Akt promotes endocardial-mesenchyme transition.

Endothelial to mesenchyme transition (EndMT) can be observed during the formation of endocardial cushions from the endocardium, the endothelial lining of the atrioventricular canal (AVC), of the developing heart at embryonic day 9.5 (E9.5). Many regulators of the process have been identified; however, the mechanisms driving the initial commitment decision of endothelial cells to EndMT have been difficult to separate from processes required for mesenchymal proliferation and migration. We have several lines of evidence that suggest a central role for Akt signaling in committing endothelial cells to enter EndMT. Akt1 mRNA was restricted to the endocardium of endocardial cushions while they were forming. The PI3K/Akt signaling pathway is necessary for mesenchyme outgrowth, as sprouting was inhibited in AVC explant cultures treated with the PI3K inhibitor LY294002. Furthermore, endothelial marker, VE-cadherin, was downregulated and mesenchyme markers, N-cadherin and Snail, were induced in response to expression of a constitutively active form of Akt1 (myrAkt1) in endothelial cells. Finally, we isolated the function of Akt1 signaling in the commitment to the transition using a transgenic model where myrAkt1 was pulsed only in endocardial cells and turned off after EndMT initiation. In this way, we determined that increased Akt signaling in the endocardium drives EndMT and discounted its other functions in cushion mesenchymal cells.

Activated Ras induces a proangiogenic phenotype in primary endothelial cells.

Angiogenic factors alter endothelial cell phenotype to promote the formation of new blood vessels, a process critical for a number of normal and pathological conditions. Ras is required for the induction of the angiogenic phenotype in response to vascular endothelial growth factor (VEGF). However, VEGF generates many signals, several of which are not dependent upon Ras activation. Our current study investigates the sufficiency of Ras activation for driving angiogenic responses. An activated Ras(V12) mutant induces prominent membrane ruffling, branching morphogenesis on three-dimensional collagen, DNA synthesis, and cell migration in primary endothelial cells. An upregulation of PI3K/AKT, Erk, and Jnk signaling pathways accompany these phenotypic changes. The inhibition of Erk blocked cell proliferation, but only partially attenuated migration. Blocking PI3K had no effect on DNA synthesis, but caused a modest reduction in cell migration. Lastly, Jnk played a significant role in both the proliferation and migration response. These effects of Ras(V12) are not the result of increased autocrine secretion of VEGF. These data suggest that the acquisition of activating Ras mutations can lead to a proangiogenic conversion in the phenotype of primary endothelial cells. Furthermore, these data raise the possibility that chronic Ras activation in endothelial cells may be sufficient to promote angiogenesis and the development of vascular anomalies.