Tumor associated neovasculature generated by the process of
Tumor-associated neovasculature, generated by the process of angiogenesis, contributes to the rapid growth of tumor by tilting the balance toward activating angiogenic factors such as vascular endothelial growth factor (VEGF) and fibroblast growth factor (FGF) and inhibiting angiogenesis inhibitors such as TSP-1 and angiostatin [18,19]. In addition to supplying surrounding tissues with oxygen and nutrients, angiogenesis activates endothelial Actinomycin D in response to inflammatory stimulus to increase the expression of adhesion molecules, including ICAM, VCAM-1, E-selectin, CD105, and CD54, and this process facilitates the interaction between neutrophils and endothelial cells . Of note, some of these adhesion molecules are not just involved in the process of cell recognition and adhesion, they can also promote angiogenesis in vitro and in vivo, as has been demonstrated with soluble VCAM-1 and E-selectin . However, the mechanism underlying this process remains not fully understood.
Cells can communicate with neighboring or distant cells through the secretion of extracellular vesicles (EVs). Those vesicles are composed of exosomes and ectosomes (30–150 nm and 100–1000 nm in diameter, respectively), with a lipid bilayer containing transmembrane proteins, which encloses the cytosolic proteins and RNA. EVs have been shown to participate in many facets of cancer progression and treatment, including metastasis, therapy-induced resistance, and angiogenesis . Importantly, in addition to proteins and mRNAs, miRNAs and other noncoding RNAs are also in active EV cargoes. Several groups have recently observed tumor cell-released EV-mediated secretion of specific miRNAs or lncRNAs in the tumor microenvironment to be responsible for cancer-promoting angiogenesis [, , , , ]. However, limited information is available on the impact of viral noncoding RNAs on promoting cancer angiogenesis in the form of tumor cell-released EVs.
Materials and methods
Discussion Recently, EVs and exosomes have been the focus of intensive research owing to their capacity to transfer proteins, miRNAs, and lncRNAs to recipient cells and their crucial roles in signal transduction pathways . A large body of evidence suggests that miRNAs and lncRNAs of human origin can be exchanged between cancer cells and mesenchymal cells [24,25,27] or from cancer stem-like cells and more aggressive cancer cells to the rest of the cancer cells [26,29,30]. Intriguingly, in the present study we provide solid evidence (in vitro, in vivo and patient samples) showing that EBERs are transferred from EBV-infected NPC cells to surrounding endothelial cells, which stimulates VCAM-1 expression via TLR3/RIG-I recognition (Fig. 9). VCAM-1 is an immunoglobulin-like molecule that plays a role in various cell-to-cell adhesion interactions, most likely by binding to integrin α4β1 (also known as very late activation antigen-4). VCAM-1 expression is maintained at low levels in endothelial cells of healthy tissues and is stimulated under inflammatory conditions by a multitude of signals such as cytokines, reactive oxygen species, oxidized low-density lipoprotein, TLR agonists, or shear stress . Owing to its involvement in inflammation and its wide distribution in human tissues and organs, VCAM-1 is indicated in autoimmune disease, cardiovascular disease, infections, and cancer . Here, we show that NPC EV-derived EBERs promote angiogenesis by facilitating VCAM-1 expression. In support of this observation, previous publications have shown soluble VCAM-1 to be angiogenic in rat corneas and to confer chemotactic activity to endothelial cells . Furthermore, VCAM-1 was found to mediate angiogenesis upon stimulation by IL-4 and IL-13 . Mechanistically, p38 and FAK, rather than ERK1/2, have been found to account for VCAM-1-stimulated angiogenesis , whereas PLC-PKC-NFκB was attributable to VCAM-1 transcription activation . In contrast, we found that EBERs stimulated ERK1/2 rather than NFκB subunit p65 phosphorylation. Furthermore, our results indicated that EBERs stimulate VCAM-1 expression via TLR3 or RIG-I, both of which could transmit downstream signaling pathways mediated by MAPK or NFκB . Of note, a recent report by Fearnley et al.  suggests a role for the canonical MAPK pathway involving ERK1/2 and hyperphosphorylation of ATF2 causing elevation in VCAM-1 gene transcription. Our current findings and this relevant published literature lead us to believe that NPC EVs promote angiogenesis via TLR3/RIG-I recognition of EBERs, which is mediated by ERK activated ATF2 rather than NFκB. Interestingly, VEGF-A stimulation of VCAM-1 expression in endothelial cells such as HUVECs has been previously reported by different groups involving multiple mechanisms [33,, , ]. Accordingly, in addition to EBERs, protein-based factors associated with EVs from NPC cells, such as VEGF-A may promote such signaling leading to increased VCAM-1 gene transcription, which exemplified the significance of VCAM-1 in NPC EVs mediated angiogenesis.