Ginsenoside Rh2 inhibits vascular endothelial growth factor‐induced corneal neovascularization

Abstract

VEGF-induced neovascularization plays a pivotal role in corneal neovascularization (CoNV). The current study investigated the potential effect of ginsenoside Rh2 (GRh2) on neovascularization. In HUVECs, pretreatment with GRh2 largely attenuated VEGF-induced cell proliferation, migration, and vessel-like tube formation in vitro. At the molecular level, GRh2 disrupted VEGF-induced VEGF receptor 2 (VEGFR2)-Grb-2-associated binder 1 (Gab1) association in HUVECs, causing inactivation of downstream AKT and ERK signaling. Gab1 knockdown (by targeted short hairpin RNA) similarly inhibited HUVEC proliferation and migration. Notably, GRh2 was ineffective against VEGF in Gab1-silenced HUVECs. In a mouse cornea alkali burn model, GRh2 eyedrops inhibited alkali-induced neovascularization and inflammatory cell infiltrations in the cornea. Furthermore, alkali-induced corneal expression of mRNAs/long noncoding RNAs in cornea were largely attenuated by GRh2. Overall, GRh2 inhibits VEGF-induced angiogenic effect via inhibiting VEGFR2-Gab1 signaling in vitro. It also alleviates angiogenic and inflammatory responses in alkali burn-treated mouse corneas.-Zhang, X.-P., Li, K.-R., Yu, Q., Yao, M.-D., Ge, H.-M., Li, X.-M., Jiang, Q., Yao, J., Cao, C. Ginsenoside Rh2 inhibits vascular endothelial growth factor-induced corneal neovascularization.

Gαi1 and Gαi3mediate VEGF-induced VEGFR2 endocytosis, signaling and angiogenesis

“…Alkali injuries are common to the human cornea, resulting in angiogenic responses. An alkali burn will lead to over-production of VEGF, which induces endothelial cell proliferation and corneal neovascularization (“CoNV”) (see our previous study 27 ). Seven days after alkali burn, a significant CoNV was detected in the burned mouse cornea ( Figure 5 A ), which was largely inhibited by eyedrops of Gαi1 and Gαi3 shRNA lentivirus ( Figure 5 A-B ) ( P < 0.05 vs. control shRNA, n=10).…”

“…Wild-type (WT), Gαi1 and Gαi3 double-knockout (DKO), Gαi1, Gαi2 or Gαi3 single-knockout (SKO) mouse embryonic fibroblasts (MEFs), as well as WT and Gab1 KO MEFs were described previously 11 , 13 , 14 . Culture of human umbilical vein endothelial cells (HUVECs) was described previously 27 . Cells were starved in 0.5% FBS medium overnight before any treatment.…”

“…As described previously 27 , the mice ocular surface was rinsed with 20 mL of 0.9% saline solution for 1 min. Then, eyedrops of shRNA lentivirus (2 μL per eye) were instilled onto the alkali-burned eyes.…”

MicroRNA-29a-3p Downregulation Causes Gab1 Upregulation to Promote Glioma Cell Proliferation

“…Two non-overlapping GV248 lentiviral shRNAs ("-a/-b", with non-overlapping sequences) against human Gab1 (NM_002039) were described previously [41]. The detailed protocol for shRNA infection and stable cell selection were described in our previous studies [14,[23][24][25][26]31].…”

“…In order to study the function of Gab1 in human glioma cells, two distant lentiviral Gab1 shRNAs with non-overlapping sequences , "shGab1a" and "shGab1b" [41], were applied. Performing qRT-PCR ( Fig.…”