Abstract 14049: Jmjd8 is an Important Regulator of Angiogenic Sprouting
Proteins containing a Jumonji C (JmjC) domain have important cellular functions by either hydroxylating or demethylating proteins. Jmjd6 and the important hypoxia-related protein FIH1 belong to the JmjC domain-only subgroup of this protein family and have important functions in endothelial cells (EC) by regulating the splicing of the VEGFR1 or the protein function of HIF1α. However, the expression and function of the other members of the JmjC domain-only subgroup is largely unknown. Therefore, we performed a siRNA-screening to determine the function and expression of some members of the subgroup namely Jmjd4, NO66 and Jmjd8, RNAi mediated inhibition of Jmjd4 and NO66 had no effect on tube formation in EC in vitro. In contrast, siRNA-mediated knockdown of Jmjd8 significantly decreased in vitro network formation of HUVECs (64%±4% reduction of cumulative tube formation). The same effect was observed by adding different siRNAs targeting Jmjd8 (66%±5% and 63±5% reduction of cumulative tube formation, p<0.05). Moreover, RNAi-mediated knockdown of Jmjd8 significantly decreased in vitro sprout formation of HUVECs using the spheroid assay (92%±3% and 90%±1% reduction of cumulative sprout length, p<0.05). To rule out proliferative or apoptotic effects BrdU staining followed by FACS analysis was performed after siRNA-mediated knockdown of Jmjd8 with different siRNAs in HUVECs. Knockdown of Jmjd8 had no effect on proliferation or apoptosis. Since epigenetic control mechanisms are important for differentiation, we analyzed the expression of Jmjd8 in differentiation. Therefore, we induced spontaneous differentiation by LIF withdrawal and additionally stimulated endothelial differentiation by adding VEGF to murine embryonic stem cells. Jmjd8 expression was significantly upregulated at day 4 (+1.64±0.12 fold) and day 6 (+1.72±0.22 fold) of spontaneous, and at day 7 of endothelial (+2.68±0.73 fold) differentiation. Moreover, analyzing zebrafish development revealed an increase of Jmjd8 expression from 24 to 72 hpf. Taken together Jmjd8 is upregulated in endothelial and zebrafish development; while the inhibition of Jmjd8 in EC impaired EC function suggesting that Jmjd8 plays an important role in the control of vasculogenesis and angiogenesis.
- © 2012 by American Heart Association, Inc.