Molecular dissection of blood vessel growth and maturation during angiogenesis


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Rachel A Daniel1, Matthew Fisher1, Miriam V Flores Merino3, Caterina Lo Presti3, Giuseppe Battaglia3, Juliane M Jurgensmeier2, Anderson Ryan2, Gillian M Tozer1

1University of Sheffield, UK, 2AstraZeneca, Alderley Edge, UK, 3Kroto Research Institute, University of Sheffield, UK

Abstract

Background

A mature vasculature can be defined as a system that effectively supplies its supported tissue with its needs for optimum growth. A complex network of angiogenic growth factors directs blood vessel growth and maturation. The initial aim of this study is to optimise a system by which growth factors, either individually or in specific combinations, can be introduced into the murine dorsal skin flap window chamber model, to study their role in vascular growth and maturation.

Method

The suitability of three materials (matrigel, polyether sponge, polyvinylpyrrolidone (PVP) hydrogel) as growth factor carriers in the window chamber was tested. ELISA was used to analyse growth factor release from hydrogels in vitro. Chemotaxis cell migration assays were used for testing effects of growth factors on endothelial cell lines in vitro. Window chamber vasculature was monitored in vivo following implantation of growth factor carriers, using intravital microscopy, and then tissue fixed for immunohistochemical analysis of the endothelial cell marker CD31.

Results and Conclusion

Of materials tested, PVP hydrogel was considered most suitable for in vivo implantation. Hydrogel allows control over growth factor release rate, also conferring a level of protection from enzymatic degradation. A 1% crosslinker hydrogel encapsulated VEGF120 and VEGF164 respectively, but ELISA showed inefficient growth factor release in vitro, with little angiogenic activity after implantation into window chambers. To increase growth factor release, a hydrogel of larger pore size (0.5% crosslinker) was evaluated,

Angiogenic activity was observed in vivo in the window chamber model implanted with 0.5% crosslinker hydrogels impregnated with VEGF164 bFGF. Increased vascular development was seen particularly around the immediate areas surrounding hydrogel implants, with the formation of microvascular clusters. Further optimisation of hydrogel growth factor release is required for the study of other growth factors and their combinations.

Acknowledgments

This work is funded by the BBSRC and AstraZeneca.