Zeb1 promotes corneal neovascularization by regulation of vascular endothelial cell proliferation

Abstract

Angiogenesis is required for tissue repair; but abnormal angiogenesis or neovascularization (NV) causes diseases in the eye. The avascular status in the cornea is a prerequisite for corneal clarity and thought to be maintained by the equilibrium between proangiogenic and antiangiogenic factors that controls proliferation and migration of vascular endothelial cells (ECs) sprouting from the pericorneal plexus. VEGF is the most important intrinsic factor for angiogenesis; anti-VEGF therapies are available for treating ocular NV. However, the effectiveness of the therapies is limited because of VEGF-independent mechanism(s). We show that Zeb1 is an important factor promoting vascular EC proliferation and corneal NV; and a couple of small molecule inhibitors can evict Ctbp from the Zeb1-Ctbp complex, thereby reducing EC Zeb1 expression, proliferation, and corneal NV. We conclude that Zeb1regulation of angiogenesis is independent of Vegf and that the ZEB1-CtBP inhibitors can be of potential therapeutic significance in treating corneal NV.

Expression and Function of ZEB1 in the Cornea

“…In summary ( Figure 3 ), ZEB1 is an important transcription factor in development; genetic knockout of Zeb1 in mice causes defects in neural, immune and respiratory systems, thereby resulting in embryo death prior to birth [ 63 ]. In adults, ZEB1 plays an important role in stem cell homeostasis, immune response, and angiogenesis [ 21 , 64 , 65 ]. However, abnormal activation may cause cellular transformation like EMT leading to tumorigenesis and fibrosis [ 8 , 56 ].…”

“…The nearest blood vessels to the cornea are also located in the limbus where an angiogenesis can be initiated by severe corneal damages leading to a pathogenesis called corneal neovascularization. As ZEB1 is required for cell proliferation in the alkali-induced mouse corneal neovascularization model, we show that Zeb1 is highly expressed in corneal epithelial basal cells, vascular endothelial cells, and infiltrated immune cells ( Figure 2 ) [ 21 ].…”

“…We have demonstrated that Zeb1 promotes corneal NV by repression of cyclin-dependent kinas (Cdk) inhibitors to promote vascular endothelial cell proliferation; and the loss-of-function of Zeb1, either by the knockdown of mRNA, and by the inhibition of its interaction with Ctbp would inhibit the corneal NV. We also found that the disruption of Zeb1 interaction with Ctbp by the ZEB1–CtBP inhibitors did upregulate the miR-200 family members, but did not repress Vegf expression, indicating that Zeb1 promotes angiogenesis is not through Vegf signal pathway [ 21 ].…”

“…As a result, no direct ZEB1 inhibitor has ever been identified though some indirect small molecules were suggested to inactivate ZEB1 by inhibition of ZEB1 and CtBP interaction such as NSC95397 [ 61 , 62 ]. We have demonstrated that CtBP interaction with ZEB1 retains ZEB1 in the nucleus while disrupt the ZEB1-CtBP complex would transport ZEB1 to the cytoplasm [ 21 ]. We have also shown that knockdown of ZEB1 by ZEB1 short hairpin (sh) RNA lentivirus in mouse retinal microvascular endothelial cells significantly reduces their proliferation, and that topical application of the ZEB1-CtBP inhibitor to the alkali-treated mouse corneas decreases corneal neovascularization [ 21 ].…”

“…We have demonstrated that CtBP interaction with ZEB1 retains ZEB1 in the nucleus while disrupt the ZEB1-CtBP complex would transport ZEB1 to the cytoplasm [ 21 ]. We have also shown that knockdown of ZEB1 by ZEB1 short hairpin (sh) RNA lentivirus in mouse retinal microvascular endothelial cells significantly reduces their proliferation, and that topical application of the ZEB1-CtBP inhibitor to the alkali-treated mouse corneas decreases corneal neovascularization [ 21 ]. These results suggest that both interfering RNA and small chemicals can be of therapeutic significance to reduce expression of ZEB1 in the target corneal cells.…”