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Purpose: To evaluate the safety and efficacy of intravitreal injections of 0.5 mg conbercept in patients with choroidal neovascularization secondary to pathological myopia (pmCNV). Methods: The 177 pmCNV patients were randomly assigned in a 3:1 ratio to receive conbercept or sham injection, respectively. The conbercept group receive conbercept intravitreal injections administered on a pro re nata (PRN) basis after 3 monthly loading doses. The sham group received three consecutive monthly sham injections and then one conbercept injection followed by PRN conbercept intravitreal injections. Results: At month 3, the mean BCVA for the two groups were improved by 12.0 letters (conbercept group, from 54.05 letters to 66.05 letters) and 0.6 letters (sham group, from 49.77 letters to 50.33 letters), respectively (p < 0.001). The mean central retinal thickness (CRT) at month 3 in the two groups decreased 62.0 μm (conbercept group, from 348.90 μm to 286.18 μm) and 4.4 μm (sham group, from 347.86 μm to 343.47 μm) (p < 0.001). At month 9, the mean BCVA improved by 13.3 letters in the conbercept group and 11.3 letters in the sham group. The mean CRT decreased 73.6 μm in the conbercept group and 55.9 μm in the sham group (p < 0.001). The most common ocular adverse events were associated with intravitreal injections, such as conjunctival haemorrhage and increased intraocular pressure. Conclusion: Intravitreal injections of 0.5 mg conbercept provided improvement in visual and anatomical outcomes in pmCNV patients with low rates of ocular and nonocular safety events. [ABSTRACT FROM AUTHOR]

To investigate the influence of age on the function and morphology of patients with myopic choroidal neovascularization (mCNV) and to evaluate the effect and prognostic factors of recurrence of Conbercept treatment on mCNV patients over 50 years. A total of 64 patients (64 eyes) with mCNV were enrolled in this retrospective study. The differences in baseline best-corrected visual acuity (BCVA) and morphological features on imaging between the younger group (˂ 50 years) and the older group (≥ 50 years) were analyzed. Of all, 21 eyes of 21 mCNV patients aged over 50 years who received Conbercept injection were further analyzed. Between the younger and the older group, significant differences were shown in mean BCVA (0.58 ± 0.28 vs 0.77 ± 0.31), subfoveal choroidal thickness (SFCT) (108.17 ± 78.32 μm vs 54.68 ± 39.03 μm) and frequency of vitreoretinal interface abnormalities (VIA) (2 vs 13), respectively (P < 0.05). After treated with Conbercept, the mean BCVA of 21 older mCNV patients increased from 0.83 ± 0.30 at baseline to 0.49 ± 0.24 at one year. Baseline BCVA, external limiting membrane damage, CNV area and CNV location correlated with the visual acuity at the 1-year follow-up. There were 7 (33.3%) recurrent cases during the follow-up and the risk of recurrence in patients with baseline central macular thickness (CMT) ≥ 262.86 μm was 14 times greater than that of patients with CMT < 262.86 μm. The risk of recurrence increased 1.84 times for every 100-μm increment in the CMT. Patients over 50 years with mCNV had a worse BCVA, thinner choroid, and higher risk of VIA than young mCNV patients. The standard Conbercept treatment strategy was safe and effective in mCNV patients over 50 years. As patients over 50 years with a greater CMT have a high risk of recurrence, more attention should be paid on these patients by following them up closely. [ABSTRACT FROM AUTHOR]

Ocular neovascularization is a comprehensive process involved in retinal vascular development and several blinding diseases such as age-related macular degeneration and retinopathy of prematurity, with vascular endothelial growth factor (VEGF) regarded as the master regulator. However, the qualified effect of anti-VEGF therapy reveals that the underlying mechanisms are still not clearly identified. To initialize angiogenesis, endothelial cells undergo a phenotype switching to generate highly migratory and invasive cells. This process shares certain similar characters observed in endothelial-mesenchymal transition (EndMT). Here, we found that SNAI1, an EndMT transcription factor, was expressed by endothelial cells in both physiological and pathological ocular neovascularization. SNAI1 overexpression triggered cell morphological change and enhanced cell motility, while loss of SNAI1 attenuated migration, invasion and sprouting. RNA sequence analysis further revealed that SNAI1 knockdown decreased the expression of genes related to cytoskeleton rearrangement and ECM remodeling. Moreover, intravitreal injection of small interfering RNA of SNAI1 suppressed new vessel formation in developing retina as well as mice model of choroidal neovascularization and oxygen-induced retinopathy. Therefore, we propose that the EndMT transcription factor SNAI1 promotes the early phase of ocular neovascularization and may provide a potential therapeutic target. [ABSTRACT FROM AUTHOR]

Retinal microglia cells contribute to vascular angiogenesis and vasculopathy induced by relative hypoxia. However, its concrete molecular mechanisms in shaping retinal angiogenesis have not been elucidated. Basigin, being involved in tumour neovasculogenesis, is explored to exert positive effects on retinal angiogenesis induced by microglia. Therefore, we set out to investigate the expression of basigin using a well-characterized mouse model of oxygen-induced retinopathy, which recapitulated hypoxia-induced aberrant neovessel growth. Our results elucidate that basigin is overexpressed in microglia, which accumulating in retinal angiogenic sprouts. In vitro, conditioned media from microglia BV2 under hypoxia treatment increase migration and tube formation of retinal capillary endothelia cells, compared with media from normoxic condition. The angiogenic capacity of BV2 is inhibited after basigin knockdown by small interfering RNAs. A new molecular mechanism for high angiogenic capacity, whereby microglia cells release basigin via up-regulation of PI3K-AKT and IGF-1 pathway to induce angiogenesis is unveiled. Collectively, our results demonstrate that basigin from hypoxic microglia plays a pivotal pro-angiogenic role, providing new insights into microglia-promoting retinal angiogenesis. [ABSTRACT FROM AUTHOR]

To investigate the role of macrophages in oxygen-induced retinal neovascularization (NV) in mice, particularly the involvement of bone marrow-derived cells (BMCs) and the underlying mechanisms, BMCs from green fluorescent protein (GFP) transgenic mice were transplanted into postnatal day (P) 1 mice after irradiation. The mice were exposed to 75 % oxygen from P7 to P12 to initiate oxygen-induced retinopathy (OIR). The macrophages were depleted by injection of clodronate-liposomes (lip) intraperitoneally. The eyes were collected at P12 and P17. Retinal flatmounts and histopathological cross-sections were performed to analyze the severity of retinal NV and BMC recruitment. BMCs immunopositive for CD31 (PECAM-1; endothelial cell marker) and α-SMA (smooth muscle cell marker) antigens were detected using a confocal microscope. Expression of vascular endothelial growth factor (VEGF) and stromal cell-derived factor-1 (SDF-1) mRNA was detected by RT-PCR. The VEGF, SDF-1, CXCR4 and CD45 protein expression was detected by western blot examination. The retinal avascular area in OIR mice at P12 was unaffected after macrophage depletion carried out twice (38.27 ± 1.92 % reduction) using clodronate-lip. The retinal avascular area and the NV area at P17 were reduced after macrophage depletion four times (79.53 ± 1.02 % reduction); these findings were supported by retinal flatmounts and histopathological cross-sections. Macrophage depletion led to significant inhibition of BMC recruitment into the NV tufts at P17, with decreased expression of retinal VEGF, SDF-1, CXCR4 and CD45. The recruited BMCs differentiated primarily into CD31-positive endothelial cells (ECs) and α-SMA-positive smooth muscle cells (SMCs). This study suggested that macrophages promoted the vasculogenesis of retinal NV, particularly the contribution of BMCs in the mouse OIR model, which might be triggered by VEGF and SDF-1 production. [ABSTRACT FROM AUTHOR]

Purpose: This study investigated the in-vivo formation process of laser-induced choroidal neovascularization (CNV) in rat using high-resolution spectral-domain optical coherence tomography (SD-OCT), and compared the results to histological methods. Methods: Brown Norway rats ( n = 60, 6-8 weeks of age) received 532-nm diode laser photocoagulation. SD-OCT and fluorescein angiography (FA) were performed in vivo 2, 5, 7, 14, and 21 days post-laser application. Haematoxylin and eosin (H&E) staining and immunohistochemistry for CD31, phosphorylated vascular endothelial factor receptor 2 (pVEGFR2) were conducted at each time point to observe the CNV in vitro. Choroidal flatmount preparations were observed using a confocal laser scanning microscope (CLSM) and a scanning electron microscope (SEM). Results: SD-OCT monitored the longitudinal morphological changes of laser-induced CNV. CNV reached its maximal size on day 7, and began a gradual reduction on day 14. FA revealed similar dynamic changes in leakage. CNV thickness, as assessed by SD-OCT, was consistent with H&E-stained sections at each time point. CLSM and SEM revealed the details of the fibrovascular membrane. CD31 and pVEGFR2 expression supported the results of SD-OCT and histology. Conclusions: SD-OCT was a convenient and reliable tool for the imaging of the CNV formation process and quantification of the lesion size in vivo. [ABSTRACT FROM AUTHOR]

Purpose: Choroidal neovascularization (CNV) is a major cause of vision loss in patients with age-related macular degeneration (AMD). Stromal cell-derived factor-1 (SDF-1)/CXC chemokine receptor 4 (CXCR4) plays a critical role in homing of bone marrow-derived cells (BMCs) to choroidal neovascularization (CNV). In this study, we investigated the contribution of hypoxia specific HIF-1α-induced SDF-1 expression in retinal pigment epithelium (RPE) cells and the potential role of SDF-1 in CNV formation. Materials and Methods: Green fluorescent protein (GFP) chimeric mice were developed by transplanting bone marrow cells of gfp++/++ transgenic mice to sublethally irradiated C57BL/6J mice. CNV was induced by laser photocoagulation. Ocular tissue was processed for immunofluorescence to detect HIF-1α and SDF-1 expression, and cell surface markers such as CXCR4, CD34 and CD31 and so on during CNV formation. In vitro, adult human RPE (hRPE) cells were cultured under conditions of chemical hypoxia using CoCl2 administration. And RNAi technique was used to knock down HIF-1α gene to observe the expression of HIF-1α and SDF-1 in hRPE cells. Results: BMCs trafficked around laser lesion adjacent to RPE layer 4 h after laser photocoagulation, where SDF-1 expression was relatively higher. With increasing expression of SDF-1, more BMCs were infiltrated into laser lesion to participate in CNV, and both reached peak at 3 d ( p < 0.05). About 81% BMCs involved in CNV were CXCR4++. Many of them acquired the surface marker of endothelial precursor cells (CD34++) and endothelial cells (CD31++). The constituent ratio of CD34++ and CD31++ BMCs increased with SDF-1 expression. In vitro, we proved that hypoxia specific-HIF-1α influenced SDF-1 expression in hRPE cells. Conclusions: These findings suggested that hypoxia-induced SDF-1 expression in RPE might be a critical initiator for recruitment of BMCs in CNV. SDF-1 might be another important factor in BMCs' differentiation into endothelial cells to participate in the CNV. [ABSTRACT FROM AUTHOR]

Abstract: Choroidal neovascularization (CNV) is one of the most frequent causes of severe and progressive vision loss, while its pathogenesis is still poorly understood. Focal adhesion kinase (FAK), a non-receptor tyrosine kinase, plays a crucial role in linking signals initiated by both the extracellular matrix (ECM) and soluble signaling factors and controls essential cellular processes. Extensive evidence has shown that FAK is activated in angiogenic response. This study aims to investigate the effect of FAK on CNV formation. The Brown-Norway (BN) rats underwent laser rupture of Bruch''s membrane to induce CNV and were then killed at 1, 3, 7, and 14 days following laser injury. Immunofluorescence and Western blot were processed to detect FAK protein. Retinal pigment epithelial (RPE) cells were cultured under hypoxia and RNA interference (RNAi) technique was used to knock down the FAK gene in RPE cells. Expression of hypoxia inducible factor-1 (HIF-1α) and vascular endothelial growth factor (VEGF) in RPE cells were investigated by RT-PCR and Western blot. Two kinds of coculture models were used to observe the effects of specific blockade of FAK in RPE cells on the proliferation and migration of choroidal microvascular endothelial cells (CECs), respectively. FAK was highly expressed in the rat RPE-choroid tissue after photocoagulation. In vitro experiment showed that FAK was involved in hypoxia signaling in cultured RPE cells. The absence of FAK effectively reduced the expression of hypoxia-induced HIF-1α and VEGF in RPE cells, resulting in the inhibition of proliferation and migration of CECs. Our results suggest that FAK pathway activation plays a role in the development of CNV, and regulates the proliferation and migration of CECs by acting through HIF-1 and then up-regulating the expression of the angiogenic factor VEGF in RPE cells. It is reasonable to propose that FAK siRNA will potentially provides a means to attenuate the strong stimuli for neovascularization in CNV-dependent disorders, which could present a therapeutically relevant strategy for the inhibition of CNV. [Copyright &y& Elsevier]

Purpose: Choroidal neovascularization (CNV) is one of the most frequent causes of severe and progressive vision loss. Prior studies have shown that bone marrow-derived cells (BMCs) play an important role in CNV, indicating that BMCs can be a potential target for inhibiting the development of CNV. It could be helpful for our understanding of CNV to study the dynamic conduct of BMCs in the CNV microenvironment. Methods: Green fluorescent protein (gfp) chimeric mice were developed by transplanting bone marrow cells from gfp+/+ transgenic mice to adult C57BL/6J mice. The chimeric mice underwent laser rupture of Bruch's membrane to induce CNV and were killed at 1, 2, 3, and 4 weeks after laser injury. The eyes were enucleated and processed for immunofluorescence to detect markers for vascular smooth muscle cells (α smooth muscle actin, α SMA), endothelial cells (CD31), or macrophages (F4/80) on gfp+ cells. All sections were qualitatively and quantitatively assessed by confocal microscopy. Results: Large number of gfp-labeled cells appeared in the lesions and integrated into CNV. Gfp+ cells, which were immunoreactive for α SMA, CD31, or F4/80, can be detected through the whole study. The constituent ratio of those three cell-types in total gfp+ cells in CNV altered as CNV developed. The maximal ratios of CD31-labeled cells and F4/80-labeled cells presented at 2 week, while the ratio of α SMA-labeled cells upgraded continuously. Conclusions: BMCs underwent a serial of changes in position and expression during the progression of CNV. Those changes may result from the interaction between BMCs and the CNV microenvironment. [ABSTRACT FROM AUTHOR]

Purpose : To investigate the potential role of endostatin, an endogenous angiogenesis inhibitor, in the prevention of choroidal angiogenesis-related disorders. Methods : Bovine choroidal endothelial cells (CEC) were cultured and treated with basic fibroblast growth factor (bFGF) alone or combined with endostatin at concentrations ranging from 0.1 to 10 µg/ml. The proliferation and migration of CECs were evaluated by using 3, (4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) colorimetric assay and modified Boyden chamber assay, respectively. For evaluating expression and secretion of matrix metalloproteinase-2 (MMP-2), CEC-conditioned media were subjected to zymography and/or Western blot analysis, and the cells were used for semiquantitative reverse transcription polymerase chain reaction (RT-PCR) analysis. Results : Endostatin did not inhibit bFGF-induced or nonstimulated CEC proliferation (p > 0.05). The bFGF-induced migration was significantly inhibited by endostatin at concentrations of 1 and 10 µg/ml (p < 0.05). The bFGF-upregulated expression of mRNA in CECs and the secretion of MMP-2 protein of CECs were both suppressed by endostatin. Conclusions: Inhibitory effect of endostatin on expression and secretion of MMP-2 and cell migration, but not on proliferation of CECs, could respond to its therapeutic action for choroidal neovascularization-dependent disorders. [ABSTRACT FROM AUTHOR]

Abstract: To investigate the influence of hyperglycemia on the severity of choroidal neovascularization (CNV) in diabetic mice, especially the involvement of bone marrow-derived cells (BMCs) and underlying molecular mechanisms. The mice were randomly divided into control group, diabetes group and diabetes treated with insulin group, which were laser treated to induce CNV. The CNV severity was evaluated by fundus fluorescein angiography, HE staining and choroidal flatmount. The BMCs recruitment and differentiation in CNV were examined in GFP chimeric mice by choroidal flatmount and immunofluorescence. The bone marrow-derived mesenchymal stem cells (BMSCs) recruitment and migration were tested in vivo and in vitro. VEGF and SDF-1 production in vivo and in vitro were tested by realtime PCR and ELISA. The CNV severity and expression of VEGF and SDF-1 were enhanced in DM mice compared with control mice and that insulin treatment decreased CNV severity in DM mice. The DM mice demonstrated more BMCs and bone marrow-derived mesenchymal stem cells (BMSCs) recruited and incorporated into CNV, increased ratio of BMCs expressing endothelial cell marker or macrophage marker, and up-regulated expression of VEGF and SDF-1 in CNV. Human BMSCs migration and expression of VEGF and SDF-1 in retinal pigment epithelial (RPE) cells increased when cultured under high glucose. This study suggested that hyperglycemia enhanced the expression of VEGF and SDF-1 in RPE cells, and promoted recruitment and incorporation of BMCs and affected differentiation of BMCs in CNV, which led to more severe CNV in diabetic mice. [Copyright &y& Elsevier]

Choroidal neovascularization (CNV) is an acknowledged pathogenic mechanism of various ocular diseases, and in situ cells and mobilized bone marrow-derived cells (BMCs) are thought to participate in this process. We aimed to evaluate the roles of integrin α5 in BMCs and vascular endothelial cells (VECs) in the CNV process mediated by SDF-1/CXCR4 signaling. Adult wild-type mice were engrafted with whole BMCs obtained from GFP transgenic mice and then laser injured to induce CNV. BMCs and RF/6A cells were cultured to discover the mechanism of CNV in vitro. BMCs were mobilized to CNV areas, which expressed elevated SDF-1 and CXCR4. When SDF-1 was intravitreally injected, the number of BMCs was profoundly increased. In the SDF-1-treated group, the levels of integrin α5 expressed on BMCs and VECs were significantly higher than those on the cells in the control group. SDF-1 significantly increased the expression and positive ratio of integrin α5, which was involved in the recruitment and differentiation of BMCs into BMC-derived VECs, and these effects were suppressed by the CXCR4 inhibitor AMD3100. The PI3K/AKT pathway rather than the ERK pathway mediated SDF-1/CXCR4 induction of integrin α5. Integrin α5 suppression efficiently prevented the production of TGF-β and bFGF but not VEGF. Inhibiting the SDF-1/CXCR4-PI3K/AKT-integrin α5 axis reduced CNV severity. Integrin α5 participates in BMC recruitment and differentiation in SDF-1/CXCR4-induced CNV and inhibition of this pathway may be a new approach to inhibit CNV. • BMCs were mobilized to CNV areas and differentiated into endothelial cells. • SDF-1/CXCR4 signal promotes integrin α5 expression in BMCs via PI3K/AKT. • Integrin α5 potentiated mobilization and differentiation of BMC to endothelial cells. • Integrin α5 exacerbated angiogenesis in choroidal lesion areas. [ABSTRACT FROM AUTHOR]