Cysteine-rich 61 (CYR61) is up-regulated in proliferative diabetic retinopathy.

To investigate the role of CYR61 as a retinal angiogenic factor in proliferative diabetic retinopathy (PDR). Effects of CYR61 on RF/6A cell proliferation, migration and angiogenesis were observed by MTT assay, Transwell assay, and tube formation assay. The expression and distribution of CYR61 on retina layers of diabetic mouse were demonstrated by immunohistochemistry. The expression of Cyr61 mRNA in diabetic mouse retina was evaluated by reverse transcription-polymerase chain reaction (RT-PCR). Vitreous CYR61 levels of PDR and non-diabetic patients were measured by enzyme-linked immunosorbent assay (ELISA). Expression and distribution of CYR61 on epiretinal membrane of PDR, proliferative vitreoretinopathy (PVR) and idiopathic epiretinal membrane were evaluated by immunohistochemistry. RF/6A cell proliferation, migration and tube formation capacity increased with increased concentration of CYR61 (p = 0.000). Anti-CYR61 antibody could inhibit cell migration and tube formation promoted by CYR61. In diabetic mouse, CYR61 was expressed in retina layers just as normal mouse, but the staining was stronger than normal in ganglion cell layer and inner plexiform layer. The Cyr61 mRNA expression in retina of diabetic mouse was more than that in normal mouse (p = 0.009). Vitreous CYR61 level was higher in patients with PDR than non-diabetic patients (p = 0.000). PDR patients with plenty of neovasculature on retina and epiretinal membranes had higher level of vitreous CYR61 than patients with little neovasculature (p = 0.001). CYR61 expressed in the cytoplasm of epiretinal membranes in PDR, especially in the wall cells of the tube-like structure. CYR61 are likely to be involved in the pathogenesis of diabetic retinopathy, and may play a role in the course of neovasculation.

Zhang X ，Yu W ，Dong F 《-》

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

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. 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. 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. 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.

You JJ ，Yang CH ，Chen MS ，Yang CM ... -  《-》

Tenascin-C promotes angiogenesis in fibrovascular membranes in eyes with proliferative diabetic retinopathy.

We previously demonstrated that tenascin-C was highly expressed in the fibrovascular membranes (FVMs) of patients with proliferative diabetic retinopathy (PDR). However, its role in the pathogenesis of FVMs has not been determined. The purpose of this study was to investigate what role tenascin-C plays in the formation and angiogenesis of FVMs. The level of tenascin-C was determined by sandwich enzyme-linked immunosorbent assay in the vitreous samples collected from patients with PDR and with a macular hole as control. The locations of tenascin-C, α- smooth muscle actin (SMA), CD34, glial fibrillary acidic protein (GFAP), and integrin αV in the FVMs from PDR patients were determined by immunohistochemistry. We also measured the in vitro expression of the mRNA and protein of tenascin-C in vascular smooth muscle cells (VSMCs) stimulated by interleukin (IL)-13. The effects of tenascin-C on cell proliferation, migration, and tube formation were determined in human retinal endothelial cells (HRECs) in culture. The mean vitreous levels of tenascin-C were significantly higher in patients with PDR than in patients with a macular hole (p<0.001). Double immunofluorescence analyses of FVMs from PDR patients showed that tenascin-C co-stained FVMs with α-SMA, CD34, and integrin αV but not with GFAP. In addition, IL-13 treatment increased both the expression and secretion of tenascin-C by VSMCs in a dose-dependent manner. Tenascin-C exposure promoted proliferation, migration, and tube formation in HRECs. Tenascin-C neutralizing antibody significantly blocked the tube formation by HRECs exposed to VSMC-IL-13-conditioned medium. Our findings suggest that tenascin-C is secreted from VSMCs and promotes angiogenesis in the FVMs associated with PDR.

Kobayashi Y ，Yoshida S ，Zhou Y ，Nakama T ，Ishikawa K ，Arima M ，Nakao S ，Sassa Y ，Takeda A ，Hisatomi T ，Ikeda Y ，Matsuda A ，Sonoda KH ，Ishibashi T ... -  《MOLECULAR VISION》

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

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. 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. 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. 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.

You JJ ，Yang CM ，Chen MS ，Yang CH ... -  《-》

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

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. 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. 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. 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.

You JJ ，Yang CH ，Huang JS ，Chen MS ，Yang CM ... -  《INVESTIGATIVE OPHTHALMOLOGY & VISUAL SCIENCE》