huvec-tube-formation-assay-to-evaluate-impact-natural-products-on

HUVEC Tube-formation Assay to Evaluate the Impact of Natural Products on Angiogenesis

Angiogenesis is a phenomenon that includes different processes, such as endothelial cell proliferation, differentiation, and migration, that lead to the ...

University of Campania "Luigi Vanvitelli"

Angiogenesis is a process encompassing several steps such as endothelial cells proliferation, differentiation and migration to form a vascular network, involving different signal transduction pathways. Among these, ERK1/2 signaling mediates VEGF-dependent signaling pathway. Here we report that the water extract of Ruta graveolens (RGWE), widely known as a medicinal plant, is able to impair in a dose-dependent manner, cell network formation without affecting cell viability. Biochemical analysis showed that the major component of RGWE is rutin, unable to reproduce RGWE effect. We found that RGWE inhibits ERK1/2 phosphorylation and that this event is crucial in cell network formation since the transfection of HUVEC with a constitutively active MEK (caMEK), the ERK1/2 activator, induces a robust cell network formation as compared to untransfected and/or mock transfected cells and, more importantly, caMEK transfected cells became unresponsive to RGWE. Moreover, RGWE inhibits VEGF and nestin gene expression, necessary for vessel formation, and the caMEK transfection induces their higher expression. In conclusion, we report that RGWE is able to significantly impair vessels network formation without affecting cell viability, preventing ERK1/2 activation and, in turn, down-regulating VEGF and nestin expression. These findings point to RGWE as a potential therapeutic tool capable to interfere with pathologic angiogenesis.

Ruta graveolens water extract inhibits cell-cell network formation in human umbilical endothelial cells via MEK-ERK1/2 pathway.

Differential DNA methylation defects of H19/IGF2 are associated with congenital growth disorders characterized by opposite clinical pictures. Due to structural differences between human and mouse, the mechanisms by which mutations of the H19/IGF2 Imprinting Control region (IC1) result in these diseases are undefined. To address this issue, we previously generated a mouse line carrying a humanized IC1 (hIC1) and now replaced the wildtype with a mutant IC1 identified in the overgrowth-associated Beckwith-Wiedemann syndrome. The new humanized mouse line shows pre/post-natal overgrowth on maternal transmission and pre/post-natal undergrowth on paternal transmission of the mutation. The mutant hIC1 acquires abnormal methylation during development causing opposite H19/Igf2 imprinting defects on maternal and paternal chromosomes. Differential and possibly mosaic Igf2 expression and imprinting is associated with asymmetric growth of bilateral organs. Furthermore, tissue-specific imprinting defects result in deficient liver- and placenta-derived Igf2 on paternal transmission and excessive Igf2 in peripheral tissues on maternal transmission, providing a possible molecular explanation for imprinting-associated and phenotypically contrasting growth disorders.

Luca Colucci-D'Amato

Laboratory of Cellular and Molecular Neuropathology,
Department of Environmental,
Biological and Pharmaceutical Sciences and Technologies,
Laboratory of Cellular and Molecular Neuropathology, Department of Environmental, Biological and Pharmaceutical Sciences and Technologies

University of Campania "Luigi Vanvitelli"

HUVEC Tube-formation Assay to Evaluate the Impact of Natural Products on Angiogenesis