title: CCN1/Cyr61-PI3K/AKT signaling promotes retinal neovascularization in oxygen-induced retinopathy.
abstract: Retinal neovascularization (RNV) is a characteristic pathological finding of retinopathy of prematurity (ROP). Cysteine-rich 61 [Cyr61, also known as CCN family member 1 (CCN1)] has been reported to mediate angiogenesis. The aim of the present study was to investigate the mechanisms of CCN1/Cyr61-phosphoinositide 3-kinase (PI3K)/AKT signaling in ROP. The contribution of CCN1 to human umbilical vein endothelial cell (HUVEC) proliferation and apoptosis under hypoxic conditions was determined using a cell counting kit‑8 (CCK-8) and Annexin V/propidium iodide (PI) staining, respectively, as well as using siRNA targeting CCN1 (CCN1 siRNA). The cells exposed to hypoxia were also treated with the PI3K/AKT inhibitor, LY294002. In addition, mouse pups with oxygen-induced retinopathy (OIR) were administered an intravitreal injection of CCN1 siRNA. RNV was assessed by magnesium-activated adenosine diphosphate-ase (ADPase) staining. RT-qPCR, western blot analysis, immunofluorescence staining and immunohistochemistry were used to detect the distribution and expression of CCN1, PI3K and AKT. Exposure to hypoxia increased the neovascularization clock hour scores (from 1.23±0.49 to 5.60±0.73, P<0.05) and the number of preretinal neovascular cells, as well as the mRNA and protein expression levels of CCN1, PI3K and AKT (all P<0.05). The injection of CCN1 siRNA decreased the neovascularization clock hour scores and the number of preretinal neovascular cells (1.53±0.72 vs. 4.76±1.04; 12.0±2.8 vs. 31.4±2.6, respectively, both P<0.05), as well as the mRNA and protein expression levels of CCN1, PI3K and AKT (protein, -45.3, -22.5 and -28.4%; mRNA, -43.7, -58.7 and -42.9%, respectively, all P<0.05) compared to the administration of scrambled siRNA under hypoxic conditions. Treatment with LY294002 decreased the mRNA and protein expression levels of CCN1 in the cells exposed to hypoxia (both P<0.05). The administration of CCN1 siRNA resulted in less severe neovascularization in the eyes of the the mouse pups with OIR. Thus, out data suggest that CCN1 plays an important role in RNV in ROP, and may thus be a potential target for the prevention and treatment of ROP.
authors.affiliation: Department of Ophthalmology, Shengjing Affiliated Hospital, China Medical University, Shenyang, Liaoning 110004, P.R. China.
authors.forename: Yu
authors.initials: Y
authors.lastname: Di
authors.affiliation: Graduate School, China Medical University, Shenyang, Liaoning 110122, P.R. China.
authors.forename: Yiou
authors.initials: Y
authors.lastname: Zhang
authors.affiliation: Department of Ophthalmology, Shengjing Affiliated Hospital, China Medical University, Shenyang, Liaoning 110004, P.R. China.
authors.forename: Qingzhu
authors.initials: Q
authors.lastname: Nie
authors.affiliation: Department of Ophthalmology, Shengjing Affiliated Hospital, China Medical University, Shenyang, Liaoning 110004, P.R. China.
authors.forename: Xiaolong
authors.initials: X
authors.lastname: Chen
mesh_terms: D000818:Animals; D000831:Animals, Newborn; D017209:Apoptosis / Q000187:drug effects / Q000235:genetics; D015153:Blotting, Western; D015687:Cell Hypoxia; D049109:Cell Proliferation / Q000187:drug effects / Q000235:genetics; D002478:Cells, Cultured; D002867:Chromones / Q000494:pharmacology; D055515:Cysteine-Rich Protein 61 / Q000235:genetics / Q000378:metabolism*; D061307:Human Umbilical Vein Endothelial Cells / Q000378:metabolism; D006801:Humans; D008297:Male; D008810:Mice, Inbred C57BL; D018613:Microscopy, Confocal; D009025:Morpholines / Q000494:pharmacology; D010100:Oxygen / Q000378:metabolism; D019869:Phosphatidylinositol 3-Kinases / Q000235:genetics / Q000378:metabolism*; D000081082:Phosphoinositide-3 Kinase Inhibitors; D051057:Proto-Oncogene Proteins c-akt / Q000235:genetics / Q000378:metabolism*; D034622:RNA Interference; D015861:Retinal Neovascularization / Q000235:genetics / Q000378:metabolism*; D012178:Retinopathy of Prematurity / Q000235:genetics / Q000378:metabolism*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D015398:Signal Transduction / Q000187:drug effects / Q000235:genetics
chemical_list: D002867:Chromones; D055515:Cysteine-Rich Protein 61; D009025:Morpholines; D010100:Oxygen; D019869:Phosphatidylinositol 3-Kinases; D000081082:Phosphoinositide-3 Kinase Inhibitors; D051057:Proto-Oncogene Proteins c-akt; C085911:2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one