Homotypic endothelial nanotubes induced by wheat germ agglutinin and thrombin

Abstract

Endothelial barrier formation is maintained by intercellular communication through junctional proteins. The mechanisms involved in maintaining endothelial communication subsequent to barrier disruption remain unclear. It is known that low numbers of endothelial cells can be interconnected by homotypic actin-driven tunneling nanotubes (TNTs) which could be important for intercellular transfer of information in vascular physiology. Here we sought insight into the triggers for TNT formation. Wheat germ agglutinin, a C-type lectin and known label for TNTs, unexpectedly caused striking induction of TNTs. A succinylated derivative was by contrast inactive, suggesting mediation by a sialylated protein. Through siRNA-mediated knockdown we identified that this protein was likely to be CD31, an important sialylated membrane protein normally at endothelial cell junctions. We subsequently considered thrombin as a physiological inducer of endothelial TNTs because it reduces junctional contact. Thrombin reduced junctional contact, redistributed CD31 and induced TNTs, but its effect on TNTs was CD31-independent. Thrombin-induced TNTs nevertheless required PKCα, a known mediator of thrombin-dependent junctional remodelling, suggesting a necessity for junctional proteins in TNT formation. Indeed, TNT-inducing effects of wheat germ agglutinin and thrombin were both correlated with cortical actin rearrangement and similarly Ca2+-dependent, suggesting common underlying mechanisms. Once formed, Ca2+ signalling along TNTs was observed.

Cellular and Molecular Networking Within the Ecosystem of Cancer Cell Communication via Tunneling Nanotubes

“…Part of the biochemical cascade that results in VTE includes activation of thrombin, a serine protease that converts fibrinogen to fibrin. A recent elegant study demonstrated the ability of thrombin to induce TNTs in endothelial cells (Pedicini et al, 2018 ), providing further support to the notion that TNTs play a previously uncovered role in this cancer-related process.…”

Secretome and Tunneling Nanotubes: A Multilevel Network for Long Range Intercellular Communication between Endothelial Cells and Distant Cells

“…While hypoxia promotes the formation of TNTs, TNTs in turn stimulate a potential positive feedback loop by mediating intercellular transfer of hypoxia-inducible factor-1α (HIF-1α) and vascular endothelial growth factor (VEGF) between connected cells, from cancer cells to ECs, to stimulate angiogenesis [ 147 ]. Thrombin induce TNTs in ECs [ 161 ]. Since cancer provides a prothrombotic state, this further supports a role for TNTs interplay between cancer cells and ECs.…”

“…TNTs can also occur between ECs ( Figure 3 ), but we still have little understanding of the role of homotypic endothelial-to-endothelial TNTs or how they are formed. It has been suggested that ECs maintain Ca 2+ signals during an injury on monolayer integrity by forming endothelial-to-endothelial TNTs [ 161 ] This induction depends on the highly sialylated CD31 protein, an adhesion molecule that is largely restricted to ECs. In cultured ECs (HUVEC) thrombin-that disrupt endothelial cell barrier and promote cell signaling and reorganization of the actin cytoskeleton readily causes the appearance of TNTs [ 161 ].…”

“…It has been suggested that ECs maintain Ca 2+ signals during an injury on monolayer integrity by forming endothelial-to-endothelial TNTs [ 161 ] This induction depends on the highly sialylated CD31 protein, an adhesion molecule that is largely restricted to ECs. In cultured ECs (HUVEC) thrombin-that disrupt endothelial cell barrier and promote cell signaling and reorganization of the actin cytoskeleton readily causes the appearance of TNTs [ 161 ]. Together these findings suggest that endothelial-to-endothelial TNTs may involve the modulation of sialylated CD31 and the action of thrombin via protein kinase C (PKC)α.…”

MAPK Signaling Is Required for Generation of Tunneling Nanotube-Like Structures in Ovarian Cancer Cells

“…TnTs can also transmit information unidirectionally or bi-directionally [ 4 , 7 , 8 ]. Additionally, TnTs can be homotypic in that they connect similar cells (tumor cell to tumor cell) [ 7 , 9 ] or heterotypic meaning they connect different cell types (like macrophages to tumor cells) [ 10 ]. High-resolution microscopy and 3-dimensional reconstructions confirmed that TnTs are present in invasive malignancies, including human ovarian adenocarcinoma [ 5 ], providing evidence that TnTs are a characteristic of highly invasive cancers with metastatic potential, such as OC.…”