Cyr61 induces the expression of monocyte chemoattractant protein-1 via the integrin ανß3, FAK, PI3K/Akt, and NF-κB pathways in retinal vascular endothelial cells.

Diabetes causes a number of metabolic and physiological abnormalities in the retina. Many of the molecular and physiological abnormalities that develop during diabetic retinopathy are due to inflammation. Monocyte chemoattractant protein-1 (MCP-1) is an important factor involved in diabetic retinopathy. In a previous study, we found that cysteine-rich 61 (Cyr61), an important angiogenic factor, also plays an important role in diabetic retinopathy. In addition to the direct effects of Cyr61, we observed that Cyr61 can induce the expression of MCP-1. However, the mechanism through which this occurs is not completely understood in chorioretinal vascular endothelial cells. We therefore investigated the effects of Cyr61 on MCP-1 expression in this cell type. Cyr61 stimulated the expression of MCP-1 at the mRNA, protein, and secreted protein levels in a dose-dependent and time-dependent manner. Both total MCP-1 levels and secreted MCP-1 levels were attenuated during the response to Cyr61 stimulation by pretreatment with integrin ανß3-blocking antibodies, a FAK inhibitor (PF573228), a PI3K inhibitor (LY294002), and an Akt inhibitor (A6730). Electrophoretic mobility shift assays revealed that the above inhibitors suppressed the activation of NF-κB. Additionally, deletion of the NF-κB-binding element in the MCP-1 gene promoter led to a decrease in expression in luciferase reporter assays. These results show that the induction of MCP-1 by Cyr61 is mediated through the activation of the integrin ανß3, FAK, PI3K/Akt, and IKK/NF-κB pathways in chorioretinal vascular endothelial cells.

Elevation of angiogenic factor Cysteine-rich 61 levels in vitreous of patients with proliferative diabetic retinopathy.

PURPOSE: Cysteine-rich 61 (Cyr61) is one of the angiogenic factors involved in proliferative diabetic retinopathy (PDR). To further investigate its role, we measure and compare the vitreous levels of Cyr61 and vascular endothelial growth factor in patients with PDR and to localize Cyr61 expression in associated proliferative epiretinal membranes. METHODS: Vitreous obtained from 56 patients with active PDR, 16 patients with active PDR pretreated with bevacizumab, 19 patients with quiescent PDR, 15 non-PDR patients with diabetic macular edema, and 25 patients with non-diabetic-related eye diseases were subjected to enzyme-linked immunosorbent assay for Cyr61 and vascular endothelial growth factor levels. Epiretinal membranes from 18 patients were stained immunohistochemically for Cyr61. RESULTS: Vitreous Cyr61 levels were significantly higher in active PDR patients, quiescent PDR patients, and diabetic macular edema patients compared with non-diabetic control patients (P < 0.01). Pretreatment of bevacizumab significantly suppressed vitreous vascular endothelial growth factor levels; however, it did not inhibit vitreous Cyr61 levels in active PDR patients. Cysteine-rich 61 was strongly detected in endothelial cells and myofibroblasts within active PDR membranes but not in idiopathic epiretinal membrane. CONCLUSION: Vitreous Cyr61 levels were related to different states of PDR and correlated with vascular endothelial growth factor levels in PDR patients. Pretreatment of bevacizumab did not inhibit vitreous Cyr61 levels in active PDR patients. Cysteine-rich 61 might mediate angiogenesis and post-angiogenic fibrosis in PDR.

Regulation of Cyr61/CCN1 expression by hypoxia through cooperation of c-Jun/AP-1 and HIF-1α in retinal vascular endothelial cells.

Hypoxia is the most important factor in the pathogenesis of diabetic retinopathy. Cysteine-rich 61 (Cyr61) is one of the angiogenic factors involved in the development of proliferative diabetic retinopathy (PDR). The aim of this study was to investigate the mechanism of hypoxia-induced Cyr61 expression in retinal vascular endothelial cells. The hypoxia-induced expression of mRNA and protein of Cyr61 was studied in monkey choroidal retinal vascular endothelial (RF/6A) cells. Luciferase reporter assays and electrophoretic mobility shift assays were used to identify the hypoxia responsible region and transcription factors in the Cyr61 promoter. Chromatin immunoprecipitation and immunoprecipitation were performed to study the role of hypoxia-inducible factor (HIF)-1α and c-Jun/activator protein-1 (AP-1) in Cyr61 transcriptional regulation. The results showed that hypoxia significantly induced Cyr61 mRNA and protein expression in RF/6A cells. The effect was mediated through phosphorylation of c-Jun. Luciferase assays, electrophoretic mobility shift assays, chromatin immunoprecipitation and immunoprecipitation showed that HIF-1α interacted with c-Jun/AP-1 and their binding on the AP-1 binding motif within the Cyr61 promoter induced the expression of Cyr61. In conclusion, hypoxia controlled the transcriptional regulation of the Cyr61 gene in RF/6A cells by cooperation of HIF-1α and c-Jun/AP-1. Cyr61 might play an important role in ischemic retinal diseases, such as PDR.

Cysteine-rich 61, a member of the CCN family, as a factor involved in the pathogenesis of proliferative diabetic retinopathy.

PURPOSE: Cysteine-rich 61 (Cyr61/CCN1) is reported to mediate angiogenesis. In this study, its role in ocular angiogenesis and proliferative diabetic retinopathy (PDR) was investigated. METHODS: The effects of Cyr61 were evaluated by determining proliferation and chemotaxis and in an assay of capillary tube formation in synthetic matrix by chorioretinal endothelial cells (RF/6A). In the same cells, Cyr61 expression under hypoxic conditions was then investigated. Interactions between Cyr61 and vascular endothelial growth factor (VEGF) were examined using endothelial cell chemotaxis, tube-formation assay, and cross-stimulation assay. A mouse model of oxygen-induced retinopathy (OIR) and a rat model of streptozocin-induced diabetes were used to evaluate Cyr61 expression in the retina. Cyr61 levels were also measured and chemotactic effects were evaluated in vitreous samples from patients with PDR. RESULTS: Cyr61 significantly induced proliferation, migration, and synthetic matrix tube formation of RF/6A cells. Hypoxia significantly induced Cyr61 mRNA and protein expression. Cyr61 induced expression of VEGF and vice versa. Anti-Cyr61 or anti-VEGF could inhibit the effects of both Cyr61 and VEGF. Intravitreal injection of anti-Cyr61 antibody significantly inhibited retinal neovascularization in the mouse OIR model. Cyr61 mRNA and protein were significantly expressed in the retina of streptozocin-induced diabetic rats. Vitreous levels of Cyr61 were elevated in patients with PDR when compared with nondiabetic patients. CONCLUSIONS: Cyr61 acts as an angiogenic mediator of ocular neovascularization in vitro and in vivo. It may interact with VEGF in a synergetic manner. Vitreous levels of Cyr61 are elevated in PDR, and it may play an important role in the disease's pathogenesis.

Fractalkine, a CX3C chemokine, as a mediator of ocular angiogenesis.

PURPOSE: Fractalkine (FKN) is a chemoattractant and adhesion molecule for leukocytes. Angiogenic effect of FKN also has been reported. This study was an investigation of FKN-mediated angiogenesis in vitro and in vivo to determine its role in ocular angiogenic disorders. METHODS: FKN effects on cultured human umbilical vein endothelial cells (HUVECs) and bovine retinal capillary endothelial cells (BRECs) were evaluated with chemotaxis assay and a synthetic matrix capillary tube formation assay in vitro. Reverse transcription-polymerase chain reaction (RT-PCR) and Western blot analysis were used to detect mRNA and protein expression of FKN and its receptor, CX3CR1, in HUVECs and BRECs. A rabbit corneal neovascularization assay and an oxygen-induced retinopathy (OIR) model of mice were used to test the angiogenic property of FKN in vivo. FKN levels of vitreous samples from patients with proliferative diabetic retinopathy were measured by enzyme-linked immunosorbent assay (ELISA). Immunodepletion of FKN in PDR vitreous samples by anti-FKN polyclonal antibody was observed in endothelial cell chemotaxis assays. RESULTS: FKN significantly induced migration of HUVECs and BRECs. FKN induced formation of endothelial cell capillary tubes on synthetic matrix. Expression of FKN and CX3CR1 was detected in HUVECs and BRECs by RT-PCR and Western blot analysis. FKN significantly induced more blood vessel growth than did the control in the rabbit corneal pocket neovascularization assay. Intravitreal injection of anti-mouse FKN antibody decreased retinal angiogenesis in the OIR model. The vitreous level of FKN was elevated in patients with PDR compared with control subjects. Immunodepletion of soluble FKN from PDR vitreous samples caused 36.6% less migration of BRECs. CONCLUSIONS: FKN is an angiogenic mediator in vitro and in vivo. The vitreous level of FKN was elevated in patients with PDR. FKN may play an important role in ocular angiogenic disorders such as PDR.