Study of the role of ccbe1 in mouse ESCs and cardiac progenitor cells

RESUMO: As doenças cardiovasculares são a principal causa de morte no mundo, sendo o ataque cardíaco e o acidente vascular cerebral os mais frequentes. O enfarte agudo do miocárdio consiste na redução ou completo bloqueio da circulação intramiocardial, levando a uma situação de isquemia e morte de cardiomiócitos. Apesar de um ligeiro aumento após uma lesão, a capacidade de renovação dos cardiomiócitos no coração dos mamíferos não é suficiente para produzir uma regeneração cardíaca apropriada. No seguimento de um enfarte agudo do miocárdio, o tecido miocárdico passa por um processo de remodelação ventricular onde a maior parte do tecido afetado é substituído por uma cicatriz não-contrátil. Para compreender como a regeneração funciona e se podemos melhorá-la é crucial perceber os mecanismos que regulam o normal desenvolvimento do coração. A proteína CCBE1 (do acrónimo em inglês collagen and calcium binding EGF-like domains 1) é indispensável para para o processamento do fator de crescimento VEGF-C (do acrónimo em inglês vascular endothelium growth factor C), essencial para a linfangiogénese embrionária. O nosso laboratório mostrou que CCBE1 também é necessário para a correta migração e proliferação das células precursoras cardíacas durante o desenvolvimento do coração nos embriões de galinha. Nos embriões de murganhos, CCBE1 também é necessário para a correta migração e proliferação dos vasos coronários. Apesar destas descobertas, não se compreende completamente o seu papel na especificação cardíaca precoce. Nesta tese mostramos que a expressão de Ccbe1 encontra-se aumentada após o surgimento dos progenitores cardíacos positivos para Isl1 e Nkx2.5 durante a diferenciação de células estaminais embriónicas de murganho in vitro. Ao avaliar os progenitores cardíacos positivos para Isl1 isolados das células estaminais em diferenciação in vitro, Ccbe1 encontra-se em níveis elevados. A disrupção da expressão de Ccbe1, quer com shRNAs quer com um anticorpo neutralizante, afeta a diferenciação das células estaminais para a linhagem da mesoderme cardíaca, resultando numa diminuição da expressão de marcadores de cardiomiócitos. Para um pleno entendimento do papel de CCBE1 na cardiogénese in vivo, gerámos uma linha de murganho knockout condicional para Ccbe1, delecionando o exão 3 do gen. Estes murganhos, em vez de morrer a E14.5 como o knockout convencional, atingem a idade adulta sem um fenótipo aparente. Uma análise mais detalhada dos corações mostrou-nos um fenótipo moderado nos murganhos recém-nascidos e no adulto, com uma diminuição da espessura do miocárdio e uma redução no rácio do peso do coração-peso do corpo, respetivamente. Este fenótipo não era uma consequência do desenvolvimento coronário, uma vez que estes murganhos não tinham nenhum defeito nesse sentido. De moto a entender com maior rigor a função de CCBE1 no desenvolvimento e na doença cardíaca, desenvolvemos uma nova linha de murganhos com sequências LoxP a volta do exão 1 do Ccbe1. Esta linha tornar-se-á útil no futuro para poder gerar knockouts condicionais, possibilitando o cruzamento com linhas Cre transgénicas. Os resultados apresentados nesta tese mostram que CCBE1 é importante para o estabelecimento dos progenitores cardíacos precoces in vitro e, consequentemente, para a correta formação de cardiomióticos. Também mostramos que, in vivo, os murganhos knockout condicionais para Ccbe1 nos precursores cardíacos apresentam defeitos no desenvolvimento cardíaco independentemente do fenótipo coronário, confirmando o seu papel na correta formação do coração.ABSTRACT: Cardiovascular diseases are the leading cause of deaths worldwide, being heart attacks and strokes the most prevalent ones. Myocardial infarction (MI) occurs when the intramyocardial blood vessel circulation is reduced or completely blocked, leading to ischemia and cardiomyocyte death. Despite a slight increase after injury, the cardiomyocyte renewal capacity of the mammalian heart is insufficient for a proper regeneration of the heart. After a MI occurs, the myocardial tissue goes through a fibrotic ventricular remodeling, in which the damaged tissue is replaced by a non-contractile scar. Unveiling the mechanisms that regulate the normal development of the heart is crucial to understand how regeneration in the adult works and whether we can enhance it to reduce the fibrotic process that occurs after MI. Collagen and calcium binding EGF-like domains 1 (CCBE1) has been identified as an indispensable protein for the processing of vascular endothelium growth factor C (VEGF-C), a crucial protein for lymphangiogenesis during embryonic development. Our lab has shown that CCBE1 is also required for the correct migration and proliferation of cardiac precursor cells during heart development in chick embryos. Also, in mouse embryos, CCBE1 is crucial for the proper formation of the coronary vessels. However, its specific role in early cardiac commitment is not fully understood. In the present thesis we demonstrate that during mouse embryonic stem cell (mESC) differentiation Ccbe1 is upregulated upon emergence of Isl1- and Nkx2.5- positive cardiac progenitors. Ccbe1 is markedly enriched in Isl1-positive cardiac progenitors isolated from mESCs differentiating in vitro or embryonic hearts developing in vivo. Disruption of Ccbe1 with either shRNAs or blockade with a neutralizing antibody resulted in impaired differentiation of mESCs along the cardiac mesoderm lineage resulting in a decreased expression of mature cardiomyocyte markers. To understand better the role of CCBE1 in cardiogenesis in vivo, we also generated a heart conditional knockout mouse for Ccbe1 targeting exon 3. These mice, instead of dying at E14.5 like the conventional knockout, reached adulthood with no apparent phenotype. Detailed analysis of the hearts showed a mild phenotype in newborn and adult mice, with decreased myocardial thickness and heart-to-body weight ratio respectively. This phenotype was independent of the known function in coronary vessel development, which developed normally. To further comprehend CCBE1 function in heart development and disease, we developed a new mouse line with LoxP sites around exon 1 of Ccbe1. This line will be useful in the future to generate conditional knockouts by crossing it with Cre transgenic lines. The data presented in this thesis demonstrates that CCBE1 is relevant for the proper establishment of the early cardiac progenitors in vitro and consequently in the proper formation of mature cardiomyocytes. We further show that, in vivo, conditional knockout mice for Ccbe1 in cardiac precursors present defects in normal heart development independently of the known coronary vessel phenotype, proving its involvement in proper cardiac establishment

CCBE1 in Cardiac Development and Disease

Funding Information: Funding: OB was a pre-doctoral fellow of Fundação para a Ciência e Tecnologia (PD/BD/105891/2015). This work was supported by Fundação para a Ciência e a Tecnologia (FCT) grants (PTDC/SAU-ENB/121095/2010, HMSP-ICT/0039/2013 and 02/SAICT/2017/029590) to JA Belo and by the Scientific Employment Stimulus to JMI (Norma Transitória 8189/2018, FCT), and iNOVA4Health – UIDB/Multi/04462/2020 and UIDP/Multi/04462/2020, a program financially supported by Fundação para a Ciência e Tecnologia / Ministério da Educação e Ciência, through national funds is acknowledged.The collagen- and calcium-binding EGF-like domains 1 (CCBE1) is a secreted protein extensively described as indispensable for lymphangiogenesis during development enhancing VEGF-C signaling. In human patients, mutations in CCBE1 have been found to cause Hennekam syndrome, an inherited disease characterized by malformation of the lymphatic system that presents a wide variety of symptoms such as primary lymphedema, lymphangiectasia, and heart defects. Importantly, over the last decade, an essential role for CCBE1 during heart development is being uncovered. In mice, Ccbe1 expression was initially detected in distinct cardiac progenitors such as first and second heart field, and the proepicardium. More recently, Ccbe1 expression was identified in the epicardium and sinus venosus (SV) myocardium at E11.5–E13.5, the stage when SV endocardium–derived (VEGF-C dependent) coronary vessels start to form. Concordantly, CCBE1 is required for the correct formation of the coronary vessels and the coronary artery stem in the mouse. Additionally, Ccbe1 was found to be enriched in mouse embryonic stem cells (ESC) and revealed as a new essential gene for the differentiation of ESC-derived early cardiac precursor cell lineages. Here, we bring an up-to-date review on the role of CCBE1 in cardiac development, function, and human disease implications. Finally, we envisage the potential of this molecule’s functions from a regenerative medicine perspective, particularly novel therapeutic strategies for heart disease.publishersversionpublishe

Tyramine and histamine risk assessment related to consumption of dry fermented sausages by the Spanish population

Tyramine and histamine are the main dietary bioactive amines related to acute adverse health effects. Dry fermented sausages can easily accumulate high levels of these hazards and are frequently consumed in Spain. The present work aims to assess the exposure to tyramine and histamine from the consumption of dry fermented sausages by the Spanish population and to assess the risk to suffer acute health effects from this exposure. A probabilistic estimation of the exposure to these hazards was derived combining probability distributions of these amines in dry fermented sausages (n = 474) and their consumption by the Spanish population. The mean dietary exposure to tyramine and histamine was 6.2 and 1.39 mg/meal, respectively. The risk of suffering hypertensive crisis or histamine intoxication by healthy population due to tyramine or histamine intake, respectively, exclusively from dry fermented sausages, can be considered negligible. For individuals under treatment with MAOI drugs, the probability to surpass the safe threshold dose (6 mg/meal) was estimated as 34%. For patients with histamine intolerance, even the presence of this amine in food is not tolerable and it could be estimated that 7000 individuals per million could be at risk to suffer the related symptoms after consuming dry fermented sausage

Growth-Promoting Effect of Cava Lees on Lactic Acid Bacteria Strains: A Potential Revalorization Strategy of a Winery By-Product

The growing trend of circular economy has prompted the design of novel strategies for the revalorization of food industry by-products. Cava lees, a winery by-product consisting of non-viable cells of Saccharomyces cerevisiae rich in β-glucans and mannan-oligosaccharides, can be used as a microbial growth promoter, with potential food safety and health applications. The aim of this study was to assess in vitro the effect of cava lees on the growth of 21 strains of lactic acid bacteria (LAB) species commonly used as starter cultures and/or probiotics. Firstly, 5% of cava lees was selected as the most effective amount for enhancing microbial counts. After screening different LAB, statistically significantly (p < 0.05) higher microbial counts were found in 12 strains as a consequence of cava lees supplementation. Moreover, a greater and faster reduction in pH was observed in most of these strains. The growth-promoting effects of cava lees on LAB strains supports the potential revalorization of this winery by-product, either to improve the safety of fermented products or as a health-promoting prebiotic that may be selectively fermented by probiotic species.info:eu-repo/semantics/publishedVersio

Revalorization of cava lees to improve the safety of fermented sausages

: The revalorization of food processing by-products not only reduces the environmental impact of their disposal, but also generates added economic value. Cava lees consist of inactive cells of Saccharomyces cerevisiae, and though regarded as a valueless winery by-product, they are rich in fiber and phenolic compounds. In this study, a challenge test was performed to assess the effect of cava lees and a phenolic extract (LPE) derived therefrom on the behaviour of technological microbiota (lactic acid bacteria used as a starter culture) and the foodborne pathogens Salmonella spp. and Listeria monocytogenes during the fermentation and ripening of pork sausages. Ten batches of fermented sausages were prepared with and without cava lees or the LPE, and with or without different strains of Latilactobacillus sakei (CTC494 or BAP110). The addition of cava lees reduced the pH values of the meat batter throughout the fermentation and ripening process. No growth-promoting effect on spontaneous lactic acid bacteria (LAB) or the starter culture was observed. In contrast, the presence of cava lees prevented the growth of the tested pathogens (Salmonella and L. monocytogenes), as did the starter culture, resulting in significantly lower counts compared to the control batch. In addition, the combination of cava lees with L. sakei CTC494 had a bactericidal effect on Salmonella. LPE supplementation did not affect the pH values or LAB counts but reduced the mean counts of Salmonella, which were 0.71 log10 lower than the control values at the end of the ripening. The LPE did not exert any additional effect to that of the starters applied alone. The revalorization of cava lees as a natural ingredient to improve the microbiological safety of fermented sausages is a feasible strategy that would promote a circular economy and benefit the environment

Loss of Ccbe1 affects cardiac-specification and cardiomyocyte differentiation in mouse embryonic stem cells

Understanding the molecular pathways regulating cardiogenesis is crucial for the early diagnosis of heart diseases and improvement of cardiovascular disease. During normal mammalian cardiac development, collagen and calcium-binding EGF domain-1 (Ccbe1) is expressed in the first and second heart field progenitors as well as in the proepicardium, but its role in early cardiac commitment remains unknown. Here we demonstrate that during mouse embryonic stem cell (ESC) differentiation Ccbe1 is upregulated upon emergence of Isl1- and Nkx2.5- positive cardiac progenitors. Ccbe1 is markedly enriched in Isl1-positive cardiac progenitors isolated from ESCs differentiating in vitro or embryonic hearts developing in vivo. Disruption of Ccbe1 activity by shRNA knockdown or blockade with a neutralizing antibody results in impaired differentiation of embryonic stem cells along the cardiac mesoderm lineage resulting in a decreased expression of mature cardiomyocyte markers. In addition, knockdown of Ccbe1 leads to smaller embryoid bodies. Collectively, our results show that CCBE1 is essential for the commitment of cardiac mesoderm and consequently, for the formation of cardiac myocytes in differentiating mouse ESCs.Fundacao para a Ciencia e Tecnologia (FCT) [SFRH/BD/82280/2011]; FCT [SFRH/BPD/46506/2008, CEDOC/2015/36/iNOVA4Health/Multi/04462, SFRH/BPD/87114/2012, PTDC/SAU-ENB/121095/2010, HMSP-ICT/0039/2013]; Fundacao para a Ciencia e Tecnologia / Ministerio da Educacao e Ciencia [UID/Multi/04462/2013]; FEDER under the PT2020 Partnership Agreemen

Inhibition of Biogenic Amines Formation in Fermented Foods by the Addition of Cava Lees

Food safety can be compromised by some bioactive compounds such as biogenic amines that can be specially found in fermented foods due to the bacterial decarboxylation of some amino acids by fermentative or spoilage bacteria. Cava lees are a winery by-product rich in fiber and phenolic compounds and previous works have raised their revalorization from a food safety point of view. The aim of the current work was to investigate whether the use of cava lees can help to control biogenic amine formation in bread and fermented sausages. In bread, only very low levels of biogenic amines (putrescine, cadaverine, and/or spermidine) were found, whose content did not vary with the addition of different amounts of lees. However, the addition of lees in fermented sausages significantly reduced the formation of tyramine and cadaverine. In sausages spontaneously fermented and inoculated with Salmonella spp., the presence of cadaverine and putrescine diminished by 62 and 78%, respectively, due to the addition of cava lees. The addition of cava lees phenolic extract also showed an anti aminogenic effect (21% for cadaverine and 40% for putrescine), although in a lesser extent than cava lees. Cava lees and their phenolic extract were shown to be an effective strategy to control the undesirable accumulation of high levels of biogenic amines during the production of fermented products

Renal denervation for resistant hypertension and heart failure with a reduced ejection fraction

Hypertension is a risk factor for the development of heart failure and has a negative impact on the survival of these patients. Although patients with these two conditions usually take different antihypertensive medications, some patients do not achieve adequate blood pressure control and their hypertension becomes resistant or refractory. In this scenario, percutaneous renal denervation has emerged in recent years as an alternative to achieve blood pressure control goals. We present the case of a 53-year-old woman with a medical history of essential hypertension, hypercholesterolemia, unipolar depression, and diabetes, who was diagnosed with dilated cardiomyopathy with reduced left ventricular ejection fraction (33%). Despite the initiation of multiple antihypertensive medications and placement of a cardiac resynchronization therapy pacemaker, the patient remained hypertensive with a left ventricular ejection fraction of 40%. At that time, percutaneous renal denervation was performed without complications, and one year after the procedure, the patient had improved better blood pressure control and the left ventricular ejection fraction increased to 51%. This case illustrates one of the clinical scenarios in which it has been suggested that renal denervation may be more beneficial, as in the situation of patients with refractory hypertension and heart failure