RESUMO
Angiogenesis contributes to the generation of the vascular bed but also affects the progression of many diseases, such as tumor growth. Many details of the molecular pathways controlling angiogenesis are still undefined due to the lack of appropriate models. We propose the proepicardial explant as a suitable model for studying certain aspects of angiogenesis. The proepicardium (PE) is a transient embryonic structure that contains a population of undifferentiated endothelial cells (ECs) forming a vascular net continuous with the sinus venosus. In this paper, we show that PE explants give rise to CD31-positive vascular sprouts in the presence of basic fibroblast growth factor (bFGF) and 2 isoforms of vascular endothelial growth factor A (VEGF-A), i.e. VEGF-A120 and VEGF-A164. Vascular sprouts exhibit differences in number, length, thickness and the number of branches, depending on the combination of growth factors used. Moreover, the ECs of the sprouts express various levels of mRNA for Notch1 and its ligand Dll4. Additionally, stimulation with bFGF/VEGF-A164 upregulates the expression of Lyve-1 antigen in the ECs in the sprouts. In summary, we present a new model for angiogenesis studies involving mouse PE as a source of ECs. We believe that our model may act as a supplementary assay for angiogenesis studies along with the existing models.
Assuntos
Indutores da Angiogênese/farmacologia , Fator 2 de Crescimento de Fibroblastos/farmacologia , Neovascularização Fisiológica/efeitos dos fármacos , Pericárdio/efeitos dos fármacos , Fator A de Crescimento do Endotélio Vascular/farmacologia , Proteínas Adaptadoras de Transdução de Sinal , Animais , Biomarcadores/metabolismo , Proteínas de Ligação ao Cálcio , Feminino , Regulação da Expressão Gênica no Desenvolvimento , Idade Gestacional , Glicoproteínas/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/genética , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Proteínas de Membrana Transportadoras , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos CBA , Pericárdio/embriologia , Pericárdio/metabolismo , Molécula-1 de Adesão Celular Endotelial a Plaquetas/metabolismo , Gravidez , Receptor EphB2/genética , Receptor EphB2/metabolismo , Receptor EphB4/genética , Receptor EphB4/metabolismo , Receptor Notch1/genética , Receptor Notch1/metabolismo , Fatores de Tempo , Técnicas de Cultura de TecidosRESUMO
The epicardium is the last layer of the vertebrate heart to form, surrounding the heart muscle during embryogenesis and providing signaling cues essential to the continued growth and differentiation of the heart. This outer layer of the heart develops from a transient structure, the proepicardial organ (PEO). Despite its essential roles, the early signals required for the formation of the PEO and the epicardium remain poorly understood. The molecular markers wt1 and tcf21 are used to identify the epicardial layer in the zebrafish heart, to trace its development and to determine genes required for its normal development. Disruption of lateral plate mesoderm (LPM) migration through knockdown of miles apart or casanova leads to cardia bifida with each bilateral heart associated with its own PEO, suggesting that the earliest progenitors of the epicardium lie in the LPM. Using a gene knockdown approach, a genetic framework for PEO development is outlined. The pandora/spt6 gene is required for multiple cardiac lineages, the zinc-finger transcription factor wt1 is required for the epicardial lineage only and finally, the cell polarity genes heart and soul and nagie oko are required for proper PEO morphogenesis.