High cell growth rates during embryogenesis and adult-pathological conditions require high nutrient supply and hence, extensive vascular remodeling or angiogenesis. Since, the molecules and signaling mechanisms regulating blood vessel remodeling in embryos and adults are similar; adult angiogenesis merely recapitulates embryonic angiogenesis. One such signaling pathway we study is the Ras-MAPK cascade. ERK1/2 and ETS1/2 are key members of this pathway.
A large body of ex-vivo work emphasizes the importance of these signaling molecules in angiogenesis. However, mouse knockout models of these genes fail to show severe vascular phenotypes. In addition, their precise role in coordinating endothelial cell function remains poorly defined. In view of the high sequence homology between ERK1 and ERK2, and ETS1 and ETS2, they likely compensate for each other. The inability of previous models to examine the function of ERK1/2 and ETS1/2 in EC led us to examine the redundant role of Erk1/2 and Ets1/2 in endothelial cells (EC) during mouse embryonic angiogenesis.
Cre-loxP technology was adopted to inactivate Erk2 in Erk1-null mice using EC specific Tie2-Cre . Disruption of Erk1/2 in EC results in embryonic lethality at E10.5 due to reduced angiogenesis. Gene expression profiling on isolated EC identified cell cycle and cell migration as the principal biological processes affected in the double mutant EC. Consistent with the microarray profiling, EC lacking ERK1/2 show highly reduced proliferation both in vivo and in vitro . In addition, analysis of proteins regulating cell migration (Paxillin and FAK) revealed a significant decrease in their expression both in vivo and in vitro in absence of ERK1/2 thus resulting in drastically reduced endothelial cell motility.
The transcription factors ETS1 and ETS2 are targets of the ras/Erk signaling pathways. Mutation of both Ets1 and Ets2 resulted in embryonic lethality at mid-gestation with striking defects in vascular branching observed. The function of these factors was EC autonomous as demonstrated using Cre/loxP technology. The work described here demonstrates that deletion of Ets1/2 in isolated adult endothelial cells results in downregulation of anti-apoptotic (Bcl-XL and cIAP2 ) and cell migration (Mmp9 ) genes in double mutants versus controls. Chromatin Immunoprecipitation (ChIP) assay revealed that both ETS1 and ETS2 were loaded at target promoters. Consistent with the reduced expression of anti-apoptotic genes, EC apoptosis was significantly increased when both Ets1 and Ets2 were deleted. These results establish essential and overlapping functions for Ets1 and Ets2 in coordinating endothelial cell functions with survival during embryonic angiogenesis.
Our approach has revealed the essential and synergistic function of ERK1/2 and ETS1/2 in coordinating EC function, and thus angiogenesis during embryonic development.