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Integrin specificity in rigidity sensitive proliferation, activation and directional migration of endothelial cells

Periodic Reporting for period 1 - ISPADMEC (Integrin specificity in rigidity sensitive proliferation, activation and directional migration of endothelial cells)

Reporting period: 2015-09-01 to 2017-08-31

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.
The specific objectives for this project can be summarized as following:
i) Preparation and characterization of substrates with tunable viscoelasticity, integrin specificity and presentation at the nanoscale.
ii) Characterization of endothelial cell physiology as a function of specific integrin engagement and substrate viscoelasticity.

Towards the achievement of the first objective, we have prepared glass surfaces with long-range quasi-hexagonal patterns of gold nanoparticles by employing the Block Copolymer Micelle Lithography (BCML) technique. In order to prepare gold-nanopatterned surfaces with additional tuneable viscoelasticity, we adapted a previous procedure to obtain nano-patterned hydrogels. We obtained hydrogels with different mechanical properties in the physiologically-relevant range (1-40 kPa).
The integrin specificity of the nano-patterned glasses and hydrogels was accomplished by functionalizing the immobilized gold nanoparticles using thiol-gold chemistry and thiol-modified cyclic-RGD or integrin-selective antagonists presenting a thiol group. The characterization of integrin specificity was carried out by means of the investigation of the EC adhesion and focal adhesion formation on the different integrin selective surfaces.
Once the surfaces were prepared and characterized, we performed endothelial cell adhesion studies on these highly integrin selective surfaces.
• We have confirmed that ECs spread efficiently on nano-patterned substrates above a threshold in ligand density.
• We have shown also that assembled FAs on nano-patterned substrates with an inter-particle distance of 30 nm exhibited differences in morphology and cellular distribution as a function of the presented ligands and hence by the corresponding integrins used by ECs to adhere.
• We examined the integrin composition of FAs on non-transformed ECs at the initial stages of cells spreading. This investigation revealed that on a5b1 integrin-selective substrates, integrin avb3 co-clustered with a5b1 integrins.
• We studied ECs adhesion to integrin selective nano-patterned substrates with different mechanical properties. We examined the distribution of a5b1 and avb3 integrins as a function of substrate elasticity. The results suggest that cell contractility did not regulate avb3 integrin recruitment to integrin a5b1-based FAs. In the absence of FAs, due to reduced contractility, there is no integrin segregation with both integrins seemingly probing the substrate at edges.
• In order to find a mechanism which could explain this integrin recruitment, we performed different biochemical experiments. Overall, our data indicate that avb3 recruitment to a5b1-based FAs is mediated by the presence of a soluble serum factor. We suggest that such a factor serves as ligand for avb3 integrin. We could also show that focal adhesion kinase (FAK) inhibition attenuates avb3 recruitment indicating a role for FAK-mediated signalling during avb3 recruitment to adhesion clusters.
A layer of endothelial cells (ECs) constitutes the endothelium along the entire circulation system by adhering to the underlying vessels or heart tissue. Normal vascular development, homeostasis and remodelling necessitate continuous and extensive crosstalk between ECs and their extracellular matrix (ECM). Integrins cluster in focal adhesions (FAs) at points of contact with the ECM, providing the essential physical and chemical link necessary for cell survival, proliferation and migration. Endothelial cell adhesion to FN is predominantly mediated by α5β1 and αvβ3 integrins. The synergy between α5β1 and αvβ3 integrins enables the cell to sense and adapt to its mechanical microenvironment, a trait that is relevant when tissues go through extensive physical remodelling during embryonic development, angiogenesis and cancer progression. However, despite significant progress, a clear mechanistic picture of how these integrins mediate distinct functions by binding the same ECM ligand is still lacking.
The ISPADMEC project exploited the advantages of well-defined nano-patterned substrates and highly selective integrin antagonists to elucidate the role of each integrin and unravel their contribution in the fundamental process of cell adhesion. Our research findings reveal new, detailed and fundamental knowledge of endothelial cell integrin biology. The insight provided by our work has implications in many fields, including bioengineers, cell and vascular biologists, among others.