The main purpose of this proposal was the development of a multifunctional synthetic platform which allows precise regulation of both biochemical and mechanical parameters in order to isolate their contribution on fundamental endothelial cells (EC) functions. The proposed work exploited advances in materials science and nanotechnology to modulate with high precision the presentation of highly selective ligands at the nanometer and micrometer length scales, on substrates with tuneable viscoelasticity and mechanics.
In particular, our project addressed the fundamental questions of how differential integrin engagement affects endothelial cell adhesion. The great interest in understanding the role of cell adhesion on the provisional ECM protein fibronectin is better appreciated if we consider its essential role in both physiological (e.g. development, wound healing) and pathological (e.g. tumour metastasis, fibrosis) situations, and the consideration of both a5b1 and avb3 integrin receptors as clinical targets for medical applications. In particular, the observation that integrin avb3 is upregulated in proliferating endothelial cells during angiogenesis and tumour growth, concurrently with fibronectin deposition, has led to clinical trials targeting avb3 which unfortunately have not yet delivered their promise, partly due to our lack of detailed understanding on integrin-related functions and cross-talk.
This project allowed us to precisely regulate both biochemical and mechanical parameters in order to isolate their contribution on fundamental endothelial cell functions and to successfully establish the combined effects of ligand presentation, integrins specifity and substrate mechanics on EC physiology in an in vitro setting.
Specifically, by using this well-defined nano-patterned platform with highly-selective peptidomimetics we could decouple individual integrin contributions and reveal an intriguing integrin cross-talk event at early stages of adhesion cluster formation, namely the recruitment of avb3 integrins onto a5b1-based FAs. We demonstrate that adhesion clusters assembled upon integrin a5b1 engagement are able to recruit avb3 integrins to these clusters, but not vice-versa, and show that this recruitment is critical for allowing efficient focal adhesion assembly and coherent cell spreading on integrin-selective susbtrates. This type of integrin cross-talk has not been previously appreciated, primarily due to the lack of approapiate tools to separate the function of each integrin. Most studies relied on genetic manipulation to alter integrin expression, but were limited by the use of ligands that bind several receptors or the lack of desired selectivity. Within this project, we could exploit the advantages of well-defined nano-patterned susbtrates and highly selective integrin antagonists to elucidate the role of each integrin and unravel their contribution in the fundamental process of cell adhesion.
