Choroidal and retinal angiogenesis are the primary causes of vision loss in patients with neovascular AMD, proliferative DR, retinopathy of prematurity, and other diseases. It should be noted that these retinal vascular disorders are always accompanied by neuronal damage[11]. Although anti-VEGF treatments have been extensively administered for neovascular eye diseases[12], they are likely to aggravate this neuronal injury and thus deteriorate visual function[13]. To solve this dilemma, a treatment that can protect neurons and simultaneously inhibit angiogenesis is urgently needed. 7,8-DHF, a naturally-occurring flavone, has been reported to possess multiple beneficial effects, such as neuroprotective effect, anti-inflammatory, and vasorelaxing[14]. Recently, growing evidence suggests that 7, 8-DHF may also inhibit pathological neovascularization[7], although this has not yet been confirmed. In the present study, we observed that 25 µM 7,8-DHF significantly prevented the proliferation, migration, and tube formation of RF/6A cells and promoted their apoptosis in vitro regardless of in the presence of VEGF, indicating that 7,8-DHF has inhibitory effects on angiogenesis in vitro. This result, together with the previous findings that low concentration (500 nM) of 7,8-DHF provides neuroprotection via the activation of TrkB receptor while high concentration (25 µM) of that via its potent antioxidant activity[15–17], make 7,8-DHF a potential candidate for the treatment of retinal vascular disease co-existing with neuropathy.
Many studies have revealed that 7,8-DHF at the concentration of 25 µM is nontoxic and even protective to several types of cells in vitro[16–18], whereas in the present experiment we discovered that 25 µM 7,8-DHF significantly inhibits the viability of RF/6A cells. The varying effects of 7, 8-DHF in these cell types may be attributed to its different pharmacological actions. VEGF is a potent proangiogenic factor that stimulates the proliferation, migration, and tube formation of endothelial cells and inhibits their apoptosis[19]. These biological VEGF signaling pathways are mainly mediated by VEGFR-2 receptor, which is abundant on the surface of vascular endothelial cells[20]. Phosphorylation of VEGFR-2 induces multiple downstream signals that subsequently promote angiogenesis in endothelial cells[21]. Thus, VEGF/VEGFR2 signal pathway is an important target for studying the angiogenesis inhibiting effects of 7, 8-DHF. In the present study, we demonstrated that although VEGFR2 expression was relatively constant, phosphorylated VEGFR2 levels were significantly decreased in RF/6A cells pretreated with 7, 8-DHF for 2 hours, in both the presence and absence of VEGF, indicating that 7, 8-DHF modulates the VEGFR2 signaling pathway by deactivating VEGFR2 rather than down-regulating its expression. There are two possible explanations for the observed 7, 8-DHF-induced signal deactivation. One is that 7, 8-DHF may down-regulate the expression of VEGF-A in RF/6A cells and thus inhibit signal transmission. According to this hypothesis, the extent of angiogenesis and level of phosphorylated VEGFR2 protein should be significantly different between the 7, 8-DHF-treated and 7, 8-DHF plus VEGF-treated groups, which contradicts our findings in this study. The other explanation is that 7, 8-DHF prevents VEGFR2 activation by interfering with the binding of VEGF to VEGFR2. This well explains why even if VEGF levels are elevated in RF/6A cells, the levels of angiogenesis and phosphorylated VEGFR2 protein remain unchanged in the presence of 7,8-DHF (25 µM). This assumption is also supported by a previous study using a combination of molecular docking, computational mapping tools, and molecular dynamics, which showed that 7, 8-DHF may exert its effects on VEGFR2 via the intracellular kinase domain[7]. Collectively, the above evidence suggests that 7, 8-DHF inhibits angiogenesis in RF/6A cells in vitro by suppressing the phosphorylation of VEGFR2, although the exact mechanism of VEGFR2 dephosphorylation induced by 7, 8-DHF remains to be elucidated.
In this study, we comprehensively investigated the antiangiogenic effects of 7, 8-DHF on RF/6A cells, which were mainly used in the study of chorioretinal neovascular diseases. However, whether this medicine has similar inhibitory effects on other types of vascular endothelial cells, such as human umbilical vein endothelial cells (HUVEC), remains to be studied. Furthermore, in vivo studies are needed to obtain a comprehensive understanding of the observed effect and the mechanism underlying the effects of this compound on choroidal and retinal angiogenesis.