The diverse angiogenic effect of IL6-family cytokines on vascular endothelial cells and the modulatory influence of STAT3 signaling

Abstract

Purpose : The role of IL6-family cytokines, potent activators of the STAT3 pathway, on angiogenesis and in vascular retinal disease is controversial. Despite STAT3 as a shared signaling pathway, some IL-6 family members like oncostatin M (OSM) seem to be proangiogenic, others like ciliary neurotrophic factor (CNTF) appear to inhibit retinal angiogenesis.
This study tries to explain those differences and the role of STAT3 in retinal angiogenesis by comparing the effect of cytokine-induced activation and knock-down in vascular endothelial cells.

Methods : Angiogenic responses of human umbilical vein cells in response to VEGF, OSM or CNTF treatment were assessed by spheroid sprouting- and migration assays. Cytokine-dependent signaling was evaluated by western blot and metabolic alterations by extracellular flux. A STAT3 knock-down was established using siRNA. Transcriptomic shifts were assessed by RNA sequencing.

Results : OSM elicits proangiogenic responses on top of VEGF-induced cell sprouting (Δ = +31%, p<0.05) and migration (Δ = +29.2%, p<0.05) whereas CNTF has antiangiogenic effects on sprouting (Δ = -21.9%, p<0.05) and a small influence on migration (Δ = +6.1%, p<0.05). Both cytokines activate STAT3 while OSM also signals through ERK and AKT. Furthermore, OSM phosphorylates STAT3 at serine 727 and shifts cells to an active metabolic state by increasing mitochondrial performance. RNA-Seq revealed a transcriptomic shift favoring migration in response to OSM but not CNTF treatment. STAT3 knock-down reduced VEGF-induced cell sprouting (Δ = -39%, p<0.05) by affecting the cytoskeleton assembly. Interestingly, STAT3 knock-down changed CNTF’s antiangiogenic effect to a proangiogenic response (Δ = +77.8%, p<0.05) while enhancing OSM’s proangiogenic drive (Δ = +369.9%, p<0.05). No other pathways showed compensatory upregulation on protein level. RNA-Seq analysis determined a clear shift to a state of proliferation and migration in OSM-stimulated knock-down cells.

Conclusions : The divergent angiogenic properties of OSM and CNTF can be explained by individual differences in signaling, influence on metabolism and transcriptomic shifts. The shared STAT3 pathway seems to be essential to keep proangiogenic responses in check. In conclusion, this study sheds light on IL6-family endothelial cell interaction and reveals a novel perspective on the role of STAT3 in angiogenesis.

This is a 2021 ARVO Annual Meeting abstract.