Experimental Study On The Mechanism Of Osteopontin Induced Corneal Neovascularization

Objective:Experiments study the role of osteopontin (OPN) on corneal fibroblasts (CFs) and human umbilical vein endothelial cells (HUVECs) co-culture model, reveal the relevant factors and pathways about OPN-induced corneal neovascularization and establish the foundation for further research on the mechanism that cell microenvironment change impact on OPN induced corneal neovascularization. Method:1. In accordance with the ethical requirements, corneal stroma lens were obtained from patients who the just completed refractive surgery. Apply Tissue blocks cell culture method to culture corneal stroma lens from femtosecond laser refractive surgery, obtain primary cultured cells. Apply inverted optical microscope, hematoxylin and eeosin (HE) staining and immunohistochemistry staining method to identify the tissue cultured cells and confirm corneal fibroblasts. Use transwell chamber system to establish HUVECs and CFs co-culture model.2. Study were divided into two experimental groups. The two experimental groups were CFs and HUVECs co-culture group, HUVECs single culture group. After individually cultured 24 h, PI3K/AKT inhibitor LY294002, ERK1/2 inhibitor PD98059, osteopontin antibody, anti-integrin anti-αvβ3 antibody, anti-VEGF antibody were respectively added to cells, two groups were induced by osteopontin after continuously culture 30 min. After incubation 24 h, endothelial cells would be stained with dye crystal violet and the proliferation and migration would be observed and taken photographs by inverted optical microscope; VEGF mRNA and protein levels was detected by enzyme linked immunosorbent assay (ELISA), Real-time polymerase chain reaction (PCR), Western blot. Moreover this study applies Western blot to observe ERK1/2, PI3K/AKT phosphorylation situation.Results:1. Stroma lens of femtosecond laser refractive surgery were cultured by tissue blocks method. The fifth day, some cells begin to climb out from corneal stroma lens tissue blocks and almostly covered the whole bottom of flasks at ninth day. Direct observation with inverted optical microscope:the cultured cells were fusiform and adherently grew, they mostly got togather surround the tissue blocks, like fish, and can overlap growth; HE staining results:the cells were fusiform or polygonal, they are composed of oval, blue and prominent nucleoli, large cell body, light purple cytoplasm; Immunohistochemistry staining results:Vimentin (+), Desmin (-), S-100 (-), Keratin (-).2. In transwell chamber system, endothelial cells were seeded in the upper chamber, corneal fibroblasts were seeded in the next chamber, separately cultured 24h before two nested together. We established successfully CFs and HUVECs co-culture mode1.3. HUVECs and CFs co-culture group and HUVECs single cultrue group were cultured 24 h, we applyed cotton swab to wipe away the upper chamber cells, fixed lower cells with 1% formaldehyde solution and stained with 0.5% crystal violet, found that the lower cells number of co-culture group were average 121, significantly more than the cells number of endothelial cell single culture group, the number of endothelial cells single culture group were only 25 or less.4. ELISA experience, OPN-induced VEGF expression in co-culture group and single culture group were significantly elevated, expression of co-culture group is higher. After adding inhibitor, each experimental hole VEGF concentration has declined (except anti-VEGF hole, all p<0.05), OPN antibody, anti-avp3 antibody,anti-VEGF antibody decreased the most obvious holes. Co-culture group were higher than the holes of singe culture group, but the difference is not obvious.5. In Fluorescence quantitative PCR experiments, we found that OPN-induced VEGF expression in co-culture group and single culture group were significantly elevated, but the CFs and HUVECs co-culture group was significantly higher. VEGF expression of CFs and HUVECs co-culture group and endothelial cells single culture group was significantly decreased when two groups pretreated with PI3K/AKT inhibitor, ERK1/2 inhibitor, osteopontin antibody, anti-VEGF antibody. Compared with the control group, VEGF expression was significantly decreased in the wells with PI3K/AKT inhibitor, ERK1/2 inhibitor, osteopontin antibody, but it decreased insignificantly in the wells with VEGF antibody. Both groups, VEGF of the wells of co-culture group with inhibitors and antibodys was higher than that of single culture group.5. In Western blot experiments, in co-culture group with OPN, VEGF protein expression was significantly higher than that single culture group (p <0.05). VEGF expression of two culture groups were significantly decreased when they were pretreated with osteopontin antibody and anti-VEGF antibody, especially the role of OPN antibody is the strongest. Meanwhile, the study found that at 0.5 h ERK1/2 and PI3K/AKT in co-culture and single culture group were significantly Phosphorylated, co-culture group was more significant (p<0.05). The phosphorylation of test wells with anti-OPN antibody and anti-avp3 integrin antibody didn’t detect.Conclusions:1. Corneal stroma lens from femtosecond laser refractive surgery is a convenient tissue for the culture of fibroblasts.2. OPN can induce endothelial cell migration significantly at co-culture group, however endothelial cell migration is not obvious at single culture.3. VEGF expression of CFs and HUVECs co-culture group induced by OPN were higher than HUVECs single culture group, CFs play a important role in the expression of VEGF, is a potential anti-angiogenesis target cells.4. OPN can induce VEGF expression of CFs and HUVECs co-culture model by ERK1/2 and PI3K/AKT signaling pathways, promote angiogenesis.