Opposing actions of Notch1 and VEGF in post-natal cardiac valve endothelial cells

Abstract

The endothelium of the cardiac valves is unique compared the rest of the vasculature in its ability to undergo an endothelial-to-mesenchymal transformation (EMT) in vitro in response to transforming growth factor-beta (TGF-beta). EMT is a critical event during embryonic valve development, and both VEGF-A and Notch1 have been shown to function in this process. Here we investigate the effects of VEGF-A and Notch1 on EMT in clonal endothelial cell (EC) populations isolated from adult aortic valve leaflets. VEGF-A inhibited TGF-beta-induced EMT. Endothelial growth, however, was not affected by VEGF-A or TGF-beta. A positive role for Notch1 was revealed in three experiments: (1) TGF-beta induced Notch1 mRNA in valve ECs, (2) a gamma-secretase inhibitor of Notch1 signaling blocked EMT, and (3) overexpression of a ligand-independent form of Notch1 induced EMT. These results demonstrate, for the first time, that VEGF-A and Notch1 play opposing roles in regulating EMT in post-natal valve endothelium.

Figure 1. VEGF inhibits TGF-β-mediated EMT

(A) Wav-1 cells were treated with 10 ng/ml VEGF, 1 ng/ml TGF-β1, or 10 ng/ml VEGF and 1 ng/ml TGF-β1 in EBM-B for 5 days or 8 days and analyzed by Western blot using mouse anti-α-SMA and goat anti-CD31. (B) Wav-1 were treated with TGF-β1 as above and with 10ng/ml VEGF-receptor specific variants Flt-1-sel and KDR-sel [12] for 6 days. Cell lysates were analyzed as in A. (C) Wav-1 cells were treated as above without or with 2μM PTK 787 and analyzed as in A.

Figure 2. VEGF and TGF-β do not alter valve EC proliferation in vitro

Wav-1 proliferation was measured in EBM-B without bFGF, EBM-B (control medium), EBM-B with VEGF, EBM-B with TGF-β1, and EBM-B with VEGF and TGF-β1 over five days. All points were performed in triplicate. Cell numbers are presented as the mean ± S.D.

Fig.3. Notch1 in EMT

(A) TGF-β1 induces expression of Notch1 and Slug. Wav-1 cells were treated with 1ng/ml TGF-β1 for indicated times and total RNA prepared for RT-PCR. (B) Pharmacologic inhibition of Notch1 activation prevents TGF-β2-induced EMT. E6 cells were treated without or with 10 μM DAPT in the presence of TGF-β2 or VEGF and TGF-β2 for 6 days. Cell lysates were analyzed by Western blot with goat anti-CD31 and mouse anti-α-SMA. (C and D) Ligand independent Notch1 (Notch1-ΔEC) expressing cells undergo EMT in aortic valve ECs. Notch1-ΔEC-GFP-positive cells were treated without or with 10 μM DAPT, 5 μM CsA or 30 ng/ml soluble TGF-β receptor type II antagonist (Sol TβRII) for 8 days. (C) Immunofluorescence was performed with anti-α-SMA. Nuclei were stained by DAPI (a-e). (D) Western analysis was performed with goat anti-CD31 and mouse anti-α-SMA.

Fig. 4. Schematic of opposing actions of Notch1 and VEGF in aortic valve ECs

Over-expression of Notch1 induces EMT, blocking Notch activation inhibits EMT, and TGF-β1, a potent stimulator of EMT, increases Notch1 expression. VEGF inhibits EMT.