Phenotypically Heterogeneous Podoplanin-expressing Cell Populations Are Associated with the Lymphatic Vessel Growth and Fibrogenic Responses in the Acutely and Chronically Infarcted Myocardium

Cardiac lymphatic vasculature undergoes substantial expansion in response to myocardial infarction (MI). However, there is limited information on the cellular mechanisms mediating post-MI lymphangiogenesis and accompanying fibrosis in the infarcted adult heart. Using a mouse model of permanent coronary artery ligation, we examined spatiotemporal changes in the expression of lymphendothelial and mesenchymal markers in the acutely and chronically infarcted myocardium. We found that at the time of wound granulation, a three-fold increase in the frequency of podoplanin-labeled cells occurred in the infarcted hearts compared to non-operated and sham-operated counterparts. Podoplanin immunoreactivity detected LYVE-1-positive lymphatic vessels, as well as masses of LYVE-1-negative cells dispersed between myocytes, predominantly in the vicinity of the infarcted region. Podoplanin-carrying populations displayed a mesenchymal progenitor marker PDGFRalpha, and intermittently expressed Prox-1, a master regulator of the lymphatic endothelial fate. At the stages of scar formation and maturation, concomitantly with the enlargement of lymphatic network in the injured myocardium, the podoplanin-rich LYVE-1-negative multicellular assemblies were apparent in the fibrotic area, aligned with extracellular matrix deposits, or located in immediate proximity to activated blood vessels with high VEGFR-2 content. Of note, these podoplanin-containing cells acquired the expression of PDGFRbeta or a hematoendothelial epitope CD34. Although Prox-1 labeling was abundant in the area affected by MI, the podoplanin-presenting cells were not consistently Prox-1-positive. The concordance of podoplanin with VEGFR-3 similarly varied. Thus, our data reveal previously unknown phenotypic and structural heterogeneity within the podoplanin-positive cell compartment in the infarcted heart, and suggest an alternate ability of podoplanin-presenting cardiac cells to generate lymphatic endothelium and pro-fibrotic cells, contributing to scar development

Phenotypically Heterogeneous Podoplanin-expressing Cell Populations Are Associated with the Lymphatic Vessel Growth and Fibrogenic Responses in the Acutely and Chronically Infarcted Myocardium

Cardiac lymphatic vasculature undergoes substantial expansion in response to myocardial infarction (MI). However, there is limited information on the cellular mechanisms mediating post-MI lymphangiogenesis and accompanying fibrosis in the infarcted adult heart. Using a mouse model of permanent coronary artery ligation, we examined spatiotemporal changes in the expression of lymphendothelial and mesenchymal markers in the acutely and chronically infarcted myocardium. We found that at the time of wound granulation, a three-fold increase in the frequency of podoplanin-labeled cells occurred in the infarcted hearts compared to non-operated and sham-operated counterparts. Podoplanin immunoreactivity detected LYVE-1-positive lymphatic vessels, as well as masses of LYVE-1-negative cells dispersed between myocytes, predominantly in the vicinity of the infarcted region. Podoplanin-carrying populations displayed a mesenchymal progenitor marker PDGFRalpha, and intermittently expressed Prox-1, a master regulator of the lymphatic endothelial fate. At the stages of scar formation and maturation, concomitantly with the enlargement of lymphatic network in the injured myocardium, the podoplanin-rich LYVE-1-negative multicellular assemblies were apparent in the fibrotic area, aligned with extracellular matrix deposits, or located in immediate proximity to activated blood vessels with high VEGFR-2 content. Of note, these podoplanin-containing cells acquired the expression of PDGFRbeta or a hematoendothelial epitope CD34. Although Prox-1 labeling was abundant in the area affected by MI, the podoplanin-presenting cells were not consistently Prox-1-positive. The concordance of podoplanin with VEGFR-3 similarly varied. Thus, our data reveal previously unknown phenotypic and structural heterogeneity within the podoplanin-positive cell compartment in the infarcted heart, and suggest an alternate ability of podoplanin-presenting cardiac cells to generate lymphatic endothelium and pro-fibrotic cells, contributing to scar development

Animal models to study cardiac regeneration

Permanent fibrosis and chronic deterioration of heart function in patients after myocardial infarction present a major health-care burden worldwide. In contrast to the restricted potential for cellular and functional regeneration of the adult mammalian heart, a robust capacity for cardiac regeneration is seen during the neonatal period in mammals as well as in the adults of many fish and amphibian species. However, we lack a complete understanding as to why cardiac regeneration takes place more efficiently in some species than in others. The capacity of the heart to regenerate after injury is controlled by a complex network of cellular and molecular mechanisms that form a regulatory landscape, either permitting or restricting regeneration. In this Review, we provide an overview of the diverse array of vertebrates that have been studied for their cardiac regenerative potential and discuss differential heart regeneration outcomes in closely related species. Additionally, we summarize current knowledge about the core mechanisms that regulate cardiac regeneration across vertebrate species

Imaging Long-Term Fate of Intramyocardially Implanted Mesenchymal Stem Cells in a Porcine Myocardial Infarction Model

The long-term fate of stem cells after intramyocardial delivery is unknown. We used noninvasive, repetitive PET/CT imaging with [18F]FEAU to monitor the long-term (up to 5 months) spatial-temporal dynamics of MSCs retrovirally transduced with the sr39HSV1-tk gene (sr39HSV1-tk-MSC) and implanted intramyocardially in pigs with induced acute myocardial infarction. Repetitive [18F]FEAU PET/CT revealed a biphasic pattern of sr39HSV1-tk-MSC dynamics; cell proliferation peaked at 33–35 days after injection, in periinfarct regions and the major cardiac lymphatic vessels and lymph nodes. The sr39HSV1-tk-MSC–associated [18F]FEAU signals gradually decreased thereafter. Cardiac lymphography studies using PG-Gd-NIRF813 contrast for MRI and near-infrared fluorescence imaging showed rapid clearance of the contrast from the site of intramyocardial injection through the subepicardial lymphatic network into the lymphatic vessels and periaortic lymph nodes. Immunohistochemical analysis of cardiac tissue obtained at 35 and 150 days demonstrated several types of sr39HSV1-tk expressing cells, including fibro-myoblasts, lymphovascular cells, and microvascular and arterial endothelium. In summary, this study demonstrated the feasibility and sensitivity of [18F]FEAU PET/CT imaging for long-term, in-vivo monitoring (up to 5 months) of the fate of intramyocardially injected sr39HSV1-tk-MSC cells. Intramyocardially transplanted MSCs appear to integrate into the lymphatic endothelium and may help improve myocardial lymphatic system function after MI

Characterization of the Scarification and Neolymphangiogenesis in Experimental Mouse Model of Acute and Chronic Myocardial Infarction: the Dual Role of Podoplanin Expressing Cells

Characterization of the cellular composition of the scar after myocardial infarction (MI) is poorly investigated; new insights in the biology of wound healing and inflammation post MI may establish undiscovered interactome in the infarcted heart that will lead new approaches for cardioprotective or regenerative therapies. Using a mouse model of permanent coronary artery ligation, we examined spatiotemporal changes in the expression of endothelial, lymphendothelial and mesenchymal markers in the acutely and chronically infarcted myocardium in order to analyze the cellular composition of the scar. We found that at the time of wound granulation, a three-fold increase in the frequency of podoplanin-labeled cells occurred in the infarcted hearts compared to non-operated and sham-operated counterparts. Podoplanin immunoreactivity detected LYVE-1-positive lymphatic vessels, as well as masses of LYVE-1-negative cells dispersed between myocytes, predominantly in the vicinity of the infarcted region. Podoplanin-carrying populations displayed a mesenchymal progenitor marker PDGFRα, and intermittently expressed Prox-1, a master regulator of the lymphatic endothelial fate. At the stages of scar formation and maturation, concomitantly with the enlargement of lymphatic network in the injured myocardium, the podoplanin-rich LYVE-1-negative multicellular assemblies were apparent in the fibrotic area, aligned with extracellular matrix deposits, or located in immediate proximity to activated blood vessels with high VEGFR-2 content. Of note, these podoplanin-containing cells acquired the expression of PDGFRβ or a hematoendothelial epitope CD34. Although Prox-1 labeling was abundant in the area affected by MI, the podoplanin-presenting cells were not consistently Prox-1-positive. The concordance of podoplanin with VEGFR-3 similarly varied. The role of podoplanin in the healing heart was never investigate, our data reveal previously unknown phenotypic and structural heterogeneity within the podoplanin-positive cell compartment in the infarcted heart, and suggest an alternate ability of podoplanin-presenting cardiac cells to generate lymphatic endothelium and pro-fibrotic cells, contributing to scar development

Phenotypically heterogeneous podoplanin-expressing cell populations are associated with the lymphatic vessel growth and fibrogenic responses in the acutely and chronically infarcted myocardium

Cardiac lymphatic vasculature undergoes substantial expansion in response to myocardial infarction (MI). However, there is limited information on the cellular mechanisms mediating post-MI lymphangiogenesis and accompanying fibrosis in the infarcted adult heart. Using a mouse model of permanent coronary artery ligation, we examined spatiotemporal changes in the expression of lymphendothelial and mesenchymal markers in the acutely and chronically infarcted myocardium. We found that at the time of wound granulation, a three-fold increase in the frequency of podoplanin-labeled cells occurred in the infarcted hearts compared to non-operated and sham-operated counterparts. Podoplanin immunoreactivity detected LYVE-1-positive lymphatic vessels, as well as masses of LYVE-1-negative cells dispersed between myocytes, predominantly in the vicinity of the infarcted region. Podoplanin-carrying populations displayed a mesenchymal progenitor marker PDGFRα, and intermittently expressed Prox-1, a master regulator of the lymphatic endothelial fate. At the stages of scar formation and maturation, concomitantly with the enlargement of lymphatic network in the injured myocardium, the podoplanin-rich LYVE-1-negative multicellular assemblies were apparent in the fibrotic area, aligned with extracellular matrix deposits, or located in immediate proximity to activated blood vessels with high VEGFR-2 content. Of note, these podoplanin-containing cells acquired the expression of PDGFRβ or a hematoendothelial epitope CD34. Although Prox-1 labeling was abundant in the area affected by MI, the podoplanin-presenting cells were not consistently Prox-1-positive. The concordance of podoplanin with VEGFR-3 similarly varied. Thus, our data reveal previously unknown phenotypic and structural heterogeneity within the podoplanin-positive cell compartment in the infarcted heart, and suggest an alternate ability of podoplanin-presenting cardiac cells to generate lymphatic endothelium and pro-fibrotic cells, contributing to scar development

Role of macrophages in cardiac homeostasis and repair following myocardial infarction

Tesis doctoral inédita leída en la Universidad Autónoma de Madrid, Facultad de Ciencias, Departamento de Biología Molecular: Fecha de lectura: 19-09-2014Recent studies show that macrophages are present in the healthy myocardium and can also be recruited in response to myocardial infarction (MI). However, the specific contribution of these cells to either cardiac homeostasis or cardiac repair following injury is not fully understood. In our study, we focus on macrophages present in the heart and examine their subsets, phenotype and functional properties in the steady state and after MI. For the analysis, we developed two different mouse models of MI: cryoinjury and left anterior descending coronary artery ligation (LADligation), and we used in vivo tracing flow cytometry, state-of-the-art confocal microscopy, transcriptional and ecocardiography analysis. We identified a population of cardiac resident macrophages located in close proximity with endothelial cells and that exhibit an M2-like/tissue-homeostatic signature consistent with anti-inflammatory and pro-angiogenic activity. These findings suggest that cardiac resident macrophages found in the healthy myocardium might have a role in maintaining cardiac homeostasis and coronary vasculature integrity. After MI, we found two distinct subsets of monocytes and macrophages that sequentially appeared in the infarcted myocardium and that showed different gene expression profiles. Ly6Chi cells found soon after myocardial injury correspond to pro-inflammatory monocytes/macrophages, whereas Ly6Clo cells found at later stages represent macrophages exhibiting anti-inflammatory and pro-angiogenic activity. Notably, the gene expression profile of these cells had features of pro-angiogenic Tie2-expressing macrophages (TEMs), which are also present in tumors and ischemic tissues. We examined the presence of TEMs in the ischemic myocardium and found that these cells are recruited in response to injury and exhibit pro-angiogenic properties. These results indicate that TEMs might be involved in the revascularization of the ischemic myocardium following MI. Our findings suggest that modulation of macrophage activation in both homeostasis and cardiac disease might help to develop novel therapeutic approaches to avoid adverse cardiac remodeling and improve cardiac repair.El corazón alberga poblaciones de macrófagos tanto en condiciones fisiológicas, como en respuesta al daño cardíaco por infarto de miocardio (IM). No obstante, la contribución específica de estas poblaciones al mantenimiento de la homeostasis y a la reparación cardíaca de corazón adulto no ha sido completamente dilucidada. Nuestro estudio se centra en la caracterización de las distintas subpoblaciones de macrófagos presentes en el corazón en homeostasis y en respuesta al IM. Para ello, se han desarrollado dos modelos distintos de inducción de IM: la criolesión y la ligación permanente coronaria y se han llevado a cabo estudios in vivo por citometría de flujo, microscopía confocal, ecocardiografía y análisis de la expresión génica. Se ha identificado la existencia de una población de macrófagos residentes cardíacos cuya localización es cercana a los pequeños y grandes vasos sanguíneos de la vasculatura coronaria. Estos macrófagos exhiben un perfil génico anti-inflamatorio caracterizado por la expresión de marcadores homeostáticos y pro-angiogénicos, lo que sugiere una función de estas células en el mantenimiento de la homeostasis cardíaca y de la integridad vascular. En respuesta al IM, se ha identificado la presencia de dos subpoblaciones de monocitos y macrófagos que aparecen de forma secuencial en el corazón y muestran distintos perfiles de expresión. La subpoblación celular Ly6Chi se compone de monocitos y macrófagos con carácter pro-inflamatorio, que se hallan presentes en fases tempranas tras el IM. En fases más tardías, se encuentra un subpoblación de macrófagos Ly6Clo que exhiben propiedades anti-inflamatorias y pro-angiogénicas similares a las mostradas por poblaciones de macrófagos Tie2+ (TEMs), previamente descritos en distintos procesos isquémicos. De acorde a este hallazgo, se ha llevado a cabo el estudio de la presencia de TEMs tras el IM. Se ha demostrado que estas células corresponden a subpoblaciones de macrófagos presentes en el miocardio dañado, que exhiben un patrón de expresión altamente pro-angiogénico y son distintas de los macrófagos Ly6Clo. Los resultados obtenidos en este estudio sugieren que en el contexto tanto de homeostasis como de enfermedad cardíaca, la modulación de la activación del macrófago puede servir como herramienta útil para el desarrollo de nuevas terapias enfocadas a la mejora de la función cardíaca

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

Cardiac lymphatics in health and disease

The lymphatic vasculature, which accompanies the blood vasculature in most organs, is indispensable in the maintenance of tissue fluid homeostasis, immune cell trafficking, and nutritional lipid uptake and transport, as well as in reverse cholesterol transport. In this Review, we discuss the physiological role of the lymphatic system in the heart in the maintenance of cardiac health and describe alterations in lymphatic structure and function that occur in cardiovascular pathology, including atherosclerosis and myocardial infarction. We also briefly discuss the role that immune cells might have in the regulation of lymphatic growth (lymphangiogenesis) and function. Finally, we provide examples of how the cardiac lymphatics can be targeted therapeutically to restore lymphatic drainage in the heart to limit myocardial oedema and chronic inflammation.Peer reviewe