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Plasminogen activator inhibitor-type I is a major biosynthetic product of retinal microvascular endothelial cells and pericytes in culture.

ABSTRACT: Previous studies have shown that a glycoprotein of Mr 47,000 (designated Gp47) is a major biosynthetic product of retinal endothelial cells in vitro (Canfield, Schor, West, Schor & Grant (1987) Biochem. J. 246, 121-129). We now present data indicating that (a) an identical protein is secreted by bovine retinal pericytes, (b) this protein is plasminogen activator inhibitor-type I (PAI-1), as revealed by immunoprecipitation with specific antibodies and reverse fibrin zymography, and (c) retinal endothelial cells and pericytes synthesize different species of matrix macromolecules, that is: type IV collagen is the major collagen secreted by endothelial cells, whereas pericytes produce predominantly type I collagen; fibronectin and thrombospondin are synthesized by both cell types. Our studies also indicate that PAI-1 is produced, albeit at considerably lower levels, by large vessel vascular cells (aortic endothelial and smooth muscle cells) and human skin fibroblasts. PAI-1 produced by human skin fibroblasts appears to be a distinct molecular species compared to its bovine counterpart as assessed by its slower mobility on SDS/polyacrylamide-gel electrophoresis. The potential significance of elevated PAI-1 production by retinal endothelial cells and pericytes, as well as their distinctive patterns of matrix biosynthesis, is discussed in terms of the involvement of these cells in the maintenance and remodelling of microvessel basement membrane.

SUBMITTER: Canfield AE

PROVIDER: S-EPMC1138540 | biostudies-other | 1989 Apr

REPOSITORIES: biostudies-other

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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: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.

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Plasminogen activator inhibitor-type I is a major biosynthetic product of retinal microvascular endothelial cells and pericytes in culture.

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