Anti-angiogenic effects of a mutant endostatin: a new prospect for treating retinal and choroidal neovascularization.
ABSTRACT: Pathological fundus angiogenesis is a major cause of vision loss in retina diseases. Endostatin, a C-terminal fragment of collagen XVIII, is an endogenous anti-angiogenic protein. The present study aimed to investigate the in vitro and in vivo anti-angiogenic properties of two proteins: an N-terminal H1D/H3D mutant endostatin (M-ES) and a polyethylene glycol propionaldehyde (PEG) covalent M-ES (PEG-M-ES).M-ES and PEG-M-ES properties were characterized in vitro using a zinc ion binding assay and a stability test. Activity assays, including migration, proliferation, and tube formation assays, were performed with human retinal microvascular endothelial cells (HRMECs) and human umbilical vein endothelial cells (HUVECs). Mouse oxygen-induced retinopathy (OIR) and choroidal neovascularization (CNV) models were used to evaluate in vivo anti-angiogenic effects. In addition, a rabbit model was used to study the retinal pharmacokinetic profile following an intravitreal injection.The results indicated that the H1D/H3D mutations of endostatin reduced the zinc binding capacity of M-ES and facilitated PEG covalent binding. PEG-M-ES was more stable and persisted longer in the retina compared with M-ES. The in vitro studies demonstrated that M-ES and PEG-M-ES inhibited HRMEC and HUVEC proliferation, migration, and tube formation more efficiently than ES. In vivo, a single intravitreal injection of M-ES and PEG-M-ES significantly decreased neovascularization in both the OIR and CNV animal models.The present study demonstrated for the first time that PEG-M-ES exhibits a long-term inhibitory effect on neovascularization in vitro and in vivo. These data suggest that PEG-M-ES may represent an innovative therapeutic strategy to prevent fundus neovascularization.
Project description:Pigment epithelium-derived factor (PEDF) is a serine proteinase inhibitor with antiangiogenic activities. To investigate whether PEDF overexpression has an impact on ocular neovascularization in vivo, we generated PEDF transgenic (PEDF-Tg) mice that ubiquitously express human PEDF driven by the ?-actin promoter. The PEDF-Tg mice under normal conditions did not show any abnormalities in retinal histologic findings or visual function. In contrast, PEDF-Tg animals with oxygen-induced retinopathy (OIR) developed significantly less severe retinal neovascularization compared with wild-type (Wt) mice with OIR. In addition, PEDF-Tg mice with OIR had significantly lower vascular leakage in the retina but higher occludin levels than the Wt mice with OIR, suggesting a protective effect on the blood-retinal barrier. Furthermore, retinal levels of proinflammatory factors were significantly lower in PEDF-Tg mice with OIR than in the Wt mice with OIR. In the laser-induced choroidal neovascularization (CNV) model, the CNV area was significantly smaller in the PEDF-Tg mice than in the Wt mice. Also, the laser burn-induced overexpression of proangiogenic and inflammatory factors was observed in the retina and retinal pigment epithelium of Wt mice but not in PEDF-Tg mice. Taken together, these results suggest that overexpression of PEDF inhibits retinal inflammation and neovascularization in both the OIR and laser-induced CNV models. The PEDF-Tg mice provide a useful model for studying the roles of angiogenic inhibitors in neovascular disorders such as diabetic retinopathy.
Project description:Choroidal neovascularization (CNV) is a key pathological feature of several leading causes of vision loss including neovascular age-related macular degeneration. Here, we show that a calreticulin anti-angiogenic domain (CAD)-like peptide 27, CAD27, inhibited in vitro angiogenic activities, including tube formation, migration of endothelial cells, and vascular sprouting from rat aortic ring explants. In a rat model of laser-induced CNV, we demonstrate that intravitreal injection of CAD27 significantly attenuated the formation of CNV lesions as measured via fundus fluorescein angiography and choroid flat-mounts (19.5% and 22.4% reductions at 10 ?g and 20 ?g of CAD27 injected, respectively). Similarly, the reduction of CNV lesions was observed in rats that had received topical applications of CAD27 (choroid flat-mounts: 17.9% and 32.5% reductions at 10 ?g/mL and 20 ?g/mL of CAD27 instilled, respectively). Retinal function was unaffected, as measured using electroretinography in both groups receiving interareal injection or topical applications of CAD27 for at least fourteen days. These findings show that CAD27 can be used as a potential therapeutic alternative for targeting CNV in diseases such as neovascular age-related macular degeneration.
Project description:Pathologic ocular neovascularization commonly results in visual impairment or even blindness in numerous fundus diseases, including proliferative diabetic retinopathy (PDR), retinopathy of prematurity (ROP), and age-related macular degeneration (AMD). MicroRNAs regulate angiogenesis through modulating target genes and disease progression, making them a new class of targets for drug discovery. In this study, we investigated the potential role of miR-18a-5p in retinal neovascularization using a mouse model of oxygen-induced proliferative retinopathy (OIR). We found that miR-18a-5p was highly expressed in the retina of pups as well as retinal endothelial cells, and was consistently down-regulated during retinal development. On the other hand, miR-18a-5p was increased significantly during pathologic neovascularization in the retinas of OIR mice. Moreover, intravitreal administration of miRNA mimic, agomiR-18a-5p, significantly suppressed retinal neovascularization in OIR models. Accordingly, agomir-18a-5p markedly suppressed human retinal microvascular endothelial cell (HRMEC) function including proliferation, migration, and tube formation ability. Additionally, we demonstrated that miR-18a-5p directly down-regulated known vascular growth factors, fibroblast growth factor 1 (FGF1) and hypoxia-inducible factor 1-alpha (HIF1A), as the target genes. In conclusion, miR-18a-5p may be a useful drug target for pathologic ocular neovascularization.
Project description:PURPOSE:Hypoxia-inducible factor (HIF)-1 is a key oxygen sensor and is believed to play an important role in neovascularization (NV). The purpose of this study is to determine the role of retinal pigment epithelium (RPE)-derived HIF-1α on ocular NV. METHODS:Conditional HIF-1α knockout (KO) mice were generated by crossing transgenic mice expressing Cre in the RPE with HIF-1α floxed mice, confirmed by immunohistochemistry, Western blot analysis, and fundus fluorescein angiography. The mice were used for the oxygen-induced retinopathy (OIR) and laser-induced choroidal neovascularization (CNV) models. RESULTS:HIF-1α levels were significantly decreased in the RPE layer of ocular sections and in primary RPE cells from the HIF-1α KO mice. Under normal conditions, the HIF-1α KO mice exhibited no apparent abnormalities in retinal histology or visual function as shown by light microscopy and electroretinogram recording, respectively. The HIF-1α KO mice with OIR showed no significant difference from the wild-type (WT) mice in retinal levels of HIF-1α and VEGF as well as in the number of preretinal neovascular cells. In the laser-induced CNV model, however, the disruption of HIF-1α in the RPE attenuated the over expression of VEGF and the intercellular adhesion molecule 1 (ICAM-1), and reduced vascular leakage and CNV area. CONCLUSIONS:RPE-derived HIF-1α plays a key role in CNV, but not in ischemia-induced retinal NV.
Project description:Purpose:Ciliary neurotrophic factor (CNTF) is a well-characterized neurotrophic factor currently in clinical trials for the treatment of macular telangiectasia type II. Our previous work showed that CNTF-induced STAT3 signaling is a potent inhibitor of pathologic preretinal neovascular tuft formation in the mouse model of oxygen-induced retinopathy. In this study, we investigated the effect of CNTF on outer retinal and choroidal angiogenesis and the mechanisms that underpin the observed decrease in outer retinal neovascularization following CNTF treatment. Methods:In the Vldlr-/- and laser-CNV mouse models, mice received a one-time injection (on postnatal day [P] 12 in the Vldlr-/- model and 1 day after laser in the Choroidal Neovascularization (CNV) model) of recombinant CNTF or CxCl10, and the extent of neovascular lesions was assessed 6 days posttreatment. STAT3 downstream targets affected by CNTF treatment were identified using quantitative PCR analysis. A proteome array was used to compare media conditioned by CNTF-treated and control-treated primary Müller cells to screen for CNTF-induced changes in secreted angiogenic factors. Results:Intravitreal treatment with recombinant CNTF led to significant reduction in neovascularization in the Vldlr-/- and laser-CNV mouse models. Treatment effect in the Vldlr-/- was long-lasting but time sensitive, requiring intravitreal treatment before P19. Mechanistic workup in vitro as well as in vivo confirmed significant activation of the STAT3-signaling pathway in Müller cells in response to CNTF treatment and upregulation of CxCl10. Intravitreal injections of recombinant CxCl10 significantly reduced outer retinal neovascularization in vivo in both the Vldlr-/- and laser-CNV mouse models. Conclusions:CNTF treatment indirectly affects outer retinal and choroidal neovascularization by inducing CxCl10 secretion from retinal Müller cells.
Project description:The purpose of this study was to investigate the impact of stanniocalcin-1 (STC-1), a photoreceptor-protective glycoprotein, on the development of choroidal neovascularization (CNV) in relation to VEGF and its main receptor (VEGFR2) expression after laser injury.In rats, CNV was induced by laser photocoagulation in both eyes, followed by intravitreal injection of STC-1 in the right eye and vehicle or denatured STC-1 injection in the left eye as control. Two weeks after laser injury, fundus autofluorescence (FAF) imaging and fundus fluorescein angiography (FFA) were performed. Fluorescein leakage from CNV was graded using a defined scale system. The size of CNV was quantified with spectral domain optical coherence tomography (SD-OCT), fluorescein-labeled choroid-sclera flat mounts, and hematoxylin-eosin staining. Protein expressions were evaluated by Western blot.Photocoagulation produced a well-circumscribed area of CNV. With STC-1 treatment, CNV lesions assessed by FAF were increased by 50% in both intensity and area. The CNV lesions were also increased with SD-OCT, flat-mount, and histologic analyses. FFA disclosed enhanced fluorescein leakage in CNV lesions in STC-1 treated eyes. The STC-1 protein was detected in the choroidal tissue and its level was increased with CNV lesions in correlation with VEGF and VEGFR2 expressions. Intravitreal administration of STC-1 significantly increased choroidal expression of both VEGF and VEGFR2 proteins.Chorodial tissue expresses STC-1, which seemingly acts as a stress response protein by enhancing pathological new blood vessel growth in laser-induced CNV. It is likely that STC-1 promotes CNV development via VEGF signaling.
Project description:Choroidal neovascularization (CNV) is a major cause of severe visual loss in patients with age-related macular degeneration (AMD). Recently, itraconazole has shown potent and dose-dependent inhibition of tumor-associated angiogenesis. We evaluated the anti-angiogenic effect of itraconazole in a rat model of laser-induced CNV. After laser photocoagulation in each eye to cause CNV, right eyes were administered intravitreal injections of itraconazole; left eyes received balanced salt solution (BSS) as controls. On day 14 after laser induction, fluorescein angiography (FA) was used to assess abnormal vascular leakage. Flattened retinal pigment epithelium (RPE)-choroid tissue complex was stained with Alexa Fluor 594-conjugated isolectin B4 to measure the CNV area and volume. Vascular endothelial growth factor receptor 2 (VEGFR2) mRNA and protein expression was determined 1, 4, 7, and 14 days after intravitreal injection by quantitative RT-PCR or Western blot. VEGF levels were analyzed by enzyme-linked immunosorbent assay (ELISA). Intravitreal itraconazole significantly reduced leakage from CNV as assessed by FA and CNV area and volume on flat mounts compared with intravitreal BSS (p = 0.002 for CNV leakage, p<0.001 for CNV area and volume). Quantitative RT-PCR showed significantly lower expression of VEGFR2 mRNA in the RPE-choroid complexes of itraconazole-injected eyes than those of BSS-injected eyes on days 7 and 14 (p = 0.003 and p = 0.006). Western blots indicated that VEGFR2 was downregulated after itraconazole treatment. ELISA showed a significant difference in VEGF level between itraconazole-injected and BSS-injected eyes on days 7 and 14 (p = 0.04 and p = 0.001). Our study demonstrated that intravitreal itraconazole significantly inhibited the development of laser-induced CNV in rats. Itraconazole had anti-angiogenic activity along with the reduction of VEGFR2 and VEGF levels. Itraconazole may prove beneficial for treating CNV as an alternative or adjunct to other therapies.
Project description:PURPOSE: Endostatin plays an important role in inhibiting corneal neovascularization (CNV). The aim of this study was to evaluate the antiangiogenic activities of lipid-mediated subconjunctival injection of the modified RGDRGD (arginine- glycin- aspartic- arginine- glycin- aspartic- endostatin gene in a rabbit model of neovascularization in vivo. METHODS: A modified human endostatin gene containing an RGDRGD motif was obtained by rapid site-directed mutagenesis. Forty New Zealand white rabbits underwent alkaline burn and developed CNV, which were randomly divided into four groups: an experimental control group, a PCI empty vector group, a PCI-endostatin group, and a PCI-RGDRGD-endostatin group. The vector, endostatin, and RGDRGD-endostatin groups received injections into the superior bulbar conjunctiva after the burn. An injection of 5 ?g was given twice at 1-week intervals. Four eyes of two rabbits received neither treatment nor alkaline burn and served as absolute normal controls. The areas of CNV were monitored after 7 and 14 days. Corneas were examined by histology, and VEGF (vascular endothelial growth factor) and CD31 (platelet endothelial cell adhesion molecule-1) expression was detected by immunohistochemistry after 7 and 14 days. Retina, liver, and kidney were examined by histology, and CD38 expression in the inflammatory cells was detected by immunohistochemistry at 90 days. RESULTS: Subconjunctival injection of both native endostatin and modified RGDRGD-endostatin genes resulted in a significant suppression of CNV in vivo, with modified RGDRGD-endostatin being more effective than native endostatin. The mean concentration of VEGF in the PCI-RGDRGD-endostatin group significantly decreased compared to the means in the other groups. Upon histological examination, the endostatin-treated and RGDRGD-endostatin-treated eyes showed significantly less neovascular area and fewer vessels than the control and vector-injected groups. Retinal, hepatic, and renal tissue sections were normal, and there was no inflammatory cell infiltration observed. CONCLUSIONS: Native and modified endostatin can significantly inhibit CNV by suppressing the expression of VEGF. However, modified endostatin with the RGDRGD motif is far more effective than the endostatin gene in antiangiogenic activity.
Project description:Netrins are a family of matrix-binding proteins that function as guidance signals. Netrin-4 displays pathologic roles in tumorigenesis and neovascularization. To answer the question whether netrin-4 acts either pro- or anti-angiogenic, angiogenesis in the retina was assessed in Ntn-4(-/-) mice with oxygen-induced retinopathy (OIR) and laser-induced choroidal neovascularization (CNV), mimicking hypoxia-mediated neovascularization and inflammatory mediated angiogenesis. The basement membrane protein netrin-4 was found to be localised to mature retinal blood vessels. Netrin-4, but not netrin-1 mRNA expression, increased in response to relative hypoxia and recovered to normal levels at the end of blood vessel formation. No changes in the retina were found in normoxic Ntn-4(-/-) mice. In OIR, Ntn-4(-/-) mice initially displayed larger avascular areas which recovered faster to revascularization. Ganzfeld electroretinography showed faster recovery of retinal function in Ntn-4(-/-) mice. Expression of netrin receptors, Unc5H2 (Unc-5 homolog B, C. elegans) and DCC (deleted in colorectal carcinoma), was found in Müller cells and astrocytes. Laser-induced neovascularization in Nnt-4(-/-) mice did not differ to that in the controls. Our results indicate a role for netrin-4 as an angiogenesis modulating factor in O2-dependent vascular homeostasis while being less important during normal retinal developmental angiogenesis or during inflammatory neovascularization.
Project description:Choroidal neovascularization (CNV) is the main pathogenesis of age-related macular degeneration (AMD), which leads to severe vision loss in many aged patients in most advanced country. CNV compromises vision via hemorrhage and retinal detachment on account of pathological neovascularization penetrating the retina. Plasma medicine represents the medical application of ionized gas "plasma" that is typically studied in the field of physical science. Here we examined the therapeutic ability of plasma-activated medium (PAM) to suppress CNV. The effect of PAM on vascularization was assessed on the basis of human retinal endothelial cell (HREC) tube formation. In mice, laser photocoagulation was performed to induce CNV (laser-CNV), followed by intravitreal injection of PAM. N-Acetylcysteine was used to examine the role of reactive oxygen species in PAM-induced CNV suppression. Fundus imaging, retinal histology examination, and electroretinography (ERG) were also performed to evaluate PAM-induced retinal toxicity. Interestingly, HREC tube formation and laser-CNV were both reduced by treatment with PAM. N-acetylcysteine only partly neutralized the PAM-induced reduction in laser-CNV. In addition, PAM injection had no effect on regular retinal vessels, nor did it show retinal toxicity in vivo. Our findings indicate the potential of PAM as a novel therapeutic agent for suppressing CNV.