ABSTRACT
Neovascular age-related macular degeneration (AMD) is treated with anti-VEGF intravitreal injections, which can cause geographic atrophy, infection, and retinal fibrosis. To minimize these toxicities, we developed a nanoparticle delivery system for recombinant Flt23k intraceptor plasmid (RGD.Flt23k.NP) to suppress VEGF intracellularly within choroidal neovascular (CNV) lesions in a laser-induced CNV mouse model through intravenous administration. In the current study, we examined the efficacy and safety of RGD.Flt23k.NP in mice. The effect of various doses was determined using fluorescein angiography and optical coherence tomography to evaluate CNV leakage and volume. Efficacy was determined by the rate of inhibition of CNV volume at 2 weeks post-treatment. RGD.Flt23k.NP had peak efficacy at a dose range of 30-60 µg pFlt23k/mouse. Using the lower dose (30 µg pFlt23k/mouse), RGD.Flt23k.NP safety was determined both in single-dose groups and in repeat-dose (three times) groups by measuring body weight, organ weight, hemoglobin levels, complement C3 levels, and histological changes in vital organs. Neither toxicity nor inflammation from RGD.Flt23k.NP was detected. No side effect was detected on visual function. Thus, systemic RGD.Flt23k.NP may be an alternative to standard intravitreal anti-VEGF therapy for the treatment of neovascular AMD.
Subject(s)
Angiogenesis Inhibitors/administration & dosage , Choroidal Neovascularization/therapy , Drug Carriers , Macular Degeneration/therapy , Plasmids/metabolism , Vascular Endothelial Growth Factor A/antagonists & inhibitors , Angiogenesis Inhibitors/chemistry , Animals , Choroid/blood supply , Choroid/metabolism , Choroid/pathology , Choroidal Neovascularization/genetics , Choroidal Neovascularization/metabolism , Choroidal Neovascularization/pathology , Complement C3/metabolism , Disease Models, Animal , Dose-Response Relationship, Drug , Drug Evaluation, Preclinical , Female , Gene Expression Regulation , Hemoglobins/metabolism , Humans , Injections, Intravenous , Intravitreal Injections , Lasers , Macular Degeneration/genetics , Macular Degeneration/metabolism , Macular Degeneration/pathology , Male , Mice , Mice, Inbred C57BL , Nanoparticles/administration & dosage , Nanoparticles/chemistry , Plasmids/chemistry , Vascular Endothelial Growth Factor A/genetics , Vascular Endothelial Growth Factor A/metabolismABSTRACT
Long-term inhibition of extracellular vascular endothelial growth factor (VEGF) in the treatment of age-related macular degeneration (AMD) may induce retinal neuronal toxicity and risk other side effects. We developed a novel strategy which inhibits retinal pigment epithelium (RPE)-derived VEGF, sparing other highly sensitive retinal tissues. Flt23k, an intraceptor inhibitor of VEGF, was able to inhibit VEGF in vitro. Adeno-associated virus type 2 (AAV2)-mediated expression of Flt23k was maintained for up to 6 months postsubretinal injection in mice. Flt23k was able to effectively inhibit laser-induced murine choroidal neovascularization (CNV). VEGF levels in the RPE/choroid complex decreased significantly in AAV2.Flt23k treated eyes. Neither retinal structure detected by Heidelberg Spectralis nor function measured by electroretinography (ERG) was adversely affected by treatment with AAV2.Flt23k. Hence AAV2.Flt23k can effectively maintain long-term expression and inhibit laser-induced CNV in mice through downregulation of VEGF while maintaining a sound retinal safety profile. These findings suggest a promising novel approach for the treatment of CNV.
Subject(s)
Choroidal Neovascularization/genetics , Dependovirus/genetics , Genetic Vectors/genetics , Protein Interaction Domains and Motifs/genetics , Recombinant Fusion Proteins , Transduction, Genetic , Vascular Endothelial Growth Factor Receptor-1/genetics , Animals , Apoptosis , Choroid/metabolism , Choroidal Neovascularization/pathology , Choroidal Neovascularization/therapy , Disease Models, Animal , Gene Expression , Genes, Reporter , Genetic Therapy , Genetic Vectors/administration & dosage , Humans , Mice , Retina/metabolism , Retinal Pigment Epithelium/metabolism , Vascular Endothelial Growth Factor A/antagonists & inhibitors , Vascular Endothelial Growth Factor A/genetics , Vascular Endothelial Growth Factor A/metabolism , Vascular Endothelial Growth Factor Receptor-1/chemistryABSTRACT
Corneal transparency is a prerequisite for optimal vision and in turn relies on an absence of blood and lymphatic vessels, which is remarkable given the cornea's proximity to vascularized tissues. Membrane-bound vascular endothelial growth factor receptor 3 (VEGFR-3), with its cognate ligand vascular endothelial growth factor C (VEGF-C), is a major mediator of lymphangiogenesis. Here, we demonstrate that the cornea expresses a novel truncated isoform of this molecule, soluble VEGFR-3 (sVEGFR-3), which is critical for corneal alymphaticity, by sequestering VEGF-C. sVEGFR-3 binds and sequesters VEGF-C, thereby blocking signaling through VEGFR-3 and suppressing lymphangiogenesis induced by VEGF-C. sVEGFR-3 knockdown leads to lymphangiogenesis and hemangiogenesis in the mouse cornea, while overexpression of sVEGFR-3 inhibits lymphangiogenesis and hemangiogenesis in a murine suture injury model. Pax6(+/-) mice spontaneously develop corneal and lymphatic vessels and are deficient in sVEGFR-3. sVEGFR-3 suppresses hemangiogenesis by blocking VEGF-C-induced phosphorylation of VEGFR-2. Overexpression of sVEGFR-3 leads to a 5-fold increase in corneal transplant survival in mouse models. sVEGFR-3 holds promise as a molecule to control and regress lymphatic-vessel-based dysfunction. Therefore, sVEGFR-3 has the potential to protect the injured cornea from opacification secondary to infection, inflammation, or transplant rejection.
