Speaker
Description
Introduction
Cycles of vascular growth and regression can be observed in physiology as well as pathology. Angiogenic phases are intermitted by vessel regression in the menstrual or hair cycle and tumor-supplying vasculature can regrow after therapeutic intervention through anti-angiogenic therapies (AATs)(1-3). While AATs based on limiting the availability of VEGF have been successfully used in the clinics, it has also long been established that their efficacy varies with tumor types and can lead to development of more aggressive and heavily vascularised phenotypes upon treatment termination(3). To this day, a multitude of in vitro angiogenesis assays have been described, however, in vitro systems for modelling the recovery of a vascular network are lacking. In this study, we establish and describe a platform to generate controlled yet complex vascular networks which are sensitive to treatment with bevacizumab and which can be replenished through the addition of new endothelial cells.
Methodology
Three-dimensional cultures were grown by seeding cells onto a commercially available 96 well-plate featuring pre-cast poly(ethylene glycol)-based hydrogels (3DProSeed, Ectica Technologies). Sequentially seeded bone marrow-derived mesenchymal stem/stromal cells (BM-MSCs) and human umbilical vein endothelial cells (HUVECs, GFP expressing) were allowed to assemble into vascular-like networks for four days in presence of 50 ng/mL FGF-2. Networks were characterized by quantifying total GFP-length and staining for extracellular matrix molecules fibronectin and laminin. Bevacizumab was supplied from the start of endothelial culture to study its influence on network formation or after the four-day formation-period to study its effect on the maintenance or ablation of vascular networks. Fresh RFP-expressing HUVECs were added to pre-formed networks and their relative localization and potential recovery of regressing GFP-HUVEC-structures was monitored.
Results
After three days of BM-MSC pre-culture and four days of co-culture with GFP-HUVECs, tightly interconnected endothelial networks had formed. The two co-cultured cell types shared a basement membrane-like layer of fibronectin and laminin and could be maintained for several days of co-culture. Vascular network formation on this platform appears to be sensitive to bevacizumab, as low concentration of this compound (10 μg/mL) prevented this morphogenic process. Interestingly, pre-formed day four-networks were seen to degrade only in presence of high bevacizumab concentrations (100 μg/mL), while no change could be observed in presence of low concentrations (10 μg/mL) compared to control conditions for at least four more days of culture. When RFP-HUVECs were added to pre-formed day four-GFP networks, they were seen to integrate into the established GFP-structures and helped to extend the longevity of the total endothelial networks.
Conclusions
Our results suggest that the herein established and characterized platform can be employed to study processes regarding both the formation and maintenance of vascular structures. Due to its easy-to-use nature, sequential seeding of cells is possible, which will be used to study if and how endothelial cells can recover an AAT-compromised or regressed vascular network.
References
1. Sugino, N. et al., Reprod Med Biol. 7(2), 91-103 (2008)
2. Mecklenburg, L. et al., J Invest Dermatol. 114(5), 909-16 (2000)
3. Cacheux, W. et al., Ann Oncol. 19(9), 1659-61 (2008)
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