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Study of the role of ccbe1 in mouse ESCs and cardiac progenitor cells
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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.
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.
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Palavras-chave
Cardiovascular disease Heart development Embryonic stem cells Cardiac progenitor cells Ccbe1