Serine Proteinase Inhibitor SERPINA3K Suppresses Corneal Neovascularization Via Inhibiting Wnt Signaling and VEGF

Abstract

Purpose: To evaluate the anti-neovascularization effects and investigate the possible mechanisms of SERPINA3K, a member of serine proteinase inhibitor family, using a specific rat model of suture-induced corneal neovascularization. Methods: A rat corneal suture model was set up and SERPINA3K was topically administered three times daily for 7 days. The clinical indications were evaluated on day 2, 5 and 7, including area of neovascularization and inflammation index. The eyeballs were collected after day 7 and the following examinations were performed: histological investigation, immunostaining, western blot and quantitative real-time polymerase chain reaction (PCR) assay. Wnt3a, a Wnt pathway ligand, was added to cultured Human Umbilical Vein Endothelial Cells (HUVEC), followed by detecting cell migration and western blot. Meanwhile, an in vitro VEGF165-stimulated HUVEC model was applied and the following measurements were conducted: cell proliferation, cell migration and tube formation. Results: SERPINA3K significantly suppressed corneal neovascularization and inhibited corneal inflammation. SERPINA3K downregulated the levels of β-catenin, non-pi-β-catenin and transcription factor 4 (TCF4), but upregulated the level of pi-β-catenin of the corneas induced by suture. SERPINA3K also decreased the gene expression and protein level of VEGF. Meanwhile, induction of Wnt3a increased the cell migration, activated the Wnt signaling and upregulated VEGF in cultured HUVEC, which were antagonized by SERPINA3K. In addition, SERPINA3K significantly inhibited VEGF165-induced cell proliferation and migration of HUVEC, SERPINA3K also specifically suppressed the VEGF165-induced tube formation of HUVEC. Conclusions: SERPINA3K has therapeutic potential for corneal neovascularization. The underlying mechanism may be through inhibiting Wnt signaling pathway and VEGF.

Down-Regulation of MicroRNA-184 Is Associated With Corneal Neovascularization

“…We demonstrated that corneal suture can induce corneal neovascularization and inflammation and proved that the corneal suture can be used as a good and specific model for corneal neovascularization. 15 In addition, we also showed that corneal suture increased levels of VEGF and key factors of the Wnt pathway, such as b-catenin. 15 In this present study, we expanded on the preceding by investigating the role of miR-184 in corneal neovascularization using the same corneal suture model, and we also performed transfection of mimics and iovs.arvojournals.org j ISSN: 1552-5783 inhibitor of miR-184 in cultured cells to further illustrate the underlying mechanisms.…”

“…The rat corneal suture procedure was described previously. 15 Briefly, after induction of general anesthesia by intraperitoneal injection with pentobarbital (40-50 mg/kg), topical application of 0.5% proparacaine ophthalmic solution was added. Three corneal sutures (10-0 nylon) were conducted in the cornea between corneal center and the limbus at the 12, 4, and 8 o'clock positions.…”

“…After 7 days, all rats were euthanized, and the whole corneas were dissected for quantitative real-time PCR assay following the method described below, in addition to other experiments and measurements reported previously. 15 Five rat corneal samples were used for each control and suture group in this present study.…”

“…Images of corneal neovascularization were captured using a slit-lamp microscope, and the method of evaluation of corneal neovascularization was reported previously. 15 Quantitative Real-Time RT-PCR for MicroRNA and mRNA Expression Analysis Total RNA was extracted from the corneas with reagent (TRIzol; Invitrogen). Complementary DNA (cDNA) was prepared using TaqMan MicroRNA Assay Kits (Applied Biosystems, Carlsbad, CA, USA) from total RNA (100 ng), including microRNAs and specific stem-loop primers for miR-184 or small nuclear RNA U6 (an endogenous small nuclear RNA [snRNA]) and a cocktail containing reverse transcription buffer, dNTP mix, recombinant RNase inhibitor, and reverse transcriptase (Multiscribe; Applied Biosystems).…”

“…15 Cells were seeded (1 3 10 4 /well) onto a 96-well plate and were allowed to attach overnight. After 24 hours, the cells were transfected by a miR-184 mimic or miR-184 inhibitor.…”