Clinical and Basic Research of the Angiogenic Factors in Proliferative Diabetic Retinopathy

Diabetic retinopathy is a common complication in patients of DM. Proliferative diabetic retinopathy (PDR) is the most common cause of blindness in DM patients. Several growth factors are involved in the pathogenesis of PDR, esp. VEGF. VEGF is a well-established factor and involved in the important pathogenesis of PDR. Although VEGF plays an important role in PDR, anti-VEGF therapy could not completely inhibit retinal angiogenesis in PDR and post-angiogenic fibrosis could not be inhibited. In addition to VEGF, several angogenic factors might be involved in retinal angiogenesis. We investigated the role of fractalkine (FKN), Cysteine-rich 61(Cyr61), and disintegrins in the pathogenesis of PDR.

Aim 1. Fractalkine, a CX3C chemokine, act as a mediator of ocular angiogenesis
Fractalkine (FKN) is a chemoattractant and adhesion molecule for leukocytes. Angiogenic effect of FKN also has been reported. We investigate FKN-mediated angiogenesis in vitro and in vivo to understand its role in ocular angiogenic disorders.
Fractalkine effects on cultured human umbilical vein endothelial cell (HUVEC) and bovine retinal capillary endothelial cell (BREC) were evaluated with chemotaxis assay and Matrigel capillary tube formation assay in vitro. Reverse transcriptase-polymerase chain reaction (RT-PCR) and Western blot analysis were used to detect mRNA and protein expression of FKN and its receptor, CX3CR1, in HUVEC and BREC. Rabbit corneal neovascularization assay and oxygen-induced retinopathy (OIR) model of mice were used to test the angiogenic property of FKN in vivo. Fractalkine levels of vitreous samples from proliferative diabetic retinopathy (PDR) patients were measured by enzyme-linked immunosorbent assay (ELISA). Immunodepletion of FKN in PDR vitreous samples by anti-FKN polyclonal antibody was observed in endothelial cells chemotaxis assays. Fractalkine significantly induced migration of HUVEC and BREC. Fractalkine induced formation of endothelial cell capillary tubes on Matrigel. Expression of FKN and CX3CR1 was detected in HUVEC and BREC by RT-PCR and Western blotting. Fractalkine significantly induced more blood vessel growth than control in rabbit corneal pocket neovascularization assay. Intravitreal injection of anti-mouse FKN antibody decreased retinal angiogenesis in the OIR model. Vitreous level of FKN was elevated in PDR patients as compared to control. Immunodepletion of soluble FKN from PDR vitreous samples caused 36.6% less migration of BREC. Take together, FKN is an angiogenic mediator in vitro and in vivo. Vitreous level of FKN was elevated in PDR patients. FKN may play an important role in ocular angiogenic disorders such as PDR.

Aim 2. Cysteine-rich 61, a member of CCN family, promotes retinal angiogenesis
Cysteine-rich 61 (Cyr61), a member of CCN family, is an angiogenic factor. We investigated Cyr61-mediated angiogenesis in vitro and in vivo to understand its role in ocular angiogenesis and diabetic retinopathy. Cyr61 effects on monkey chorioretinal endothelial cell (RF/6A) were evaluated using proliferation assay, chemotaxis assay, and Matrigel capillary tube formation assay in vitro. In addition, we investigated Cyr61 expression under hypoxic conditions by RT-PCR and Western blotting. A mouse model of oxygen-induced retinopathy (OIR) and a rat model of streptozocin-induced diabetes were used to test the angiogenic property of Cyr61 in vivo. Cyr61 levels were also measured in vitreous samples from proliferative diabetic retinopathy (PDR) patients by enzyme-linked immunosorbent assay (ELISA). Immunodepletion of Cyr61 in PDR vitreous samples by anti-Cyr61 polyclonal antibody was evaluated in an endothelial cells chemotaxis assay. Cyr61 significantly induced the proliferation and migration of RF/6A, and the formation of endothelial cell capillary tubes on Matrigel. Hypoxia significantly induced Cyr61 mRNA and protein expression Cyr61 was significantly expressed in neovascularized retina in the mouse OIR model and in the streptozocin-induced diabetic rat model. Intravitreal injection of anti-mouse Cyr61 antibody in the OIR model significantly suppressed retinal neovascularization. Vitreous levels of Cyr61 were elevated in PDR patients when compared with non-diabetic patients. Immunodepletion of Cyr61 from PDR vitreous samples significantly inhibited the migration of RF/6A cells. Cyr61 is an angiogenic mediator in vitro and in vivo. Vitreous levels of Cyr61 are elevated in PDR patients. Cyr61 may promote retinal angiogenesis, especially in PDR.

Aim 3. Elevation of cysteine-rich 61 levels in vitreous of patients with proliferative diabetic retinopathy
Cysteine-rich 61 (Cyr61) is one of angiogenic factors involved in proliferative diabetic retinopathy (PDR). To further investigate its role, we measure and compare the vitreous levels of Cyr61and vascular endothelial growth factor (VEGF) 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 (NPDR) patients with diabetic macular edema (DME) and 25 patients with other non-diabetic-related eye diseases, were subjected to enzyme-linked immunosorbent assay for Cyr61 and VEGF levels. Epiretinal membrane (ERM) from 6 patients with idiopathic ERMs, 5 patients with active PDR and 7 patients with active PDR pretreated with bevacizumab were stained immunohistochmically for Cyr61. Vitreous Cyr61 levels were significantly higher in active PDR patients than that in quiescent PDR patients, or nondiabetic control patients. Vitreous levels of Cyr61 were significantly elevated in NPDR patients with DME. The correlations between the vitreous levels of Cyr61 and the vitreous levels of VEGF were strong in active PDR, quiescent PDR, and DME groups. Pretreatment of bevacizumab significantly suppressed vitreous VEGF levels, however, did not inhibit vitreous Cyr61 levels in active PDR patients. Cyr61 was strongly detected in endothelial cells and myofibroblasts within active PDR membranes but not in idiopathic ERM. The localization of Cyr61 in myofibroblasts and endothelial cells suggests local autocrine-paracrine mechanism for induction of angiogenesis and post-angiogenic fibrosis in PDR.

Aim 4. Regulation of Cyr61/CCN1 expression by hypoxia through cooperation of c-Jun/AP-1 and HIF-1α in retinal vascular endothelial cells
Hypoxia is one of the major factors 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 assay were performed to study the role of hypoxia-inducible factor (HIF)-1α and c-Jun/AP-1 in Cyr61 transcriptional regulation. 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. The hypoxia induced transcriptional regulation of Cyr61, a hypoxia-inducible factor, in RF/6A cells was controlled by cooperation of HIF-1α and c-Jun/AP-1. Cyr61 may have an important role in ischemic retinal diseases, such as PDR.

Aim 5. Suppression of Ocular Neovascularization by Disintegrins Derived From Snake Venom
Integrins play an important role in the pathogenesis of angiogenesis. Recently, disintegrins derived from snake venom was showed to inhibit integrins-mediated angiogesis in oncology. We investigated the possibility that disintegrins inhibited angiogenesis in intraocular angiogenesis. Four disintegrins (GRGDS, Kistrin, Echistatin, and Flavoriden) were used to perform chemotaxis, matrigel tube formation, and adhesion assay in bovine retinal vascular endothelial cells (BRECs). Chemical injury-induced corneal neovascularization model was investigated the inhibition effect of disintegrins. To test the anti-angiogenic property in oxygen-induced retinopathy (OIR) model, intravitreal injection of disintegrins was performed to inhibit the in vivo angiogenesis. Vitreous samples of proliferative diabetic retinopathy (PDR) patients were collected during pars plana vitrectomy. Vitreous samples of PDR patients pretreated with disintegrins were used for chemotaxis assays to determine the role of disintegrins. Disintegrins significantly inhibited bFGF-induced migration, bFGF-induced tube formation, and adhesion in BRECs. Topical treatment of kistrin could inhibit chemical injury-induced corneal neovascularization of rats. Intravitreal injection of disintegrins significantly inhibited retinal neovascularization of mice in the OIR model. Vitreous samples from the PDR patients pretreated with disintegrins induced less migration of retinal endothelial cells. These results establish disintegrins derived from snake venom as an anti-angiogenic mediator in vitro and in vivo and suggest that these may play an important role in the ocular angiogenesis, such as PDR. It might be a new strategy for a new potential target for treating these diseases.
Take together, we found the two angiogenic factors, fractalkine and Cyr61, mediated as an important role in ocular angiogenesis. In the in-vitro and in-vivo experiments, we investigated the factors were important factors in ocular angiogenesis. Elevated vitreous levels of fractalkine and Cyr61 further proved their role in retinal angiogenesis. To further investigate the mechanism of Cyr61 expression, we found that hypoxia, an important fractor involved in PDR, could induce and regulate the transcription of the Cyr61 gene through cooperation of transcription factor HIF-1α and c-Jun/AP-1 in retinal vascular endothelial cells. Integrins mediated as important role in angiogenesis. Disintegrins derived from snake venom could effectively inhibit activation of integrins. We investigated the anti-angiogenic effect of disintegrins in ocular angiogenesis, and found that disintegrins could inhibit angiogenesis by inhibition of integrin activation through induction of apoptosis of endothelial cells. Therefore, these finding might give us a new strategy to treat ocular angiogenesis, esp. PDR.