At the molecular level, we found that TWIST1 regulates expression of
Vegfr2, which may mediate in part the angiogenic effects of TWIST1 in retinopathy and CNV. Vascular endothelial growth factor and its receptors are well known as crucial regulators of angiogenesis.
50 Inhibition of VEGF/VEGFR2 signaling pathway suppresses not only tumor angiogenesis but also neovascular eye diseases characterized by abnormal retinal and choroid angiogenesis.
42,51,52 Here we found that
Vegfr2 mRNA levels were upregulated in pathological NV tufts in OIR retinas and in laser-induced CNV membrane, both enriched with
Twist1 expression. Loss of
Twist1 resulted in inhibition of
Vegfr2 in the retinas, and TWIST1 suppression decreased VEGFR2 levels in HRMECs. This transcriptional regulation of VEGFR2 by TWIST1, as a known transcription factor, may potentially be achieved through binding of its HLH domain to a consensus E-box sequence as a known TWIST1 binding motif,
53 which is predicted in the promoter region of
VEGFR2. TWIST1 may thus modulate pathological angiogenesis in the eye at least in part through regulation of VEGFR2 expression and thereby the VEGF/VEGFR2 signaling pathway (
Fig. 6). These results of TWIST1-mediated VEGFR2 expression are consistent with our previous finding in mouse lungs
8 and complement a previous report showing that TWIST1 increases secretion of VEGF in breast cancer,
19 suggesting that TWIST1 may act in part through both the ligand and the receptors of the VEGF/VEGFR axis to influence angiogenesis in multiple pathological conditions. Suppression of TWIST1, however, does not show as strong inhibition of pathological NV in OIR as VEGFR2 inhibition,
54 which may reflect partial regulation of VEGFR2 by TWIST1, as well as other unknown factors that are potentially altered in
Twist1 knockout retinas, to compensate for the loss of
Twist1 and downregulation of
Vegfr2. In addition, consistent with our previous finding of
Twist1 regulation of
Tie2 in pulmonary vessels,
8 increased
Tie2 levels were found in
Twist1-enriched pathologic OIR vessels, and decreased retinal levels of
Tie2 were found in
Twist1 conditional knockout retinas (
Supplementary Fig. S1), further suggesting that additional factors other than VEGFR2 might be at work in mediating the effects of TWIST1 on pathologic ocular angiogenesis.