New Insight on 2D In Vitro Angiogenesis Models: All That Stretches Is Not a Tube

HIGHLIGHTS: Tube formation on Matrigel(TM) and tube formation in co-culture with MSCs are two different stages of angiogenesis. uPA, uPAR, Jagged1, and Notch2 are common upregulated genes for ECs on Matrigel(TM), in co-culture and in dividing/migrating cells. EndMT activated at a much greater extent in ECs in a co-culture model than in a Matrigel(TM) assay. Only in the Matrigel(TM) assay are the Notch and Hippo pathway-related genes upregulated. ABSTRACT: A Matrigel-based tube formation assay is a simple and widely accepted 2D angiogenesis model in vitro. Extracellular matrix (EM) proteins and growth factors (GFs) from Matrigel(TM) exclusively trigger endothelial cell (EC) tubular network (ETN) formation. Co-culture of ECs with mesenchymal stromal cells (MSCs) is another and more reliable in vitro angiogenesis assay. MSCs modulate ETN formation through intercellular interactions and as a supplier of EM and GFs. The aim of the present study was to compare the expression profile of ECs in both models. We revealed upregulation of the uPA, uPAR, Jagged1, and Notch2 genes in dividing/migrating ECs and for ECs in both experimental models at 19 h. The expression of endothelial–mesenchymal transition genes largely increased in co-cultured ECs whereas Notch and Hippo signaling pathway genes were upregulated in ECs on Matrigel(TM). We showed that in the co-culture model, basement membrane (BM) deposition is limited only to cell-to-cell contacts in contrast to Matrigel(TM), which represents by itself fully pre-assembled BM matrix. We suggest that ETN in a co-culture model is still in a dynamic process due to immature BM whereas ECs in the Matrigel(TM) assay seem to be at the final stage of ETN formation.