Proteomes and Biomarkers of the Neurovascular Unit

Introduction

Brain endothelial cells (BECs) that form the blood–brain barrier (BBB) exhibit a unique polarized phenotype characterized by firmly sealed tight junctions, thick and elaborate luminal glycocalyx, and a multitude of transporters and ion pumps responsible for controlled exchange of water and nutrients between blood and brain compartments. Adjoining pericytes embedded in the vascular basement membrane, astrocytes projecting end feet that envelope capillaries and microvessels, and microvessel-innervating neurons form the neurovascular unit (NVU). Both the specialized phenotype of the BEC and functional regulation of the NVU are dependent on integrated molecular interactions of this cellular network .

The NVU is an important player in brain pathologies spanning brain tumors and cerebrovascular, neuroinflammatory, and neurodegenerative diseases . Brain vessels undergo a profound molecular and functional remodeling in response to pathology; the NVU proteome changes associated with this remodeling can be analyzed using a variety of advanced mass spectrometry (MS) techniques. The key techniques include isotope labeling–based or label-free proteomics methods, glycoproteomics, and targeted quantitative MS. It should be noted that the application of these techniques in both research and clinical environments typically requires “core facilities” grouping together expensive equipment and specialized expertise.

The technical objective of discovery proteomics is the broadest possible protein coverage that includes low abundant and rare proteins. In contrast, targeted proteomics focuses on a limited number of proteins, usually selected from discovery approaches, that can be monitored and quantified (in absolute concentrations) in multiplexed fashion over a time course or in patient’s samples. Various physiological and pathological aspects of the NVU have been dissected using these MS-based approaches ( Table 73.1 ).

The generic workflows used for MS-based analyses of the NVU are shown in Fig. 73.1 . Isolation of various NVU components that have been subjected to various conditions or pathology is followed by sample preparation to extract proteins. For labeling-based MS methods, paired samples are isotopically labeled followed by mixing of the pairs for differential protein expression analysis. Protein solubilization and proteolytic digestion into MS-analyzable/quantifiable peptides is followed by liquid chromatography (LC)-coupled MS analysis (tandem MS for discovery proteomics or selected-reaction monitoring for targeted quantification). Finally, protein identification/quantification, data analysis, and integration are performed using various bioinformatics tools.

The advances in applying MS-based approaches to interrogate NVU are summarized in this chapter, as both a resource and inspiration for future creative application of these techniques in the cerebrovascular research field.

Neurovascular Unit “Carta”

Discovery proteomic analyses of the NVU in various experimental paradigms are being assembled currently into a large repository called the NVU “Carta” ( Fig. 73.2 ). The NVU “Carta” aims to: (1) catalog and map protein composition of BBB endothelial cells to facilitate understanding of their unique phenotype including tight junctions and polarized expression of transporters and receptors, important for physiological homeostasis and drug delivery; (2) identify protein–protein interactions (interactome) among different constituents of the NVU involved in BEC phenotype induction, maintenance, and modulation; (3) identify NVU-specific biomarkers and therapeutic targets for cerebrovascular diseases.

To achieve these goals, three general research paradigms have been applied: (1) assembly of proteomes of isolated pure cellular and subcellular components of the NVU followed by their in silico reconstruction into models/networks that illustrate NVU functions; (2) proteomic analyses of whole brain vascular tissues (consisting of mixtures of various cellular/acellular NVU components), often from brains affected by pathology; (3) profiling of brain endothelium–specific biomarkers in accessible body fluids as descriptors of cerebrovascular pathologies using both discovery and targeted proteomic approaches. We will briefly illustrate advances in NVU analyses using each of these approaches.