased obviously (P ＜ 0.05 ).all in comparison with the normal group rats.Conclusion Different expression levels of GABABR2 in parietal cortex,prefrontal cortex,hypothalamus and hippocampus are relative to aggressive behaviors,which might be one of the mechanism for GABA in aggressive behaviors.

The Effects of Ephrin Receptor B2 on Angiogenesis and Neuroplasticity in Areas Remote From the Cortical Infarction / 国际脑血管病杂志

Cerebral infarction is not only a focal damage,but also causes secondary damage in areas remote from the ischemic territory,which will retard the recovery of neurological function.Ephrin receptor B2 (EphB2) plays an important role in the development and repair mechanism of central nervous system.Blocking the effect of EphB2 in brain by using a specific inhibitor may enhance proliferation and migration of endogeneous neuronal stem cells after experimental cerebral infarc- tion,improve neurological function and influence angiogenesis and neuroplasticity in remote sites.It is expected to become a new approach for decreasing damages in areas remote from the cerebral infarction and promoting the recovery of neurological function.