Edema stemming from leaky blood vessels is common in eye diseases such as age-related macular degeneration and diabetic retinopathy. Whereas therapies targeting vascular endothelial growth factor A (VEGFA) can suppress leakage, side-effects include vascular rarefaction and geographic atrophy. By challenging mouse models representing different steps in VEGFA/VEGF receptor 2 (VEGFR2)-induced vascular permeability, we show that targeting signaling downstream of VEGFR2 pY949 limits vascular permeability in retinopathy induced by high oxygen or by laser-wounding. Although suppressed permeability is accompanied by reduced pathological neoangiogenesis in oxygen-induced retinopathy, similarly sized lesions leak less in mutant mice, separating regulation of permeability from angiogenesis. Strikingly, vascular endothelial (VE)-cadherin phosphorylation at the Y685, but not Y658, residue is reduced when VEGFR2 pY949 signaling is impaired. These findings support a mechanism whereby VE-cadherin Y685 phosphorylation is selectively associated with excessive vascular leakage. Therapeutically, targeting VEGFR2-regulated VE-cadherin phosphorylation could suppress edema while leaving other VEGFR2-dependent functions intact.

Understanding the mechanisms that underlie age-related macular degeneration (AMD) has led to the identification of key molecules. Hypoxia-inducible transcription factors (HIFs) have been associated with choroidal neovascularization and the progression of AMD into the neovascular clinical phenotype (nAMD). HIFs regulate the expression of multiple growth factors and cytokines involved in angiogenesis and inflammation, hallmarks of nAMD. This knowledge has propelled the development of a new group of therapeutic strategies focused on gene therapy. The present review provides an update on current gene therapies in ocular angiogenesis, particularly nAMD, from both basic and clinical perspectives.

Human embryonic stem cell (hESC)-derived retinal pigment epithelial (RPE) cells could replace lost tissue in geographic atrophy (GA) but efficacy has yet to be demonstrated in a large-eyed model. Also, production of hESC-RPE has not yet been achieved in a xeno-free and defined manner, which is critical for clinical compliance and reduced immunogenicity. Here we describe an effective differentiation methodology using human laminin-521 matrix with xeno-free and defined medium. Differentiated cells exhibited characteristics of native RPE including morphology, pigmentation, marker expression, monolayer integrity, and polarization together with phagocytic activity. Furthermore, we established a large-eyed GA model that allowed in vivo imaging of hESC-RPE and host retina. Cells transplanted in suspension showed long-term integration and formed polarized monolayers exhibiting phagocytic and photoreceptor rescue capacity. We have developed a xeno-free and defined hESC-RPE differentiation method and present evidence of functional integration of clinically compliant hESC-RPE in a large-eyed disease model.

Human embryonic stem cell-derived retinal pigment epithelial (hESC-RPE) cells could serve as a replacement therapy in advanced stages of age-related macular degeneration. However, allogenic hESC-RPE transplants trigger immune rejection, supporting a strategy to evade their immune recognition. We established single-knockout beta-2 microglobulin (SKO-B2M), class II major histocompatibility complex transactivator (SKO-CIITA) and double-knockout (DKO) hESC lines that were further differentiated into corresponding hESC-RPE lines lacking either surface human leukocyte antigen class I (HLA-I) or HLA-II, or both. Activation of CD4+ and CD8+ T-cells was markedly lower by hESC-RPE DKO cells, while natural killer cell cytotoxic response was not increased. After transplantation of SKO-B2M, SKO-CIITA, or DKO hESC-RPEs in a preclinical rabbit model, donor cell rejection was reduced and delayed. In conclusion, we have developed cell lines that lack both HLA-I and -II antigens, which evoke reduced T-cell responses in vitro together with reduced rejection in a large-eyed animal model.

Purpose: Subretinal suspension transplants of human embryonic stem cell-derived retinal pigment epithelial cells (hESC-RPE) have the capacity to form functional monolayers in naive eyes. We explore hESC-RPE integration when transplanted in suspension to a large-eyed model of geographic atrophy (GA). Methods: Derivation of hESC-RPE was performed in a xeno-free and defined manner. Subretinal bleb injection of PBS or sodium iodate (NaIO₃) was used to induce a GA-like phenotype. Suspensions of hESC-RPE were transplanted to the subretinal space of naive or PBS-/NaIO₃-treated rabbits using a transvitreal pars plana technique. Integration of hESC-RPE was monitored by multimodal real-time imaging and by immunohistochemistry. Results: Subretinal blebs of PBS or NaIO₃ caused different degrees of outer neuroretinal degeneration, RPE hyperautofluorescence, focal RPE loss, and choroidal atrophy; that is, hallmark characteristics of GA. In nonpretreated naive eyes, hESC-RPE integrated as subretinal monolayers with preserved overlying photoreceptors, yet not in areas with outer neuroretinal degeneration and native RPE loss. When transplanted to eyes with PBS-/NaIO₃-induced degeneration, hESC-RPE failed to integrate. Conclusions: In a large-eyed preclinical model, subretinal suspension transplants of hESC-RPE did not integrate in areas with GA-like degeneration.