Netrin And Receptors In Guidance Corneal Angiogenesis

Purposes To investigate the effects of netrin-1 and its receptors UNC5B in microvascularmodels in vitro. To detect the different expression of netrin-1 receptors in Human umbilicalvein endothelial cells (HUVECs) and vascularized cornea. To investigate the effect ofnetrin-1 in corneal neovascularization (CNV).MethodsIn vitro study: HUVECs were isolated and cultured. The CCK-8 assay, transwell migrationassay, microvascular models (tube formation assay)were used to examine the proliferation,migration and tube formation of HUVECs that treated with different concentrations ofnetrin-1 between 10 ng/mL to 5000ng/mL in vitro. RT-PCR was used to detect theexpression of UNC5A,UNC5B,UNC5C,UNC5D,DCC and neogenin on HUVECs. TheshRNA targeting UNC5B was constructed and transfected into HUVECs throughLipofectamine 2000. The CCK-8 assay, transwell migration assay, microvascular modelswere used to examine the proliferation, migration and tube formation of HUVECs thattreated with different concentrations of netrin-1 between 10 ng/ml to 5000ng/mL in vitro.In vivo study: Corneal pocket assay was used in the study of CNV. The basic fibroblastgrowth factor (bFGF) (50ng/ul) , netrin-1(100ug/ml), bFGF and netrin-1(100ug/ml),netrin-1(5000ug/ml), bFGF and netrin-1(5000ug/ml), the gelfoam sponges and agarosepills were implanted in Group A-F, respectively. The formation of CNV was observed byslit microscope. Real time PCR demonstrated that UNC5A,UNC5B,UNC5C,UNC5D,DCC and neogenin were expressed in bFGF induced rat vascularized corneas.ResultsIn vitro study: Netrin-1 has a promotive effect at the concentration less than 500ng/mL,while has an inhibitive effect at the concentration of 1000ng/mL～5000ng/mL. 50ng/mL of netrin-1 can maximally enhance the cellular proliferation, and 100ng/mL of netrin-1 canmaximally promote the cellular migration. 1000～5000ng/mL of netrin-1 has an inhibitiveeffect in tube formation of HUVECs. Only UNC5B was detected on HUVEC. TheUNC5B-shRNA expression plasmid was successfully constructed and transfected intoHUVECs. The proliferation, migration and tube formation of HUVECs are decreased inUNC5B RNAi group.In vivo study. The CNV in Group A began to grow in 4d and peakd on the 7d by cornealpocket assay. There wasn't difference of CNV's area between Group A and Group B(P＞0.05). The CNV's area of Group C was larger than its area of Group A and B. TheCNV's area of Group D, E and F were smaller than its area of Group A. Only UNC5B andNeogenin were expressed in rat cornea. The expression of UNC5B was higher in nomalcornea than in vascularized cornea. The expression of neogenin was higher in vascularizedcornea than in normal cornea.Conclusions The study in vtro indicates that netrin-1 has dual function in angiogenesis.The promotive or inhibitive effect depends on the concentration of itself. Only UNC5B wasdetected in HUVEC. UNC5B is an inhibitive receptor in neovascularization. Netrin-1 hasdual function on angiogenesis by corneal pocket assay. Only UNC5B and neogenin wereexpressed in rat cornea. The expression of UNC5B was higher in nomal cornea than invascularized cornea. The expression of neogenin was higher in vascularized cornea than innormal cornea.