Oncostatin M Reduces Pathological Neovascularization in the Retina Through Müller Cell Activation.

Purpose: Continuous vision loss due to vasoproliferative eye disease still represents an unsolved issue despite anti-vascular endothelial growth factor (VEGF) therapy. The impact of signal transducer and activator of transcription 3 (STAT3) signaling on retinal angiogenesis and its potential use as a therapeutic target remain controversial. In vitro, oncostatin M (OSM), as a strong STAT3 activator, possesses robust proangiogenic activity. This study investigated to what extent the proangiogenic effects of OSM translate to the in vivo setting of vasoproliferative eye disease. Methods: The in vitro effect of OSM on endothelial cells was investigated in the spheroid sprouting assay and through RNA sequencing. The mouse model for oxygen-induced retinopathy (OIR) was used to evaluate the impact of OSM in vivo. Signaling patterns were measured by western blot and retinal cryosections. Primary Müller cell cultures were used to evaluate the effect of OSM on the Müller cell secretome. Murine retinal vascular endothelial cells were isolated from OIR retinas using fluorescence-activated cell sorting (FACS) and were used for RNA sequencing. Results: Although OSM induced pro-angiogenic responses in vitro, in the OIR model intravitreal injection of OSM reduced retinal neovascularization by 65.2% and vaso-obliteration by 45.5% in Müller cells. Injecting OSM into the vitreous activated the STAT3 signaling pathway in multiple retinal cell types, including Müller cells. In vitro, OSM treatment increased CXCL10 secretion. RNA sequencing of sorted vascular endothelial cells at OIR P17 following OSM treatment indicated downregulation of angiogenesis- and mitosis-associated genes. Conclusions: In vivo, OSM reveals a beneficial angiomodulatory effect by activating Müller cells and changing their secretome. The data highlight contradictions between cytokine-induced effects in vitro and in vivo depending on the cell types mediating the effect.

2D and 3D in vitro angiogenesis assays highlight different aspects of angiogenesis.

In angiogenesis research, scientists need to carefully select appropriate in vitro models to test their hypotheses to minimize the risk for false negative or false positive study results. In this study, we investigate molecular differences between simple two-dimensional and more complex three-dimensional angiogenesis assays and compare them to in vivo data from cancer-associated angiogenesis using an unbiased transcriptomic analysis. Human umbilical vein endothelial cells were treated with VEGF in 2D wound healing and proliferation assays and the 3D spheroid sprouting assay. VEGF-induced transcriptomic shifts were assessed in both settings by bulk RNA sequencing. Immunocytochemistry was used for protein detection. The data was linked to the transcriptomic profile of vascular endothelial cells from a single cell RNA sequencing dataset of various cancer tissue compared to adjacent healthy tissue control. VEGF induced a more diverse transcriptomic shift in vascular endothelial cells in a 3D experimental setting (767 differentially expressed genes) compared to the 2D settings (167 differentially expressed genes). Particularly, VEGF-induced changes in cell-matrix interaction, tip cell formation, and glycolysis were pronounced in the 3D spheroid sprouting experiments. Immunocytochemistry for VCAM1 and CD34 confirmed enhanced expression in response to VEGF-treatment in 3D settings. In vivo, vascular endothelial cells within various cancer tissue were characterized by strong transcriptomic changes in cell-matrix interaction and glycolysis similar to the 3D setting. Consequently, 3D assays may better address certain key aspects of angiogenesis in comparison to fast and scalable 2D assays. This should be taken into consideration within the context of each research question.