Integration of Endothelial Cells in Multicellular Spheroids Prevents Apoptosis and Induces Differentiation

Abstract

Single endothelial cells (EC) seeded in suspension culture rapidly undergo apoptosis. Addition of survival factors, such as VEGF and FGF-2, does not prevent apoptosis of suspended EC. However, when cells are allowed to establish cell–cell contacts, they become responsive to the activities of survival factors. These observations have led to the development of a three-dimensional spheroid model of EC differentiation. EC spheroids remodel over time to establish a differentiated surface layer of EC and a center of unorganized EC that subsequently undergo apoptosis. Surface EC become quiescent, establish firm cell–cell contacts, and can be induced to express differentiation antigens (e.g., induction of CD34 expression by VEGF). In contrast, the unorganized center spheroid cells undergo apoptosis if they are not rescued by survival factors. The responsiveness to the survival factor activities of VEGF and FGF-2 was not dependent on cell shape changes since it was retained after cytochalasin D treatment. Taken together, these findings characterize survival factor requirements of unorganized EC and indicate that polarized surface EC differentiate to become independent of exogenous survival factors. Furthermore, they demonstrate that spheroid cell culture systems are useful not just for the study of tumor cells and embryonic stem cells but also for the analysis of differentiated functions of nontransformed cells.

Distinctive role of integrin-mediated adhesion in TNF-induced PKB/Akt and NF-κB activation and endothelial cell survival

“…HUVEC multicellular spheroids form and survive in the absence of integrin ligation Korff and Augustin (1998) reported that integration of HUVEC in multicellular spheroids prevented anoikis, thereby allowing the study of non-matrix adherent HUVEC over prolonged periods of time. Anti-VEcadherin monoclonal antibody (mAb) or Ca 2 þ depletion prevented spheroid formation for at least 24 h, whereas blocking mAbs against a2, a3, a5, a6, b1, aVb3, aVb5 integrins or PECAM-1, or a peptidic aVb3/aVb5 inhibitor (EMD121974), alone or in combination, did not (Supplementary Figure S2a, and not shown).…”

“…This work was initiated to investigate whether integrindependent adhesion may modulate endothelial cell response to TNF and to identify the associated intracellular signaling events. As a main model, we used the endothelial cell spheroid model reported by Korff and Augustin (1998). It allows bypassing anoikis that rapidly occurs in single-cell suspensions (Meredith et al, 1993), thereby allowing to keep HUVEC viable for the duration of the experiment under conditions of integrin-independent but VE-cadherin-dependent adhesion (Spagnuolo et al, 2004).…”

Inhibition of human lung carcinoma cell growth by apoptosis induction using Semliki Forest virus recombinant particles

“…22 A total of 10 4 H358a cells in 0.1 ml of complete medium were plated in 96-well plates, whereas Gene Therapy 10 5 cells in 1 ml were used in 24-well plates. To prevent cell attachment, plates were coated with 1% medium agar.…”

High efficient adenoviral-mediated VEGF and Ang-1 gene delivery into osteogenically differentiated human mesenchymal stem cells

“…The preparation of cell spheroids was performed as described previously (Korff & Augustin, 1998). Briefly, subconfluent monolayers of HUVECs were trypsinized and suspended in endothelial cell basal medium (ECBM, Promocell, Heidelberg, Germany)…”