Five vascular endothelial growth factor receptor (VEGFR) ligands (VEGF-A, -B, -C, -D, and placental growth factor [PlGF]) constitute the VEGF family. VEGF-A binds VEGF receptors 1 and 2 (VEGFR1/2), whereas VEGF-B and PlGF only bind VEGFR1. Although much research has been conducted on VEGFR2 to elucidate its key role in retinal diseases, recent efforts have shown the importance and involvement of VEGFR1 and its family of ligands in angiogenesis, vascular permeability, and microinflammatory cascades within the retina. Expression of VEGFR1 depends on the microenvironment, is differentially regulated under hypoxic and inflammatory conditions, and it has been detected in retinal and choroidal endothelial cells, pericytes, retinal and choroidal mononuclear phagocytes (including microglia), Müller cells, photoreceptor cells, and the retinal pigment epithelium. Whilst the VEGF-A decoy function of VEGFR1 is well established, consequences of its direct signaling are less clear. VEGFR1 activation can affect vascular permeability and induce macrophage and microglia production of proinflammatory and proangiogenic mediators. However the ability of the VEGFR1 ligands (VEGF-A, PlGF, and VEGF-B) to compete against each other for receptor binding and to heterodimerize complicates our understanding of the relative contribution of VEGFR1 signaling alone toward the pathologic processes seen in diabetic retinopathy, retinal vascular occlusions, retinopathy of prematurity, and age-related macular degeneration. Clinically, anti-VEGF drugs have proven transformational in these pathologies and their impact on modulation of VEGFR1 signaling is still an opportunity-rich field for further research.

Age-related macular degeneration (AMD) is the leading cause of blindness in developed countries. The polymorphism rs10490924 in the ARMS2 gene is highly associated with AMD and linked to an indel mutation (del443ins54), the latter inducing mRNA instability. At present, the function of the ARMS2 protein, the exact cellular sources in the retina and the biological consequences of the rs10490924 polymorphism are unclear. Recombinant ARMS2 was expressed in Pichia pastoris, and protein functions were studied regarding cell surface binding and complement activation in human serum using fluoresence-activated cell sorting (FACS) as well as laser scanning microscopy (LSM). Biolayer interferometry defined protein interactions. Furthermore, endogenous ARMS2 gene expression was studied in human blood derived monocytes and in human induced pluripotent stem cell-derived microglia (iPSdM) by PCR and LSM. The ARMS2 protein was localized in human genotyped retinal sections and in purified monocytes derived from AMD patients without the ARMS2 risk variant by LSM. ARMS2 expression in monocytes under oxidative stress was determined by Western blot analysis. Here, we demonstrate for the first time that ARMS2 functions as surface complement regulator. Recombinant ARMS2 binds to human apoptotic and necrotic cells and initiates complement activation by recruiting the complement activator properdin. ARMS2-properdin complexes augment C3b surface opsonization for phagocytosis. We also demonstrate for the first time expression of ARMS2 in human monocytes especially under oxidative stress and in microglia cells of the human retina. The ARMS2 protein is absent in monocytes and also in microglia cells, derived from patients homozygous for the ARMS2 AMD risk variant (rs10490924). ARMS2 is likely involved in complement-mediated clearance of cellular debris. As AMD patients present with accumulated proteins and lipids on Bruch’s membrane, ARMS2 protein deficiency due to the genetic risk variant might be involved in drusen formation.

Purpose: The Heavy Silicone Oil versus Standard Silicone Oil Study (HSO study) is designed to answer the question whether a heavier‐than‐water tamponade improves the prognosis of eyes with proliferative vitreoretinopathy (PVR) of the lower retina. Methods: The HSO Study is a multicentre, randomized, prospective, controlled clinical trial stratified by surgeon comparing two endotamponades within a two‐arm parallel‐group design. Patients with inferiorly and posteriorly located PVR grade C‐A6 were randomized to either HSO or standard silicone oil as a tamponading agent. The main end‐point criteria are complete retinal attachment at 12 months and change in visual acuity (VA) 12 months postoperatively compared to the preoperative VA. Results: Forty‐six patients treated with HSO were compared to 47 patients treated with standard silicone oil. There was no difference among the groups regarding baseline data. Three patients in the HSO and five patients in the standard silicone oil group fulfilled intraoperative exclusion criteria. There was no significant difference between both groups regarding anatomical success. Neither noninferiority nor superiority was shown with regard to final acuity. Conclusions: The HSO Study is the first randomized prospective clinical trial to compare heavy and standard silicone oil in patients with PVR of the lower retina. The intermediate results failed to demonstrate superiority of a heavy tamponade.

Background: Chimeric antigen receptor (CAR)-based T cell therapy is in early clinical trials to target the neuroectodermal tumor, neuroblastoma. No preclinical or clinical efficacy data are available for retinoblastoma to date. Whereas unilateral intraocular retinoblastoma is cured by enucleation of the eye, infiltration of the optic nerve indicates potential diffuse scattering and tumor spread leading to a major therapeutic challenge. CAR-T cell therapy could improve the currently limited therapeutic strategies for metastasized retinoblastoma by simultaneously killing both primary tumor and metastasizing malignant cells and by reducing chemotherapy-related late effects. Methods: CD171 and GD2 expression was flow cytometrically analyzed in 11 retinoblastoma cell lines. CD171 expression and T cell infiltration (CD3⁺) was immunohistochemically assessed in retrospectively collected primary retinoblastomas. The efficacy of CAR-T cells targeting the CD171 and GD2 tumor-associated antigens was preclinically tested against three antigen-expressing retinoblastoma cell lines. CAR-T cell activation and exhaustion were assessed by cytokine release assays and flow cytometric detection of cell surface markers, and killing ability was assessed in cytotoxic assays. CAR constructs harboring different extracellular spacer lengths (short/long) and intracellular co-stimulatory domains (CD28/4-1BB) were compared to select the most potent constructs. Results: All retinoblastoma cell lines investigated expressed CD171 and GD2. CD171 was expressed in 15/30 primary retinoblastomas. Retinoblastoma cell encounter strongly activated both CD171-specific and GD2-specific CAR-T cells. Targeting either CD171 or GD2 effectively killed all retinoblastoma cell lines examined. Similar activation and killing ability for either target was achieved by all CAR constructs irrespective of the length of the extracellular spacers and the co-stimulatory domain. Cell lines differentially lost tumor antigen expression upon CAR-T cell encounter, with CD171 being completely lost by all tested cell lines and GD2 further down-regulated in cell lines expressing low GD2 levels before CAR-T cell challenge. Alternating the CAR-T cell target in sequential challenges enhanced retinoblastoma cell killing. Conclusion: Both CD171 and GD2 are effective targets on human retinoblastoma cell lines, and CAR-T cell therapy is highly effective against retinoblastoma in vitro. Targeting of two different antigens by sequential CAR-T cell applications enhanced tumor cell killing and preempted tumor antigen loss in preclinical testing.

Purpose: To demonstrate superiority of intravitreal ranibizumab 0.5 mg compared to focal and peripheral laser treatment in patients with radiation retinopathy for choroidal melanoma. Methods: Inclusion criteria were as follows: patients with radiation retinopathy and visual acuity impairment due to radiation maculopathy accessible for laser therapy, age ≥ 18 years, and BCVA less than 20/32. The main objective was to study the change in best-corrected visual acuity (BCVA) over 6 months from ranibizumab 0.5 mg (experimental) compared to focal laser of the macula and panretinal laser treatment of the ischemic retina (control) in patients with radiation retinopathy in choroidal melanoma. The secondary objectives of the radiation retinopathy study were to compare functional and anatomical results between ranibizumab and laser group over 12 months and to measure the frequency of vitreous hemorrhage and rubeosis iridis. Results: The intention-to-treat analysis included 31 patients assigned to ranibizumab (n = 15) or laser treatment (n = 16). In terms of BCVA at month 6, ranibizumab was superior to laser treatment, with an advantage of 0.14 logMAR, 95% CI 0.01 to 0.25, p = 0.030. The positive effect of ranibizumab disappeared after treatment was discontinued. Similar results without statistically significant difference were found with respect to macular thickness. In both groups, no change was observed at month 6 in the size of ischemia in the macula or periphery compared to baseline. There was 1 case of vitreous hemorrhage in the laser group and no case of rubeosis iridis over time. Conclusions: This study showed a statistically significant improvement in visual acuity and clear superiority of ranibizumab compared to laser treatment up to 26 weeks, but this effect disappeared at week 52 after completion of intravitreal treatment. Ranibizumab and PRP are considered equivalent in terms of the non-appearance of proliferative radiation retinopathy during the study. Trial registration: EudraCT Number: 2011-004463-69

Purpose: The cellular immune response driven by mononuclear phagocytes (MPs) is crucial for choroidal neovascularization (CNV) progression. Case reports show that a switch from pure anti-vascular endothelial growth factor-A (VEGF-A) intravitreal treatment to aflibercept, a drug with combined anti-VEGF-A and anti-placenta growth factor (PlGF) activity, can be beneficial for patients who do not respond to anti-VEGF-A alone. Since MPs harbor VEGFR1, we hypothesize that the interplay of P1GF/vascular endothelial growth factor receptor 1 (VEGFR1) in immune cells plays a pivotal role for CNV. Methods: CNV was induced with laser, and immune cells and neovascularization were analyzed in vivo and ex vivo. Immunohistochemistry was employed for protein detection. Differential expression of angiogenic factors and macrophage polarization markers were assessed by quantitative PCR (qPCR). One day after laser, intravitreal injection of aflibercept or anti-PlGF was performed. Results: In the early inflammatory phase after laser, Plgf but not Vegfa was significantly upregulated. VEGF-A upregulation is limited to the scar, whereas PlGF shows a wider distribution. M1 (proinflammatory) macrophage markers were upregulated in the early phase of CNV. However, M2 (proangiogenic) markers showed more inconsistent dynamics. We demonstrated that both aflibercept and anti-PlGF treatments decrease the overall amount of activated subretinal MPs, and especially of those expressing PlGF. These data correlated with a reduction in leakage associated to CNV. Aflibercept showed a stronger reduction in both parameters. Conclusions: The results hint at an interplay between PlGF/VEGFR1 and MPs that is important in the early phase of CNV. A combined inhibition of VEGF-A and PlGF is superior to a specific anti-PlGF treatment in terms of subretinal MP recruitment.