tative of at least 3 independent experiments. Statistical differences between groups were determined using 2-tailed unpaired Student's t test, Mann-Whitney U test, or 1-way ANOVA with post hoc Tukey's honestly significant difference test. Correlation was tested using Pearson's method. Statistical analysis was performed using Microsoft Office and Prism 6.0 software (Graph-Pad).

Conflict of interest: AS and GLS are cofounders of and hold equity in HIF Therapeutics Inc. This arrangement has been reviewed and approved by Johns Hopkins University in accordance with its conflict-of-interest policies.

Age-related macular degeneration (AMD), Neovascular AMD (nvAMD), Nonneovascular AMD (nnvAMD), Vascular endothelial growth factor (VEGF), anti-VEGF therapy, choroidal neovascularization, treat-and-extend, pro re nata, proteomics, complement, complement factor 3 (C3), complement factor H (CFH), complement factor H-related (CHFR) proteins, apolipoprotein-B100 (ApoB100), drusen Cao et al.,2

Most patients with neovascular age-related macular degeneration (nvAMD), the leading cause of severe vision loss in elderly US citizens, respond inadequately to current therapies targeting a single angiogenic mediator, vascular endothelial growth factor (VEGF). Here, we report that aqueous fluid levels of a second vasoactive mediator, angiopoietin-like 4 (ANGPTL4), can help predict the response of patients with nvAMD to anti-VEGF therapies. ANGPTL4 expression was higher in patients who required monthly treatment with anti-VEGF therapies compared with patients who could be effectively treated with less-frequent injections. We further demonstrate that ANGPTL4 acts synergistically with VEGF to promote the growth and leakage of choroidal neovascular (CNV) lesions in mice. Targeting ANGPTL4 expression was as effective as targeting VEGF expression for treating CNV in mice, while simultaneously targeting both was more effective than targeting either factor alone. To help translate these findings to patients, we used a soluble receptor that binds to both VEGF and ANGPTL4 and effectively inhibited the development of CNV lesions in mice. Our findings provide an assay that can help predict the response of patients with nvAMD to anti-VEGF monotherapy and suggest that therapies targeting both ANGPTL4 and VEGF will be a more effective approach for the treatment of this blinding disease.

Statistical analysis was performed with Microsoft Excel, version 16.0.16327.20248, or Prism, version 8.0, software (GraphPad). Statistical differences between 2 or multiple heterogenous groups were determined by unpaired Student's t test or 1-way or 2-way ANOVA. Analysis of data was performed using Excel.Study approval. All animal experiments were performed in accordance with the Animal Care and Use Program at Johns Hopkins University and were approved by the Institutional Animal Care and Use Committee (IACUC). All studies involving patients or patient tissue were approved by the Institutional Review Board of Johns Hopkins University School of Medicine. All participants provided written informed consent prior to enrolment in the study.