Abstract
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.
Highlights
Embryonic development as well as vascular homeostasis in adulthood strongly depends on the intercellular communication between different cell types of the vascular system
Our results further highlight the multipotency of pericyte action in the vasculature by disclosing the co-culture-induced upregulation of molecules involved in blood–brain barrier (BBB) establishment and CNS homeostasis as well as in the regulation of cerebral blood flow
We further show that co-culture induces significant changes in the inflammatory profile of PCs
Summary
Embryonic development as well as vascular homeostasis in adulthood strongly depends on the intercellular communication between different cell types of the vascular system. An essential role in the communication between ECs and PCs is dedicated to the direct contacts between the two cell types, so-called peg–socket interactions harboring gap junctions and adhesion plaques [3,8]. These direct contacts allow the exchange of small metabolites, ions and second messengers between two neighboring cells. One PC is thought to contact several ECs, thereby being responsible for the important task of integrating EC responses [3] Both cell types make fundamental contributions to the composition of the basement membrane and secrete extracellular matrix (ECM) proteins such as laminin, collagen and fibronectin among many others [9]. Basement membrane composition is highly versatile and undergoes dynamic changes along with different developmental stages and according to the cellular environment [10]
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