Effect of Pakl on embryonic vascular development and the related mechanisms / 第二军医大学学报

ABSTRACT

Objective To investigate the effects of p21-activated kinase 1 (Pakl) on embryonic vascular development and to discuss the underlying mechanisms. Methods In vitro transcription reactions were performed with the linearized PCS2-caPakl templates and SP6 RNA polymerase. Overexpression of constitutively active Pakl (caPakl) mRNA in one-cell stage embryos of zebrafish was achieved by microinjection. The morphological changes of the embryos were observed under an inverted microscope and the ultrastructure of vessel was confirmed by transmission electron microscope. PCS2-caPak1 was used to transfect into human umbilical vein endothelial cells (HUVECs) via LipofectamineLTX; cell viability was tested by MTT based assay; and apoptosis of HUVECs was analyzed by flow cytometry with Annexin V /7-AAD double-staining. Results Obvious cerebral hemorrhage was observed in the embryo of zebrafish at 30-36 h after fertilization, and the hemorrhage phenotype was observed in the caPakl overexpression group, with the incidence of hemorrhage being 25%-30%. In the caPakl overexpression group, transmission electron microscope found damaged vascular endothelial cells in the bleeding sites. Inverted microscope also showed abnormal morphology of cultured cells with caPakl overexpression, rounded up and started to detach from the dishes. MTT assay showed significantly decreased cell survival in the caPakl overexpression group (P<0. 0l), and flow cytometry demonstrated a significantly higher rate of cell apoptosis (P<0. 0l). Conclusion Pakl overexpression may affect embryonic vasculogenesis through inducing apoptosis of vascular endothelial cells in zebrafish.