Genetic perturbation of IFN-α transcriptional modulators in human endothelial cells uncovers pivotal regulators of angiogenesis

Abstract

Interferon-α (IFN-α) comprises a family of 13 cytokines involved in the modulation of antiviral, immune, and anticancer responses by orchestrating a complex transcriptional network. The activation of IFN-α signaling pathway in endothelial cells results in decreased proliferation and migration, ultimately leading to suppression of angiogenesis. In this study, we knocked-down the expression of seven established or candidate modulators of IFN-α response in endothelial cells to reconstruct a gene regulatory network and to investigate the antiangiogenic activity of IFN-α. This genetic perturbation approach, along with the analysis of interferon-induced gene expression dynamics, highlighted a complex and highly interconnected network, in which the angiostatic chemokine C-X-C Motif Chemokine Ligand 10 (CXCL10) was a central node targeted by multiple modulators. IFN-α-induced secretion of CXCL10 protein by endothelial cells was blunted by the silencing of Signal Transducer and Activator of Transcription 1 (STAT1) and of Interferon Regulatory Factor 1 (IRF1) and it was exacerbated by the silencing of Ubiquitin Specific Peptidase 18 (USP18). In vitro sprouting assay, which mimics in vivo angiogenesis, confirmed STAT1 as a positive modulator and USP18 as a negative modulator of IFN-α-mediated sprouting suppression. Our data reveal an unprecedented physiological regulation of angiogenesis in endothelial cells through a tonic IFN-α signaling, whose enhancement could represent a viable strategy to suppress tumor neoangiogenesis.

Keywords: Angiogenesis; CXCL10; IFN-α; Regulatory network; Transcriptional modulators.

Graphical abstract

Fig. 1

K-means clustering of $-\mathrm{\Delta }Ct$ temporal profiles. Five temporal patterns of modulations were identified in response to IFN-α stimulation (0 h) and subsequent wash-out (8 h). Single gene profiles are depicted in gray, while cluster average profiles are colored. N indicates the cluster size.

Fig. 2

IFN-α regulatory network inference. Multiple-output FFL regulatory subnetwork reconstructed from the silencing of the seven IFN-α modulators. Lines represent influence regulations in the IFN-α stimulation (2 h/8 h; solid line) and wash-out (12 h; dashed lines) phase. Arrow styles stand for: arrow, activation; $⊣$, repression; dot, unspecified sign of regulation.

Fig. 3

Measurement of CXCL10 levels in supernatants of HUVECs following silencing of STAT1, IFIH1, USP18 and IRF1. Measurement of CXCL10 levels following STAT1, IFIH1, USP18 and IRF1 siRNA-mediated silencing in HUVECs from 3 different donors. ** p<0.01 siCTRL vs. siCTRL + IFN-α; § p<0.05, §§ p<0.01, §§§ p<0.001 siSTAT1/IFIH1/USP18/IRF1 + IFN-α vs. siCTRL + IFN-α.

Fig. 4

Modulation of STAT1, USP18, IFIH1, GBP1, and IRF1 affects in vitro EC sprouting. (a) Representative pictures of spheroids obtained from 4 independent sprouting assays on HUVECs treated with 20 ng/ml VEGF, with or without 600 IU/ml IFN-α, upon STAT1, USP18, IFIH1, GBP1 and IRF1 silencing or scrambled siRNA treatment (siCTRL). (b) Quantification of sprout areas from 4 independent sprouting assays in the absence and in the presence of IFN-α. *** p<0.001 siSTAT1/USP18/IFIH1/GBP1/IRF1 vs. siCTRL; § p<0.05, §§ p<0.01, §§§ p<0.001 VEGF + IFN-α vs. VEGF alone.