A collagen IV-derived peptide disrupts α5ß1 integrin and potentiates Ang2/Tie2 signaling.

The angiopoietin (Ang)/Tie2 signaling pathway is essential for maintaining vascular homeostasis, and its dysregulation is associated with several diseases. Interactions between Tie2 and α5ß1 integrin have emerged as part of this control; however, the mechanism is incompletely understood. AXT107, a collagen IV-derived peptide, has strong antipermeability activity and has enabled the elucidation of this previously undetermined mechanism. Previously, AXT107 was shown to inhibit VEGFR2 and other growth factor signaling via receptor tyrosine kinase association with specific integrins. AXT107 disrupts α5ß1 and stimulates the relocation of Tie2 and α5 to cell junctions. In the presence of Ang2 and AXT107, junctional Tie2 is activated, downstream survival signals are upregulated, F-actin is rearranged to strengthen junctions, and, as a result, endothelial junctional permeability is reduced. These data suggest that α5ß1 sequesters Tie2 in nonjunctional locations in endothelial cell membranes and that AXT107-induced disruption of α5ß1 promotes clustering of Tie2 at junctions and converts Ang2 into a strong agonist, similar to responses observed when Ang1 levels greatly exceed those of Ang2. The potentiation of Tie2 activation by Ang2 even extended to mouse models in which AXT107 induced Tie2 phosphorylation in a model of hypoxia and inhibited vascular leakage in an Ang2-overexpression transgenic model and an LPS-induced inflammation model. Because Ang2 levels are very high in ischemic diseases, such as diabetic macular edema, neovascular age-related macular degeneration, uveitis, and cancer, targeting α5ß1 with AXT107 provides a potentially more effective approach to treat these diseases.

AAV8-vectored suprachoroidal gene transfer produces widespread ocular transgene expression.

There has been great progress in ocular gene therapy, but delivery of viral vectors to the retinal pigmented epithelium (RPE) and retina can be challenging. Subretinal injection, the preferred route of delivery for most applications, requires a surgical procedure that has risks. Herein we report a novel gene therapy delivery approach, suprachoroidal injection of AAV8 vectors, which is less invasive and could be done in an outpatient setting. Two weeks after suprachoroidal injection of AAV8.GFP in rats, GFP fluorescence covered 18.9% of RPE flat mounts and extended entirely around sagittal and transverse sections in RPE and photoreceptors. After 2 suprachoroidal injections of AAV8.GFP, GFP fluorescence covered 30.5% of RPE flat mounts. Similarly, widespread expression of GFP occurred in nonhuman primate and pig eyes after suprachoroidal injection of AAV8.GFP. Compared with subretinal injection in rats of RGX-314, an AAV8 vector expressing an anti-VEGF Fab, suprachoroidal injection of the same dose of RGX-314 resulted in similar expression of anti-VEGF Fab and similar suppression of VEGF-induced vascular leakage. Suprachoroidal AAV8 vector injection provides a noninvasive outpatient procedure to obtain widespread transgene expression in retina and RPE.

Tyrosine kinase blocking collagen IV-derived peptide suppresses ocular neovascularization and vascular leakage.

Vascular endothelial growth factor (VEGF)-neutralizing proteins provide benefit in several retinal and choroidal vascular diseases, but some patients still experience suboptimal outcomes, and the need for frequent intraocular injections is a barrier to good outcomes. A mimetic peptide derived from collagen IV, AXT107, suppressed subretinal neovascularization (NV) in two mouse models predictive of effects in neovascular age-related macular degeneration (NVAMD) and inhibited retinal NV in a model predictive of effects in ischemic retinopathies. A combination of AXT107 and the current treatment aflibercept suppressed subretinal NV better than either agent alone. Furthermore, AXT107 caused regression of choroidal NV. AXT107 reduced the VEGF-induced vascular leakage that underlies macular edema in ischemic retinopathies and NVAMD. In rabbit eyes, which are closer to the size of human eyes, intraocular injection of AXT107 significantly reduced VEGF-induced vascular leakage by 86% at 1 month and 70% at 2 months; aflibercept significantly reduced leakage by 69% at 1 month and did not reduce leakage at 2 months, demonstrating the longer effectiveness of AXT107. AXT107 reduced ligand-induced phosphorylation of multiple receptors: VEGFR2, c-Met, and PDGFRß. Optimal signaling through these receptors requires complex formation with ß3 integrin, which was reduced by AXT107 binding to αvß3 AXT107 also reduced total VEGFR2 levels by increasing internalization, ubiquitination, and degradation. This biomimetic peptide is a sustained, multitargeted therapy that may provide advantages over intraocular injections of specific VEGF-neutralizing proteins.

Periocular gene transfer of pigment epithelium-derived factor inhibits choroidal neovascularization in a human-sized eye.

Gene transfer provides a potential way to achieve sustained delivery of therapeutic proteins to the eye. Studies in rodents have suggested that periocular injection of adenoviral vectors containing expression cassettes for antiangiogenic proteins results in high intraocular levels of the proteins and suppression of choroidal neovascularization (CNV). However, the differences in size and scleral thickness between mouse and human eyes make it difficult to ascertain if periocular gene transfer is a feasible approach for treating human choroidal diseases. To address this issue, we tested the effect of periocular injection of an expression cassette for pigment epithelium-derived factor (PEDF) packaged in adenoviral vector (AdPEDF.11) in a CNV model in pigs, which have eyes that are very similar to humans in size and scleral thickness. Periocular injection of beta-galactosidase (AdLacZ.11) resulted in prominent transduction of periocular tissues, as was seen in mice. Periocular injection of AdPEDF.11 caused increased levels of PEDF in the choroid and significantly reduced the amount of CNV at rupture sites in Bruch's membrane. These data suggest that periocular gene transfer may be feasible for treatment of human choroidal diseases.

Intraocular expression of endostatin reduces VEGF-induced retinal vascular permeability, neovascularization, and retinal detachment.

Endostatin, a proteolytic fragment of collagen XVIII, is an endogenous inhibitor of tumor angiogenesis that also inhibits choroidal neovascularization. In this study, we assessed the effects of increased intraocular expression of endostatin on vascular endothelial growth factor (VEGF)-induced changes in the retina. After subretinal injection of a pair of gutless adenoviral vectors (AGV) designed to provide tamoxifen-inducible expression of endostatin, diffuse endostatin immunoreactivity was induced thoroughout the retina by administration of tamoxifen. Induction of endostatin in double transgenic mice with doxycycline-induced expression of VEGF in the retina resulted in significant suppression of leakage of intravascular [3H]mannitol into the retina. The ability of endostatin to reduce VEGF-induced retinal vascular permeability was confirmed by using [3H]mannitol leakage and two other parameters, fluorescein leakage and retinal thickness, after subretinal injection of a bovine immunodeficiency lentiviral vector coding for endostatin (BIV-vectored endostatin, or BIVendostatin). Subretinal injection of BIVendostatin resulted in more discrete, less intense staining for endostatin in the retina than that seen with the inducible AGV system, which suggested lower levels and allowed visualization of sites where endostatin was concentrated. Endostatin staining outlined retinal blood vessels, which suggested endostatin binding to a component of vessel walls. More prolonged or higher level expression of VEGF in the retina resulted in neovascularization and retinal detachment, both of which were also significantly reduced by BIVendostatin. These data suggest that endostatin may be an endogenous inhibitor of vasopermeability as well as neovascularization. In patients with diabetic retinopathy, endostatin gene transfer may provide a way to decrease the risk of three causes of visual loss: macular edema, neovascularization, and retinal detachment.

Periocular injection of microspheres containing PKC412 inhibits choroidal neovascularization in a porcine model.

PURPOSE: Oral administration of PKC412, a kinase inhibitor that blocks several isoforms of protein kinase C (PKC) and receptors for vascular endothelial growth factor (VEGF), platelet-derived growth factor, and stem cell factor, inhibits ocular neovascularization in a murine model. The purpose of this study was to determine whether sustained local delivery of PKC412 in a human-sized eye inhibits choroidal neovascularization (CNV). METHODS: Laser photocoagulation was used to rupture Bruch's membrane in young domestic pigs, and then a periocular injection of control microspheres or microspheres containing 25% or 50% PKC412 was given. After 10 days the integrated area of CNV at Bruch's membrane rupture sites was measured by image analysis. The levels of PKC412 in choroid, retina, and vitreous were measured either 10 or 20 days after periocular injection of 50% PKC microspheres or at 20 days after injection of 25% PKC412 microspheres. RESULTS: The areas of CNV at Bruch's membrane rupture sites were significantly smaller in eyes that received a periocular injection of microspheres containing 25% (P=0.0042) or 50% (P=0.0012) PKC412 than those in eyes injected with control microspheres. Ten days after periocular injection of 50% PKC412 microspheres, PKC412 was detected in the choroid, but not in the retina or vitreous. Twenty days after periocular injection of 50% PKC412, high levels of PKC412 were measured in the choroid, vitreous, and retina. Levels were lower but still substantial in all three compartments 20 days after periocular injection of 25% microspheres. CONCLUSIONS: Sustained local delivery of PKC412 provides a promising approach for treatment of CNV.

Intraocular injection of an aptamer that binds PDGF-B: a potential treatment for proliferative retinopathies.

Platelet-derived growth factor-B (PDGF-B) has been implicated in the pathogenesis of proliferative retinopathies and other scarring disorders in the eye. In this study, we sought to test the therapeutic potential of an aptamer that selectively binds PDGF-B, ARC126, and its PEGylated derivative, ARC127. Both ARC126 and ARC127 blocked PDGF-B-induced proliferation of cultured fibroblasts with an IC50 of 4 nM. Pharmacokinetic studies in rabbits showed similar peak vitreous concentrations of approximately 110 microM after intravitreous injection of 1 mg of either ARC126 or ARC127, but the terminal half-life was longer for ARC127 (98 versus 43 h). Efficacy was tested in rho/PDGF-B transgenic mice that express PDGF-B in photoreceptors and develop severe proliferative retinopathy resulting in retinal detachment. Compared to eyes injected with 20 microg of scrambled aptamer in which five of six developed detachments (three total and two partial), eyes injected with ARC126 (no detachment in five of six and one partial detachment), or ARC127 (no detachment in six of six) had significantly fewer retinal detachments. They also showed a significant reduction in epiretinal membrane formation. These data demonstrate that a single intravitreous injection of an aptamer that specifically binds PDGF-B is able to significantly reduce epiretinal membrane formation and retinal detachment in rho/PDGF-B mice. These striking effects in an aggressive model of proliferative retinopathy suggest that ARC126 and ARC127 should be considered for treatment of diseases in which PDGF-B has been implicated, including ischemic retinopathies such as proliferative diabetic retinopathy, proliferative vitreoretinopathy (PVR), and choroidal neovascularization.

Vascular targeting of ocular neovascularization with a vascular endothelial growth factor121/gelonin chimeric protein.

Tumors provide an extremely abnormal microenvironment that stimulates neovascularization from surrounding vessels and causes altered gene expression within vascular cells. Up-regulation of vascular endothelial growth factor (VEGF) receptors has allowed selective destruction of tumor vessels by administration of a chimeric protein consisting of VEGF121 coupled to the toxin gelonin (VEGF/rGel). We sought to determine whether there is sufficient up-regulation of VEGF receptors in endothelial cells participating in ocular neovascularization to permit a similar strategy. After intravenous injection of 45 mg/kg VEGF/rGel, but not uncoupled recombinant gelonin (rGel), there was immunofluorescent staining for rGel within choroidal neovascularization in mice and regression of the neovascularization occurred, demonstrating successful vascular targeting via the systemic circulation. Intraocular injection of 5 ng of VEGF/rGel also caused significant regression of choroidal neovascularization and regression of retinal neovascularization in two models, transgenic mice with expression of VEGF in photoreceptors and mice with ischemic retinopathy, whereas injection of 5 ng of rGel had no effect. These data suggest that the strategy of vascular targeting can be applied to nonmalignant neovascular diseases and could serve as the basis of a new treatment to reduce established ocular neovascularization.

Intraocular gutless adenoviral-vectored VEGF stimulates anterior segment but not retinal neovascularization.

Vascular endothelial growth factor (VEGF) and insulin-like growth factor-1 (IGF-1) have been implicated as important stimulatory factors for retinal neovascularization. In this study, we used intraocular gene transfer with gutless adenoviral (AGV) vectors to determine the effect of increased intraocular expression of VEGF, IGF-1, or sphingosine kinase (SPK), which produces sphingosine-1-phosphate, another angiogenic factor. Retinal neovascularization did not occur from intravitreous AGV-vectored VEGF, IGF-1, SPK, or combined VEGF and IGF-1, except occasionally adjacent to the retinal penetration site from the injection. However, corneal and iris neovascularization occurred after 2 weeks in all eyes injected with AGV.VEGF, but not those injected with only AGV.IGF-1 or AGV.SPK. These data suggest that the superficial capillary bed of the retina is relatively insensitive to VEGF, IGF-1, or SPK in adult mice, except when combined with retinal trauma. However, AGV-vectored VEGF is sufficient to consistently cause severe corneal and iris neovascularization. This provides a model for anterior segment neovascularization, which unlike previous models is relatively inexpensive and is not plagued by spontaneous regression, and therefore, may be useful for identification of new treatments.