RESUMO
The importance of vascular endothelial growth factor-A (VEGF) and subsequent Notch signaling in cardiac outflow tract development is generally recognized. Although genetic heterogeneity and mutations of these genes in both humans and mouse models relate to a high susceptibility to develop outflow tract malformations such as tetralogy of Fallot and peripheral pulmonary stenosis, no etiology has been proposed so far. Using immunohistochemistry, in situ hybridization, and quantitative RT-PCR on embryonic hearts, we have shown spatiotemporal increase and abnormal patterning of Vegf/VEGF/(phosphorylated) VEGFR-2, (cleaved) Notch1, and Jagged2 in the outflow tract of Vegf120/120 mouse embryos. This coincides with hyperplasia of specifically the outflow tract cushions and a high degree of subpulmonary myocardial apoptosis that, in later stages, manifest as pulmonary stenosis and ventricular septal defects. We postulate that increase of VEGF and Notch signaling during right ventricular outflow tract development can lead to abnormal development of both cushion and myocardial structures. Defective right ventricular outflow tract development as presented provides new insight in the etiology of tetralogy of Fallot.
Assuntos
Embrião de Mamíferos/anormalidades , Miocárdio/metabolismo , Receptor Notch1/genética , Transdução de Sinais/genética , Tetralogia de Fallot/genética , Fator A de Crescimento do Endotélio Vascular/genética , Animais , Aorta Torácica/anormalidades , Aorta Torácica/patologia , Modelos Animais de Doenças , Embrião de Mamíferos/metabolismo , Embrião de Mamíferos/patologia , Regulação da Expressão Gênica no Desenvolvimento , Ventrículos do Coração/anormalidades , Ventrículos do Coração/patologia , Imuno-Histoquímica , Hibridização In Situ , Proteína Jagged-2 , Proteínas de Membrana/metabolismo , Camundongos , Camundongos Mutantes , Miocárdio/patologia , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , RNA Mensageiro/metabolismo , Receptor Notch1/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Fator A de Crescimento do Endotélio Vascular/metabolismo , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/metabolismoRESUMO
AIMS: Currently, many potential cardiac revascularization therapies target the vascular endothelial growth factor (VEGF) pathway, with variable success. Knowledge regarding the role of the VEGF/Notch/ephrinB2 cascade in (ab)normal coronary development will provide information on the subtle balance of VEGF signalling in coronary maturation and might enhance our therapeutic possibilities. METHODS AND RESULTS: The effect of VEGF isoforms on coronary development was explored in vivo using immunohistochemistry and RT-qPCR on Vegf120/120 mouse embryos solely expressing VEGF120. In vitro, human arterial coronary endothelial cells were treated with VEGF121 or VEGF165 upon which RT-qPCR was performed. In vivo, mutant coronary arterial endothelium showed a decrease in protein expression of arterial markers such as cleaved Notch1, Delta-like4, and ephrinB2 concomitant with an increase of venous markers such as chicken ovalbumin upstream promoter transcription factor II. The venous endothelium showed the opposite effect, which was confirmed on the mRNA level. In vitro, mRNA expression of arterial markers highly depended on the VEGF isoform used, with VEGF165 having the strongest effect. Also, coronary arteriogenesis was anomalous in the mouse embryos with decreased arterial and increased venous medial development as shown by staining for smooth muscle alpha-actin, Delta-like1, and Notch3. CONCLUSION: We demonstrate that VEGF isoform-related spatiotemporal cardiac alterations in the VEGF/Notch/ephrinB2 cascade lead to disturbed coronary development. This knowledge can contribute to optimizing therapies targeting VEGF signalling by enabling balancing between angiogenesis and vascular maturation.