Akt promotes endocardial-mesenchyme transition

Abstract

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.

Figure 1

Akt promotes EndMT during endocardial cushion formation. (A and B) E10.5 Akt1 mRNA is expressed in the endocardial cushion (*) of the AVC, but not in the myocardium (M) of the ventricle (V), atria (A) or outflow track (OFT). The image was taken under 4× (A) and 10× magnification (B). (C-F) AVC Explants were treated with DMSO (C and E) or pharmacological inhibitor of PI3K (LY294002) (D and F). The mesenchyme outgrowth (arrows) detected in explants was significantly reduced in explants treated with L294003, both in the brightfield images (C and D) DMSO treated as well as (E and F) α SMA stained fluorescent images. What outgrowth there was with LY294002 was the cobblestone morphology of an endothelial monolayer (D, note bracket). These images were representative of multiple explants (n>10) in independent experiments.

Figure 2

EndMT markers were induced in VE-cadherin:tTA/TET:myrAkt1 Endothelial cells. (A) Protein lysates were harvested from VE-cadherin:tTA/TET:myrAkt1 endothelial cells and were treated with or without TET for 72 hours. In the presence of TET myrAkt1 is suppressed, while induction occurs in the absence of TET. An increase in N-Cadherin and Snail1 was observed by Western Blot when myrAkt1 is expressed (-TET). (B) RT-PCR of RNA isolated from VE-cadherin:tTA/TET:myrAkt1 endothelial cells shows a significant decrease in VE-cadherin mRNA expression when myrAkt1 was expressed, *student T-test p-value = 0.015. (C) Endothelial cells cultured in the absence of tetracycline (increased myrAkt1) were stained with VE-cadherin (green, b) and N-cadherin (red, c). The merged images reveal populations of cells, which had lost VE-cadherin expression (a, arrows). These studies were representative of multiple experiments (n>2) in independent experiments.

Figure 3

The VE-cadherin-tTA transgene drives responder expression only in the endocardium, not the cardiac cushion mesenchyme. (A) E11.5 VE-cadherin:tTA/TET:myrAkt embryos stained with phospho-Akt showed increased Akt activation in the endocardium. (B) LacZ staining of E11.5 VE-cadherin:tTA/TET:LacZ embryos confirmed the endothelial specificity of the VE-cadherin promoter. (C) In ex vivo heart cushion explants (e), endothelial layer monolayer (m) outgrowth expressed lacZ but the mesenchymal outgrowth cells (red arrowheads) did not, again confirming the VE-cadherin-tTA gene would not be expressed and thus not drive TET-promoter expression in the mesenchyme. The figures shown are representative images from n>5 double transgenic explants assessed. Each litter gives 25% double transgenic offspring.

Figure 4

myrAkt promotes mesenchyme outgrowth. (A-D) AVC explants (e) cultured on 3-D collagen gels had increased mesenchyme outgrowth of E9.5 VE-cadherin:tTA/TET:myrAkt AVCs (arrows) (B) when compared to control (A). This was more pronounced in E10.5 VE-cadherin:tTA/TET:myrAkt AVCs explants (D), where explants were still sprouting mesenchyme (arrows), while outgrowth of the control AVC explants was composed of an endothelial monolayer (m) (C). (E) Scoring of relative differences in mesenchymal invasion showed an increase in mesenchyme outgrowth of VE-cadherin:tTA/TET:myrAkt AVCs at E9.5 and E10.5 compared to control explants. The figures shown are representative images from n>5 double transgenic explants assessed. Each litter gives 25% double transgenic offspring.