Counterbalancing anti-adhesive effects of Tenascin-C through fibronectin expression in endothelial cells

www.nature.com/scientificreports OPEN Received: 22 May 2017 Accepted: 12 September 2017 Published: xx xx xxxx Counterbalancing anti-adhesive effects of Tenascin-C through fibronectin expression in endothelial cells Agata Radwanska1, Dominique Grall1, Sébastien Schaub1, Stéphanie Beghelli-de la Forest Divonne1,2, Delphine Ciais1, Samah Rekima1, Tristan Rupp3,4, Anne Sudaka1,2, Gertraud Orend3,4 & Ellen Van Obberghen-Schilling 1,2 Cellular fibronectin (FN) and tenascin-C (TNC) are prominent development- and disease-associated matrix components with pro- and anti-adhesive activity, respectively. Whereas both are present in the tumour vasculature, their functional interplay on vascular endothelial cells remains unclear. We have previously shown that basally-oriented deposition of a FN matrix restricts motility and promotes junctional stability in cultured endothelial cells and that this effect is tightly coupled to expression of FN. Here we report that TNC induces FN expression in endothelial cells. This effect counteracts the potent anti-adhesive activity of TNC and leads to the assembly of a dense highly-branched subendothelial matrix that enhances tubulogenic activity. These findings suggest that pro-angiogenic remodelling of the perivascular matrix may involve TNC-induced upregulation of FN in endothelial cells. Angiogenesis, the sprouting of new vasculature from a pre-existing vascular network, is an essential process during development, maintenance of tissues and metastatic spread of cancer. This multi-step process is tightly regulated and spatiotemporally controlled by various soluble cytokines, membrane-bound proteins, cell-matrix and cell-cell interactions and hemodynamic forces. In recent years it has become clear that dynamic remodelling of the extracellular matrix (ECM) is essential for all stages of angiogenesis. Through adhesive interactions with integrins expressed on the endothelial cell surface, the ECM orchestrates complex signalling cascades within the cells and affects many fundamental aspects of their biology, including proliferation, migration, cytoskeletal organization, cell shape, survival, and ultimately blood vessel stabilization (reviewed in1). Tenascin-C (TNC) and alternatively spliced forms of fibronectin (FN) are principle ECM components of the angiogenic vasculature of tumours, yet barely detected in quiescent adult vessels (reviewed in2). Genetic studies in mice and fish have pointed to a fundamental role for FN and its primary receptor α5β1 integrin in early blood vessel development and vascular physio-pathology (reviewed in3,4). FN-null mice die at embryonic day 9.5 with severe cardiovascular defects5 and α5 null mice display the most severe vascular defects of all the null phenotypes of α-encoding integrin genes6. Although TNC knockout mice do not display an embryonic lethal phenotype7,8, TNC expression is highly associated with angiogenesis in a wide range of disease states, including cancer9–11. Adhesive and counter-adhesive effects are attributed respectively to FN and TNC. One mechanism by which TNC modulates cell adhesion-dependent processes involves its direct interaction with FN, which leads to interference of FN binding to syndecan-412. TNC can also interact with cognate integrins on the surface of cells13 (and references therein). Endothelial cells express TNC-binding integrin αvβ33. αvβ3 is upregulated in tumour-associated blood vessels where it has been found to play both pro- and anti-angiogenic roles in tumour angiogenesis, depending on the context14. FN matrix assembly, or fibrillogenesis, is a complex process (reviewed in15,16) driven by α5β1 integrin that takes place at specialized integrin-based structures called fibrillar adhesions at the cell-matrix interface17–19. In the context of blood vessel remodelling, FN deposited by endothelial cells forms a pericellular network of fibrils 1 Université Côte d’Azur, CNRS, INSERM, iBV, France. 2Centre Antoine Lacassagne, Nice, 06189, France. 3Inserm, U1109, MN3T laboratory, The Microenvironmental Niche in Tumorigenesis and Targeted Therapy, Strasbourg, F-67000, France. 4 University Strasbourg, Strasbourg, F-67000, France. Correspondence and requests for materials should be addressed to E.V.O.-S. (email: ) Scientific Reports | 7: 12762 | DOI:10.1038/s41598-017-13008-9 1 www.nature.com/scientificreports/ that provides a mechanically ideal support for promoting neovessel development20. Moreover, the FN scaffold can modulate angiogenic signalling by sequestering and increasing the bioavailability of diffused factors, as it binds most of the growth factors from the platelet-derived growth factor, vascular endothelial growth factor (VEGF) and fibroblast growth factor families21–23. Cellular FN variants are expressed around tumour blood vessels24–26 and we have previously shown that FN assembly by endothelial cells is a cell-autonomous process coupled to expression of the protein27. Here we show that vascular endothelial cells respond to a direct anti-adhesive effect of TNC by enhancing FN expression and assembly. Results Different localization of FN and TNC in angiogenic blood vessels of human tumours. To deter- mine the expression and relative localization of FN and TNC in the vasculature of human tumours, we performed immunostaining (Fig. 1 and Supplementary Fig. S1) on adjacent sections of head and neck squamous cell carcinomas (HNSCC). Double immunofluorescence staining of FN and CD31 confirmed the association of FN with a subset of tumour-associated microvessels (yellow arrows). TNC was present around the same vessels (TNC-FN co-staining). Whereas FN directly ensheathed the endothelial cells, TNC was localized on the abluminal side of the vascular basement membrane. These results are consistent with previous observations24 and suggest that TNC is derived from perivascular cells. However, some vessels displayed little or no FN staining and TNC appeared to be in direct contact with cells lining the vessels (Fig. 1, white arrow). Together these observations reflect the heterogeneity of the tumour vasculature and raise questions concerning the dynamic regulation of matrix protein expression by vascular endothelial cells. FN and TNC expression in endothelial cells. To study the functional interplay of FN and TNC at the cellular level, we first examined their expression in cultured endothelial cells of different origins (Fig. 2). The amount of soluble (secreted) and cell-associated protein was determined by Western analysis of conditioned culture medium and total cell extracts, respectively. As shown in Fig. 2a (top), different levels of FN were expressed by the endothelial cells examined (BAEC > HUVEC > HMEC). In each case, the majority of the expressed protein was present in cell lysates. We were unable to detect TNC expression by endothelial cells, under conditions in which it was detected in both conditioned medium and lysates from telomerase-immortalized fibroblasts (TI (...truncated)