Search

Diabetic retinopathy (DR) is a leading cause of vision loss in working-age adults. Despite an established standard of care for advanced forms of DR, some patients continue to lose vision after treatment. This may be due to the development of diabetic macular ischemia (DMI), which has no approved treatment. Neuropilin-1 (Nrp-1) is a coreceptor with two ligand-binding domains, with semaphorin-3A (Sema3A) binding to the A-domain and vascular endothelial growth factor-A (VEGF-A) binding to the B-domain. Sema3A directs a subset of neuronal growth cones as well as blood vessel growth by repulsion; when bound to Nrp-1, VEGF-A mediates vascular permeability and angiogenesis. Modulating Nrp-1 could therefore address multiple complications arising from DR, such as diabetic macular edema (DME) and DMI. BI-Y is a monoclonal antibody that binds to the Nrp-1 A-domain, antagonizing the effects of the ligand Sema3A and inhibiting VEGF-A-induced vascular permeability. This series of in vitro and in vivo studies examined the binding kinetics of BI-Y to Nrp-1 with and without VEGF-A 165 , the effect of BI-Y on Sema3A-induced cytoskeletal collapse, the effect of BI-Y on VEGF- A 165 -induced angiogenesis, neovascularization, cell integrity loss and permeability, and retinal revascularization. The data show that BI-Y binds to Nrp-1 and inhibits Sema3A-induced cytoskeletal collapse in vitro, may enhance revascularization of ischemic areas in an oxygen-induced retinopathy mouse model, and prevents VEGF-A-induced retinal hyperpermeability in rats. However, BI-Y does not interfere with VEGF-A-dependent choroidal neovascularization. These results support further investigation of BI-Y as a potential treatment for DMI and DME. SIGNIFICANCE STATEMENT: Diabetic macular ischemia (DMI) is a complication of diabetic retinopathy (DR) with no approved pharmacological treatment. Diabetic macular edema (DME) commonly co-occurs with DMI in patients with DR. This series of preclinical studies in mouse and rat models shows that neuropilin-1 antagonist BI-Y may enhance the revascularization of ischemic areas and prevents vascular endothelial growth factor-A (VEGF-A)-induced retinal hyperpermeability without affecting VEGF-A-dependent choroidal neovascularization; thus, BI-Y may be of interest as a potential treatment for patients with DR., Competing Interests: The authors have declared a conflict of interest. All authors are employees of Boehringer Ingelheim., (Copyright © 2023 by The Author(s).)

Retinopathies are multifactorial diseases with complex pathologies that eventually lead to vision loss. Animal models facilitate the understanding of the pathophysiology and identification of novel treatment options. However, each animal model reflects only specific disease aspects and understanding of the specific molecular changes in most disease models is limited. Here, we conducted transcriptome analysis of murine ocular tissue transduced with recombinant Adeno-associated viruses (AAVs) expressing either human VEGF-A, TNF-α, or IL-6. VEGF expression led to a distinct regulation of extracellular matrix (ECM)-associated genes. In contrast, both TNF-α and IL-6 led to more comparable gene expression changes in interleukin signaling, and the complement cascade, with TNF-α-induced changes being more pronounced. Furthermore, integration of single cell RNA-Sequencing data suggested an increase of endothelial cell-specific marker genes by VEGF, while TNF-α expression increased the expression T-cell markers. Both TNF-α and IL-6 expression led to an increase in macrophage markers. Finally, transcriptomic changes in AAV-VEGF treated mice largely overlapped with gene expression changes observed in the oxygen-induced retinopathy model, especially regarding ECM components and endothelial cell-specific gene expression. Altogether, our study represents a valuable investigation of gene expression changes induced by VEGF, TNF-α, and IL-6 and will aid researchers in selecting appropriate animal models for retinopathies based on their agreement with the human pathophysiology., (© 2022. The Author(s).)

The therapeutic potential of pluripotent stem cells is great as they promise to usher in a new era of medicine where cells or organs may be prescribed to replace dysfunctional tissue. At the forefront are efforts in the eye to develop this technology as it lends itself to in vivo monitoring and sophisticated non-invasive imaging modalities. In the retina, retinal pigment epithelium (RPE) is the most promising replacement cell as it has a single layer, is relatively simple to transplant, and is associated with several eye diseases. However, after transplantation, the cells may transform and cause complications. This transformation may be partially due to incomplete maturation. With the goal of learning how to mature RPE, we compared induced pluripotent stem cell-derived RPE (iPSC-RPE) cells with adult human primary RPE (ahRPE) cells and the immortalized human ARPE-19 line. We cultured ARPE-19, iPSC-RPE, and ahRPE cells for one month, and evaluated morphology, RPE marker staining, and transepithelial electrical resistance (TEER) as quality control indicators. We then isolated RNA for bulk RNA-sequencing and DNA for genotyping. We genotyped ahRPE lines for the top age-related macular degeneration (AMD) and proliferative vitreoretinopathy (PVR) risk allele polymorphisms. Transcriptome data verified that both adult and iPSC-RPE exhibit similar RPE gene expression signatures, significantly higher than ARPE-19. In addition, in iPSC-RPE, genes relating to stem cell maintenance, retina development, and muscle contraction were significantly upregulated compared to ahRPE. We compared ahRPE to iPSC-RPE in a model of epithelial-mesenchymal transition (EMT) and observed an increased sensitivity of iPSC-RPE to producing contractile aggregates in vitro which resembles incident reports upon transplantation. P38 inhibition was capable of inhibiting iPSC-RPE-derived aggregates. In summary, we find that the transcriptomic signature of iPSC-RPE conveys an immature RPE state which may be ameliorated by targeting "immature" gene regulatory networks., Competing Interests: EM, HK, CV, WR, BS, SK, HN, and RB as employees of Boehringer Ingelheim Pharma GmbH. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Markert, Klein, Viollet, Rust, Strobel, Kauschke, Makovoz, Neubauer, Bakker and Blenkinsop.)

Purpose: Semaphorin 3A (Sema3A) is a promising therapeutic target for macular edema in age-related macular degeneration, diabetic retinopathy, and retinal vein occlusion (RVO). Anti-vascular endothelial growth factors (anti-VEGFs) are the current standard of care for many retinal diseases. This study investigated the Sema3A neutralizing antibody BI-X and/or anti-VEGF therapy (aflibercept) in an RVO mouse model. Treatment efficacy was examined and grouped by timing subsequent to the RVO mouse model induction: efficacy against the onset of intraretinal edema 1 day postinduction and protective effects at 7 days postinduction., Methods: We examined the changes in expression of Sema3A in the retina of an RVO mouse model. In addition, changes in expression of tumor necrosis factor (TNF)-α and semaphorin-related proteins (neuropilin-1 and plexin A1) in the retina upon treatment were analyzed by Western blotting. The effects of BI-X and/or aflibercept were evaluated using measures of retinal edema, blood flow, and thinning of the inner nuclear layer., Results: Induction of vein occlusion in the RVO mouse model significantly increased Sema3A expression in the retina, particularly in the inner nuclear layer. BI-X was effective as a monotherapy and in combination with anti-VEGF therapy, demonstrating a beneficial effect on intraretinal edema and retinal blood flow. Moreover, in the RVO mouse model, BI-X monotherapy normalized the changes in expression of TNF-α and semaphorin-related proteins., Conclusions: These findings support targeting Sema3A to treat intraretinal edema and retinal ischemia.

Purpose: Semaphorin 3A (Sema3A) is an axonal guidance molecule that inhibits angiogenesis by vasorepulsion and blocks revascularization in the ischemic retina. BI-X is an intravitreal anti-Sema3A agent under clinical investigation in patients with proliferative diabetic retinopathy (PDR) and diabetic macular ischemia (DMI)., Methods: Surface plasmon resonance was used to determine binding affinity of BI-X to human and murine Sema3A. In vitro, human retinal microvascular endothelial cells (HRMECs) were used to assess effects of BI-X on cell permeability and cytoskeletal collapse induced by Sema3A. In vivo, intravitreal BI-X or an anti-trinitrophenol control antibody was administered in both eyes in mice with oxygen-induced retinopathy (OIR). Retinal flat mounts were prepared, and avascular area and tip cell density were determined using confocal laser-scanning microscopy., Results: Dissociation constants for BI-X binding to human and murine Sema3A were 29 pM and 27 pM, respectively. In vitro, BI-X prevented HRMEC permeability and cytoskeletal collapse induced by Sema3A. In vivo, BI-X increased tip cell density by 33% (P < 0.001) and reduced avascular area by 12% (not significant). A significant negative correlation was evident between avascular area and tip cell density (r2 = 0.4205, P < 0.0001)., Conclusions: BI-X binds to human Sema3A with picomolar affinity and prevents cell permeability and cytoskeletal collapse in HRMECs. BI-X also enhances revascularization in mice with OIR., Translational Relevance: BI-X is a potent inhibitor of human Sema3A that improves revascularization in a murine model of OIR; BI-X is currently being investigated in patients with laser-treated PDR and DMI.

Purpose: Inflammation is implicated in the etiology of diverse retinopathies including uveitis, age-related macular degeneration or diabetic retinopathy. Tumor necrosis factor alpha (TNF-α) is a well-known proinflammatory cytokine that is described as a biomarker for inflammation in diverse retinopathies and therefore emerged as an interesting target to treat inflammation in the eye by neutralizing anti-TNF-α antibodies., Methods: Recently, we have demonstrated that Adeno-associated virus (AAV)-mediated expression of human TNF-α in the murine eye induces retinal inflammation including vasculitis and fibrosis, thereby mimicking human disease-relevant pathologies. In a proof-of-mechanism study, we now tested whether AAV-TNF-α induced pathologies can be reversed by neutralizing TNF-α antibody treatment., Results: Strikingly, a single intravitreal injection of the TNF-α antibody golimumab reduced AAV-TNF-α-induced retinal inflammation and retinal thickening. Furthermore, AAV-TNF-α-mediated impaired retinal function was partially rescued by golimumab as revealed by electroretinography recordings. Finally, to study TNF-α-induced vasculitis in human in vitro cell culture assays, we established a monocyte-to-endothelium adhesion co-culture system. Indeed, also in vitro TNF-α induced monocyte adhesion to human retinal endothelial cells, which was prevented by golimumab., Conclusions: Overall, our study describes valuable in vitro and in vivo approaches to study the function of TNF-α in retinal inflammation and demonstrated a preclinical proof-of-mechanism treatment with golimumab., Translational Relevance: The AAV-based model expressing human TNF-α allows us to investigate TNF-α-driven pathologies supporting research in mechanisms of retinal inflammation.