Abstract 19057: Disruption of Endothelial Frizzled 5 Signalling Inhibits Angiogenesis and Induces PKC-Mediated Upregulation of Vascular Regression Associated Genes
Introduction: The process of neo vessel formation is tightly regulated and imbalances in signalling can be a causative or a progressive factor in many vascular defect associated diseases. In previous studies, Frizzled 5 (Fzd5) was described to be obligatory in embryonic vascular development, as Fzd5 knock-out mice showed a lethal deficiency in placenta and yolk sac angiogenesis. However, the exact molecular mechanism behind this observation remains unclear.
Hypothesis: In the present study, we aimed to decipher the function of Fzd5 in endothelial cells, and its involvement in regulating angiogenesis.
Methods and results: Short interference RNA (siRNA) based knockdown of Fzd5 in human umbilical vein endothelial cells (HUVECs), co-cultured with pericytes in a 3D collagen matrix, resulted in a more than two-fold reduction of endothelial tube formation (n=4, p<0.05). The involvement of apoptosis as a causal factor for the poor angiogenic phenotype in the Fzd5 knockdown condition was excluded based on TUNEL staining. However, endothelial proliferation and migration, two essential components of angiogenesis, were severely impaired after knockdown of Fzd5 (both n=4, p<0.05). Diminished proliferation in absence of Fzd5 was induced by a G1 cell cycle arrest, as determined by FACS (G1 siControl 30±4% siFzd5 51±5%, n=3, p<0.05). Known Fzd/Wnt transduction cascades were studied in order to define the molecular cause for the observed phenotype. No alterations were observed in the canonical Wnt/β-catenin pathway and the non-canonical Wnt/Ca2+ pathway after knockdown of Fzd5. However, qPCR analysis on Fzd5 siRNA treated endothelial cells showed an upregulation of vascular regression associated factors Flt1 and Angpt2 of 51% and 65%, respectively (n=10, p<0.001). This upregulation was dose-dependently suppressed by the PKC inhibitor staurosporine.
Conclusions: In conclusion, the inhibitory effect of Fzd5 silencing on angiogenesis in vitro is associated with impaired endothelial proliferation and migration. Wnt/β-catenin signalling and Wnt/Ca2+ signalling are not affected by knockdown of Fzd5 in HUVECs. Instead, there appears to be a role for PKC in Fzd5 signal transduction, as it is involved in the Fzd5 knockdown mediated upregulation of Angpt2 and Flt1.
Author Disclosures: M. Brandt: None. C.G. van Dijk: None. I. Chrifi: None. M.C. Verhaar: None. D.J. Duncker: None. C. Cheng: None.
- © 2016 by American Heart Association, Inc.