Long Non-Coding RNA LINC02802 Regulates In Vitro Sprouting Angiogenesis by Sponging microRNA-486-5p.

In the last several years, accumulating evidence indicates that noncoding RNAs, especially long-noncoding RNAs (lncRNAs) and microRNAs, play essential roles in regulating angiogenesis. However, the contribution of lncRNA-mediated competing-endogenous RNA (ceRNA) activity in the control of capillary sprouting from the pre-existing ones has not been described so far. Here, by exploiting the transcriptomic profile of VEGF-A-activated endothelial cells in a consolidate three-dimensional culture system, we identified a list of lncRNAs whose expression was modified during the sprouting process. By crossing the lncRNAs with a higher expression level and the highest fold change value between unstimulated and VEGF-A-stimulated endothelial cells, we identified the unknown LINC02802 as the best candidate to take part in sprouting regulation. LINC02802 was upregulated after VEGF-A stimulation and its knockdown resulted in a significant reduction in sprouting activity. Mechanistically, we demonstrated that LINC02802 acts as a ceRNA in the post-transcriptional regulation of Mastermind-like-3 (MAML3) gene expression through a competitive binding with miR-486-5p. Taken together, these results suggest that LINC02802 plays a critical role in preventing the miR-486-5p anti-angiogenic effect and that this inhibitory effect results from the reduction in MAML3 expression.

Corrigendum to "Genetic perturbation of IFN-α transcriptional modulators in human endothelial cells uncovers pivotal regulators of angiogenesis" [Comput Struct Biotechnol J. 2020 Dec 2;18:3977-3986. doi: https://doi.org//10.1016/j.csbj.2020.11.048. eCollection 2020].

[This corrects the article DOI: 10.1016/j.csbj.2020.11.048.].

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

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.

A regulatory microRNA network controls endothelial cell phenotypic switch during sprouting angiogenesis.

Angiogenesis requires the temporal coordination of the proliferation and the migration of endothelial cells. Here, we investigated the regulatory role of microRNAs (miRNAs) in harmonizing angiogenesis processes in a three-dimensional in vitro model. We described a microRNA network which contributes to the observed down- and upregulation of proliferative and migratory genes, respectively. Global analysis of miRNA-target gene interactions identified two sub-network modules, the first organized in upregulated miRNAs connected with downregulated target genes and the second with opposite features. miR-424-5p and miR-29a-3p were selected for the network validation. Gain- and loss-of-function approaches targeting these microRNAs impaired angiogenesis, suggesting that these modules are instrumental to the temporal coordination of endothelial migration and proliferation. Interestingly, miR-29a-3p and its targets belong to a selective biomarker that is able to identify colorectal cancer patients who are responding to anti-angiogenic treatments. Our results provide a view of higher-order interactions in angiogenesis that has potential to provide diagnostic and therapeutic insights.

Prevention of hypoxia by myoglobin expression in human tumor cells promotes differentiation and inhibits metastasis.

As a tumor grows, it requires increased amounts of oxygen. However, the tumor blood vessels that form to meet this demand are functionally impaired, leading to regions of hypoxia within the tumor. Such hypoxia is one of the hallmarks of malignancy and is thought to promote a number of tumorigenic properties. Here, we sought to determine how tumors without hypoxia would progress by engineering A549 human lung carcinoma cells to ectopically express myoglobin (Mb), a multifunctional heme protein that specializes in oxygen transport, storage, and buffering. Mb expression prevented the hypoxic response in vitro and delayed tumor engraftment and reduced tumor growth following xenotransplantation into mice. Experimental tumors expressing Mb displayed reduced or no hypoxia, minimal HIF-1alpha levels, and a homogeneously low vessel density. Mb-mediated tumor oxygenation promoted differentiation of cancer cells and suppressed both local and distal metastatic spreading. These effects were primarily due to reduced tumor hypoxia, because they were not observed using point-mutated forms of myoglobin unable to bind oxygen and they were abrogated by expression of a constitutively active form of HIF-1alpha. Although limited to xenograft models, these data provide experimental proof of the concept that hypoxia is not just a side effect of deregulated growth but a key factor on which the tumor relies in order to promote its own expansion.