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Analysis of differentially expressed secreted proteins in cell culture supernatant of hBMEC in E.coli infection

ABSTRACT: The blood brain barrier (BBB) acts both a physical as well as a metabolic barrier isolating the Central Nervous System (CNS) from systemic circulation, blocking intravascular toxin entry and promoting toxic substance elimination to blood stream. The BBB is formed by four main cellular elements: endothelial cells, astrocyte end-feet, microglial cells, and pericytes. There is strong evidence that astrocytes are able to regulate the phenotypes of endothelial cells by directly contacting or secreting a range of chemical agents, resulting in BBB permeability changes and so on. Conversely, endothelium-derived leukemia inhibitory factor (LIF) has been shown to induce astrocytic differentiation, from studies in normal condition of cell culture. However, in pathological conditions, the specific regulatory mechanism that endothelial cells exert on astrocytes is still less known. In our recently study, a transwell-based, endothelial-astrocyte co-culture model was established to investigate this regulatory relationship in bacterial infection. An extraintestinal pathogenic Escherichia. coli strain was used to infect human brain microvascular endothelial cells (hBMEC) planted in the upper chamber, astrocytes co-cultured in the bottom well were actived without direct contact with bacteria or endothelial cell. The transcription levels of some chemokines and cytokines such as TNF-α and CXCL10 were significantly upregulated. We hypothesized that the secreted proteins from endothelial cells is the main cause of astrocyte activation and the culture supernatant of hBMEC infected by E.coli was obtained to perform the mass spectrometry analysis. In summary, the proteomic identification results showed that 2,692 common proteins were identified in the two groups of control and infected hBMEC, including 140 differentially expressed proteins. The bioinformatics analysis indicated that differentially expressed proteins are mainly classified into structural molecular activity and constituent of ribosome. KEGG pathway enrichment analysis showed that many proteins are involved in focal adhesion as well as a bunch of signaling pathways, PI3K-Akt, Rap1, Ras, MAPK and NF-kappaB signaling pathways, for example. The present study laid an important foundation for the subsequent study of secreted proteins of hBMEC in bacterial infections.

INSTRUMENT(S): Q Exactive HF

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Brain Microvascular Endothelial Cell, Cell Culture

DISEASE(S): Bacterial Meningitis

SUBMITTER: Dong Huo

LAB HEAD: Xiangru Wang

PROVIDER: PXD021427 | Pride | 2023-03-08

REPOSITORIES: Pride

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Project description:The blood-brain barrier (BBB) consists of specific physical barriers, enzymes and transporters, which together maintain the necessary extracellular environment of the central nervous system (CNS). The main physical barrier is found in the CNS endothelial cell, and depends on continuous complexes of tight junctions combined with reduced vesicular transport. Other possible constituents of the BBB include extracellular matrix, astrocytes and pericytes, but the relative contribution of these different components to the BBB remains largely unknown. Here we demonstrate a direct role of pericytes at the BBB in vivo. Using a set of adult viable pericyte-deficient mouse mutants we show that pericyte deficiency increases the permeability of the BBB to water and a range of low-molecular-mass and high-molecular-mass tracers. The increased permeability occurs by endothelial transcytosis, a process that is rapidly arrested by the drug imatinib. Furthermore, we show that pericytes function at the BBB in at least two ways: by regulating BBB-specific gene expression patterns in endothelial cells, and by inducing polarization of astrocyte end-feet surrounding CNS blood vessels. Our results indicate a novel and critical role for pericytes in the integration of endothelial and astrocyte functions at the neurovascular unit, and in the regulation of the BBB. The brain microvascular fragments were isolated from mice with different genotypes, each represented by 3-4 biological replicates. Genotypes 1-2: Platelet derived growth factor-B (PDGF-B) retention-motif knockout (pdgfbret/ret) represent the pericyte-deficient situation, and the heterozygous mice (pdgfbret/+) are used as controls. Genotypes 3-4: Hypomorphic PDGF-B mutants that rescue pdgfb-/- null mice, in which a one copy of a conditionally silent human PDGF-B transgene targeted to the Rosa 26 locus (R26P) is turned on by endothelial-specific expression of Cre recombinase. In this data set these mice are named as Tie2Cre, R26P+/0, pdgfb-/- (representing the pericyte-deficient situation). Mice wt for pdgfb (pdgfb+/+) and carrying one silent copy of R26P (R26P+/0), are used as controls. Genotype 5: Adult Notch3+/+ wildtype (WT).

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Analysis of differentially expressed secreted proteins in cell culture supernatant of hBMEC in E.coli infection

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