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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.

SUBMITTER: Kurmann L

PROVIDER: S-EPMC8074760 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:BACKGROUND:Pericytes of the blood-brain barrier (BBB) are embedded within basement membrane between brain microvascular endothelial cells (BMECs) and astrocyte end-feet. Despite the direct cell-cell contact observed in vivo, most in vitro BBB models introduce an artificial membrane that separates pericytes from BMECs. In this study, we investigated the effects of pericytes on BMEC barrier function across a range of in vitro platforms with varied spatial orientations and levels of cell-cell contact. METHODS:We differentiated RFP-pericytes and GFP-BMECs from hiPSCs and monitored transendothelial electrical resistance (TEER) across BMECs on transwell inserts while pericytes were either directly co-cultured on the membrane, indirectly co-cultured in the basolateral chamber, or embedded in a collagen I gel formed on the transwell membrane. We then incorporated pericytes into a tissue-engineered microvessel model of the BBB and measured pericyte motility and microvessel permeability. RESULTS:We found that BMEC monolayers did not require co-culture with pericytes to achieve physiological TEER values (> 1500 Ω cm2). However, under stressed conditions where TEER values for BMEC monolayers were reduced, indirectly co-cultured hiPSC-derived pericytes restored optimal TEER. Conversely, directly co-cultured pericytes resulted in a decrease in TEER by interfering with BMEC monolayer continuity. In the microvessel model, we observed direct pericyte-BMEC contact, abluminal pericyte localization, and physiologically-low Lucifer yellow permeability comparable to that of BMEC microvessels. In addition, pericyte motility decreased during the first 48 h of co-culture, suggesting progression towards pericyte stabilization. CONCLUSIONS:We demonstrated that monocultured BMECs do not require co-culture to achieve physiological TEER, but that suboptimal TEER in stressed monolayers can be increased through co-culture with hiPSC-derived pericytes or conditioned media. We also developed the first BBB microvessel model using exclusively hiPSC-derived BMECs and pericytes, which could be used to examine vascular dysfunction in the human CNS.

Project description:Many pathological conditions of the brain are associated with structural abnormalities within the neurovascular system and linked to pericyte (PC) loss and/or dysfunction. Since crosstalk between endothelial cells (ECs) and PCs greatly impacts the function of the blood-brain barrier (BBB), effects of PCs on endothelial integrity and function have been investigated extensively. However, the impact of ECs on the function and activity of PCs remains largely unknown. Hence, using co-cultures of human brain vascular PCs with human cerebral microvascular ECs on opposite sides of porous Transwell inserts which facilitates direct EC-PC contact and improves EC barrier function, we analyzed EC-driven transcriptomic changes in PCs using microarrays and changes in cytokines/chemokines using proteome arrays. Gene expression analysis (GEA) in PCs co-cultured with ECs versus PCs cultured alone showed significant upregulation of 1'334 genes and downregulation of 964 genes. GEA in co-cultured PCs revealed increased expression of five prominent PC markers as well as soluble factors, such as transforming growth factor beta, fibroblast growth factor, angiopoietin 1, brain-derived neurotrophic factor, all of which are involved in EC-PC crosstalk and BBB induction. Pathway enrichment analysis of modulated genes showed a strong impact on many inflammatory and extracellular matrix (ECM) pathways including interferon and interleukin signaling, TGF-β and interleukin-1 regulation of ECM, as well as on the mRNA processing pathway. Interestingly, while co-culture induced the mRNA expression of many chemokines and cytokines, including several CCL- and CXC-motif ligands and interleukins, we observed a decreased expression of the same inflammatory mediators on the protein level. Importantly, in PCs, ECs significantly induced interferon associated proteins (IFIT1, IFI44L, IF127, IFIT3, IFI6, IFI44) with anti-viral actions; downregulated prostaglandin E receptor 2 (prevent COX-2 mediated BBB damage); upregulated fibulin-3 and connective tissue growth factor essential for BBB integrity; and multiple ECMs (collagens and integrins) that inhibit cell migration. Our findings suggest that via direct contact, ECs prime PCs to induce molecules to promote BBB integrity and cell survival during infection and inflammatory insult. Taken together, we provide first evidence that interaction with ECs though porous membranes induces major changes in the transcriptomic and proteomic profile of PCs. ECs influence genes involved in diverse aspects of PC function including PC maturation, cell survival, anti-viral defense, blood flow regulation, immuno-modulation and ECM deposition.

Project description:Autoreactive T lymphocytes crossing the blood-brain barrier (BBB) into the central nervous system (CNS) play a crucial role in the initiation of demyelination and neurodegeneration in multiple sclerosis (MS). Recently, extracellular vesicles (EV) secreted by BBB endothelial cells (BBB-EC) have emerged as a unique form of cell-to-cell communication that contributes to cerebrovascular dysfunction. However, the precise impact of different size-based subpopulations of BBB-EC-derived EV (BBB-EV) on the early stages of MS remains unclear. Therefore, our objective was to investigate the content and function of distinct BBB-EV subpopulations in regulating BBB integrity and their role in T cell transendothelial migration, both in vitro and in vivo. Our study reveals that BBB-ECs release two distinct size based EV populations, namely small EV (sEV; 30-150 nm) and large EV (lEV; 150-300 nm), with a significantly higher secretion of sEV during inflammation. Notably, the expression patterns of cytokines and adhesion markers differ significantly between these BBB-EV subsets, indicating specific functional differences in the regulation of T cell migration. Through in vitro experiments, we demonstrate that lEV, which predominantly reflect their cellular source, play a major role in BBB integrity loss and the enhanced migration of pro-inflammatory Th1 and Th17.1 cells. Conversely, sEV appear to protect BBB function by inducing an anti-inflammatory phenotype in BBB-EC. These findings align with our in vivo data, where the administration of sEV to mice with experimental autoimmune encephalomyelitis (EAE) results in lower disease severity compared to the administration of lEV, which exacerbates disease symptoms. In conclusion, our study highlights the distinct and opposing effects of BBB-EV subpopulations on the BBB, both in vitro and in vivo. These findings underscore the need for further investigation into the diagnostic and therapeutic potential of BBB-EV in the context of MS.

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

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