Transcryptomic Analysis of Human Brain-Microvascular Endothelial Response to -Pericytes: Cell Orientation Defines Barrier Function

Brigitte Leeners,Bruno Imthurn,Omolara O Ogunshola,Lisa Kurmann,Raghvendra K Dubey,Michal Okoniewski

doi:10.3390/cells10040963

Brigitte Leeners, Bruno Imthurn + Show 4 more

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https://doi.org/10.3390/cells10040963

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Abstract

Pericytes facilitate blood–brain barrier (BBB) integrity; however, the mechanisms involved remain unclear. Hence, using co-cultures of human cerebral microvascular endothelial cells (ECs) and vascular pericytes (PCs) in different spatial arrangements, as well as PC conditioned media, we investigated the impact of PC-EC orientation and PC-derived soluble factors on EC barrier function. We provide the first evidence that barrier-inducing properties of PCs require basolateral contact with ECs. Gene expression analysis (GEA) in ECs co-cultured with PCs versus ECs alone showed significant upregulation of 38 genes and downregulation of 122 genes. Pathway enrichment analysis of modulated genes showed significant regulation of several pathways, including transforming growth factor-β and interleukin-1 regulated extracellular matrix, interferon and interleukin signaling, immune system signaling, receptor of advanced glycation end products (RAGE), and cytokine–cytokine receptor interaction. Transcriptomic analysis showed a reduction in molecules such as pro-inflammatory cytokines and chemokines, which are known to be induced during BBB disruption. Moreover, cytokine proteome array confirmed the downregulation of key pro-inflammatory cytokines and chemokines on the protein level. Other molecules which influence BBB and were favorably modulated upon EC-PC co-culture include IL-18 binding protein, kallikrein-3, CSF2 CSF3, CXCL10, CXCL11 (downregulated) and IL-1-R4; HGF, PDGF-AB/BB, PECAM, SERPIN E1 (upregulated). In conclusion, we provide the first evidence that (1) basolateral contact between ECs and PCs is essential for EC barrier function and integrity; (2) in ECs co-cultured with PCs, the profile of BBB disrupting pro-inflammatory molecules and cytokines/chemokines is downregulated; (3) PCs significantly modulate EC mechanisms known to improve barrier function, including TGF-β regulated ECM pathway, anti-inflammatory cytokines, growth factors and matrix proteins. This human PC-EC co-culture may serve as a viable in vitro model for investigating BBB function and drug transport.

Highlights

In the Supplementary Materials, we provide measurements that demonstrate the usability of the system and show that for example hydrocortisone, a well-known barrier-inducer [21], increases barrier function as measured with this system
We examined the effect of different spatial pericyte-endothelial cell (PC-endothelial cells (ECs)) arrangements on barrier function
Studies with co-culture models often make use of pure animal or a mixture of animal and human cell types, which may be of a poor predictive value for translation into human physiology [28,29]

Summary

Introduction

In addition to a single layer of endothelial cells (ECs), astrocytes, pericytes (PCs), microglia and neurons hold critical functions in maintaining the integrity of the vessel wall [1,2]. PCs have long been an overlooked player in the field of BBB research. These mural cells have gained increasing attention as several studies have revealed the importance of PCs with regard to a properly functioning endothelial barrier. In vivo studies using viable pericyte-deficient mouse models provide evidence that PCs are critical for BBB formation and PC loss results in severe vessel leakage [1,2]

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