Study the effect of different protein pre-coatings on the endothelial deposited and remodeled pericellular matrix by a non-canonical amino-acid tagging: relevance for BBB modelling.

The blood-brain barrier (BBB) is a fundamental structure that regulates the selective passage of molecules between systemic blood and the brain parenchyma, ensuring cerebral protection and homeostasis allowing for the normal functions of the central nervous system (CNS) [1]. Its selective permeability represents a significant obstacle for pharmacological drugs permeability in brain drug delivery, which leads to the failure of most molecules directed to CNS. In certain pathological conditions, the BBB integrity is often compromised, which actively contributes to the progression of various diseases such as brain tumours, ischemic and haemorrhagic strokes, Alzheimer’s Disease (AD) among others [2], [3]. The development of reliable in vitro BBB model represents a crucial challenge in biomedical research, essential for studying cellular interactions and developing innovative therapies. The BBB, aside from the endothelial monolayer and the basal membrane, is also constituted by vast cell types and other components such as astrocytes end-feet which are embedded in the basal membrane, neurons, pericytes, microglia and, together with the extracellular matrix and the previously described components, constitute the neurovascular unit responsible of the CNS homeostasis. Even presenting a complex constitution, most in vitro BBB models tend to focus on the study of the endothelial monolayer due to their active role on the control of the brain-blood traffic [4]. Comprised of brain endothelial cells (BECs), this monolayer is characterized by the presence of a vast number of specific characteristics, e.g. tight junctions, the arrest of pinocytic vesicles activity, the expression of efflux transporters and enzymes that metabolize bioactive substances [3]. Each of these features defines their barrier capability. In close contact and anchored in the basal membrane (BM), endothelial cells (ECs) express cellular junctions, such as tight, adherent and gap junctions which are specialized structures that regulate ions and other hydrophilic molecules’ passage between cells and intra and extracellular space being the key component in blocking smaller molecules from transversing the BBB without being first recognized and transported across this barrier [3]. The ECs capacity to recognize BM proteins bioactive domains guides the dynamic protein deposition orchestra, essential for maintaining the structural and functional integrity of the BBB. Incomplete or disruptions in these dynamics are known to lead to increased permeability and contribute to various neurological disorders. To understand and evaluate the role of the different BM proteins on endothelial pericellular matrix deposition we use human umbilical vein endothelial cells (HUVEC), an easily accessible, versatile, and widely used in vitro model for studying vascular processes, cultured on different coatings (laminin, fibronectin, collagen type I, collagen type IV). We will apply an innovative technique Bioorthogonal Noncanonical Amino Acid Tagging (BONCAT) [5] to study the effect of different protein coatings on the spatiotemporal visualization of nascent pericellular synthetized proteins. Moreover, via this co-staining, we will identify and evaluate the different pre-coating proteins’ effects on specific endothelial markers such as CD144, CD31, Zonula Occludens-1 (ZO-1), claudin-5, and occludin. The results of this study will provide the foundation for designing in vitro BBB models on optimized substrates. The proposed approach will facilitate the development of surfaces enriched with specific active peptides, improving the fidelity and reproducibility of in vitro models and opening new perspectives in pharmacological and bioengineering fields.