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

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.

Influence of Traumatic Brain Injury on Extracellular Tau Elimination at the Blood-Brain Barrier

“…Banks and colleagues found that full-length tau and various truncated tau proteins readily crossed the BBB bidirectionally and entered the blood following injection into the brain (Banks et al, 2016), consistent with our intracranial tau injection studies. In line with these BBB ndings, our studies employed a recently described indirect co-culture BBB model (Kurmann et al, 2021) consisting of brain endothelia and pericytes, and found the BBB transit of each tau species was greater than that observed for LyD (basolateral-to-apical). In comparing the tau species, the aggregate enriched tau showed increased BBB transcytosis compared to monomeric tau, potentially explaining the enhanced elimination of these species in the tau residence studies above.…”

Transcriptomic Analysis of Human Brain -Microvascular Endothelial Cell Driven Changes in -Vascular Pericytes

“…In a previous study we investigated the transcriptomic changes between ECs cultured alone and in presence of PCs in the same co-culture model as was used in the present study [ 18 ]. Interestingly, the number of DRGs observed in co-cultured vs. mono-cultured PCs (2’298) was more than ten-fold higher than the number of observed DRGs in ECs (160).…”

“…Since pericyte–endothelial cell (PC–EC) crosstalk essentially contributes to the formation and stabilization of the endothelial barrier, the impact of these mural cells on EC function has been extensively investigated by us [ 18 ] and others [ 2 , 25 , 26 ]. We recently demonstrated that basolateral contact between ECs and PCs regulates multiple genes and cytokines/chemokines in human brain microvascular ECs [ 18 ].…”

“…Most research has focused on assessing the effects of mural and glial cells on endothelial integrity and barrier function, including a recent publication from our lab [ 18 ]. However, much less is known about the impact of ECs on mural cell function, including PCs.…”

“…To accomplish our goals, transcriptomic analysis using microarray was performed on PCs cultured alone or in co-culture with ECs on opposite sides of permeable Transwell inserts, which allows ECs and PCs to have direct cell–cell contact and permits the exchange of paracrine factors through the pores of the membrane, as shown by Kang et al [ 16 ]. In the present study, we specifically investigated EC driven changes in PC gene expression using the EC–PC co-culture model that facilitates EC-barrier function, as we previously demonstrated [ 18 ]. Additionally, we performed cytokine proteome arrays, to elucidate whether the observed transcriptional changes in the inflammatory profile of PCs are also reflected on the protein level.…”