Impaired pericyte recruitment and abnormal retinal angiogenesis as a result of angiopoietin-2 overexpression

Angiopoietin-2 (Ang2) is among the relevant growth factors induced by hypoxia and plays an important role in the initiation of retinal neovascularizations. Ang2 is also involved in incipient diabetic retinopathy, as it may cause pericyte loss. To investigate the impact of Ang2 on developmental and hypoxia-induced angiogenesis, we used a transgenic mouse line overexpressing human Ang2 in the mouse retina. Transgenic mice displayed a reduced coverage of capillaries with pericytes (-14%; p < 0.01) and a 46% increase of vascular density of the capillary network at postnatal day 10 compared to wild type mice. In the model of oxygen-induced retinopathy (OIR), Ang2 overexpression resulted in enhanced preretinal (+103%) and intraretinal neovascularization (+29%). Newly formed intraretinal vessels in OIR were also pericyte-deficient (-26%; p < 0.01). The total expression of Ang2 in transgenic mice was seven-fold, compared with wild type controls. Ang2 modulated expression of genes encoding VEGF (+65%) and Ang1 (+79%) in transgenic animals. These data suggest that Ang2 is involved in pericyte recruitment, and modulates intraretinal, and preretinal vessel formation in the eye under physiological and pathological conditions.

Nonpeptide somatostatin receptor agonists specifically target ocular neovascularization via the somatostatin type 2 receptor

PURPOSE: To define the molecular pharmacology underlying the antiangiogenic effects of nonpeptide imidazolidine-2,4-dione somatostatin receptor agonists (NISAs) and evaluate the efficacy of NISA in ocular versus systemic delivery routes in ocular disease models. METHODS: Functional inhibitory effects of the NISAs and the somatostatin peptide analogue octreotide were evaluated in vitro by chemotaxis, proliferation, and tube-formation assays. The oxygen-induced retinopathy (OIR) model and the laser model of choroidal neovascularization (CNV) were used to test the in vivo efficacy of NISAs. Transscleral permeability of a candidate NISA was also measured. RESULTS: NISAs inhibited growth factor-induced HREC proliferation, migration and tube formation with submicromolar potencies (IC(50), 0.1-1.0 microM) comparable to octreotide. In the OIR model, systemic administration of the NISAs RFE-007 and RFE-011 inhibited retinal neovascularization in a dose-dependent manner, comparable to octreotide. In the CNV model, intravitreal RFE-011 resulted in a 56% reduction (P < 0.01) in CNV lesion area, whereas systemic administration resulted in a 35% reduction (P < 0.05) in lesion area. RFE-011 demonstrated transscleral penetration. CONCLUSIONS: Micromolar concentrations of octreotide and NISAs are necessary for antiangiogenic effects, whereas nanomolar concentrations are effective for endocrine inhibition. This suggests that the antiangiogenic activity of NISAs and octreotide is mediated by an overall much less efficient downstream coupling mechanism than is growth hormone release. As a result, the intravitreal or transscleral route of administration should be seriously considered for future clinical studies of SSTR2 agonists used for treatment of ocular neovascularization to ensure efficacious concentrations in the target retinal and choroidal tissue.

Decreased hypoxia-induced neovascularization in angiopoietin-2 heterozygous knockout mouse through reduced MMP activity

BACKGROUND/AIMS: Proliferative diabetic retinopathy is characterized by the formation of retinal neovascularization. Angiopoietin-2 (Ang-2) and matrix metalloproteinase (MMP) play a critical role in angiogenesis. However, the precise location and function of Ang-2 during formation of retinal neovascularizations driven by hypoxia in relation to MMP activity have not been elucidated. In this study, we investigated the response of Ang-2 heterozygous knockout retinas (Ang2(+/-) mouse) to hypoxia and its link to MMP activity in an oxygen-induced retinopathy (OIR) model. METHODS: Pre-retinal neovascularizations were quantitated in vertical sections. Intra-retinal angiogenesis was assessed by whole mount immunofluorescence staining of retinas. MMP activity was examined in retinal protein lysate and whole mount retinal in situ zymography. RESULTS: Ang2(+/-) retinas subjected to the OIR model showed 33% reduced neovascularization and 271% increased avascular zones at postnatal day 17. In the OIR model, Ang-2 was modestly expressed in pre-retinal neovascularizations and venules, but strongly in arterioles and capillary sprouts. MMPs were activated in close association to where Ang-2 is expressed. MMP activity was substantially decreased in Ang2(+/-) retinas. CONCLUSIONS: Our present data suggest the spatially concomitant expression of Ang2 and MMPs, and that Ang2 modulates hypoxia-induced neovascularization by regulating MMP activity.