Dataset Information

Comprehensive evaluation of blood-brain barrier-forming micro-vasculatures: Reference and marker genes with cellular composition.

ABSTRACT: Primary brain microvessels (BrMV) maintain the cellular characters and molecular signatures as displayed in vivo, and serve as a vital tool for biomedical research of the blood-brain barrier (BBB) and the development/optimization of brain drug delivery. The variations of relative purities or cellular composition among different BrMV samples may have significant consequences in data interpretation and research outcome, especially for experiments with high-throughput genomics and proteomics technologies. In this study, we aimed to identify suitable reference gene (RG) for accurate normalization of real-time RT-qPCR analysis, and determine the proper marker genes (MG) for relative purity assessment in BrMV samples. Out of five housekeeping genes, ?-actin was selected as the most suitable RG that was validated by quantifying mRNA levels of alpha-L-iduronidase in BrMV isolated from mice with one or two expressing alleles. Four marker genes highly/selectively expressed in BBB-forming capillary endothelial cells were evaluated by RT-qPCR for purity assessment, resulting in Cldn5 and Pecam1 as most suitable MGs that were further confirmed by immunofluorescent analysis of cellular components. Plvap proved to be an indicator gene for the presence of fenestrated vessels in BrMV samples. This study may contribute to the building blocks toward overarching research needs on the blood-brain barrier.

SUBMITTER: Dai M

PROVIDER: S-EPMC5953434 | biostudies-literature | 2018

REPOSITORIES: biostudies-literature

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Comprehensive evaluation of blood-brain barrier-forming micro-vasculatures: Reference and marker genes with cellular composition.

Dai Mei M Lin Yi Y El-Amouri Salim S SS Kohls Mara M Pan Dao D

PloS one 20180515 5

Primary brain microvessels (BrMV) maintain the cellular characters and molecular signatures as displayed in vivo, and serve as a vital tool for biomedical research of the blood-brain barrier (BBB) and the development/optimization of brain drug delivery. The variations of relative purities or cellular composition among different BrMV samples may have significant consequences in data interpretation and research outcome, especially for experiments with high-throughput genomics and proteomics techno ...[more]

PMID: 29763456

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Project description:The blood-brain barrier (BBB) dysfunction is an initial event of various neuroinflammatory diseases. However, the absence of reliable markers and mechanisms for BBB damage greatly limits the diagnosis and treatment of neuroinflammatory diseases. Soluble CD146 (sCD146) is mainly derived from vascular endothelial cells (ECs) and highly elevated in inflammatory settings. Based on a small cohort, our previous study showed that sCD146 is elevated in the cerebrospinal fluid (CSF) of multiple sclerosis (MS), which is accompanied with BBB damage. Nevertheless, whether sCD146 monitors and regulates the BBB dysfunction remains unknown. Methods: Coupled serum and CSF samples from patients with or without neuroinflammatory diseases were collected via multicenter collaborations. sCD146 was measured by sandwich ELISA using anti-CD146 antibodies AA1 and AA98, both of which were generated in our laboratory. The correlations between sCD146 and other clinical parameters or inflammatory factors were analyzed by Spearman's correlation coefficient analysis. The role of sCD146 on BBB function was examined in an in vitro BBB model. Results: Between July 20, 2011, and February 31, 2017, we collected coupled serum and CSF samples from 823 patients, of which 562 (68.3%) had neuroinflammatory diseases, 44 (5.3%) had remitting MS, and 217 (26.4%) had non-inflammatory neurological diseases (NIND). We found that sCD146 in CSF, but not in serum, is abnormally elevated in neuroinflammatory diseases (37.3 ± 13.3 ng/mL) compared with NIND (4.7 ± 2.9 ng/mL) and remitting MS (4.6 ± 3.5 ng/mL). Abnormally elevated CSF sCD146 is significantly correlated with the hyperpermeability-related clinical parameters of BBB and neuroinflammation-related factors. Moreover, CSF sCD146 shows higher sensitivity and specificity for evaluating BBB damage. Using an in vitro BBB model, we found that sCD146 impairs BBB function by promoting BBB permeability via an association with integrin ?v?1. Blocking integrin ?v?1 significantly attenuates sCD146-induced hyperpermeability of the BBB. Conclusion: Our study provides convincing evidence that CSF sCD146 is a sensitive marker of BBB damage and neuroinflammation. Furthermore, sCD146 is actively involved in BBB dysfunction.

Dataset Information

Comprehensive evaluation of blood-brain barrier-forming micro-vasculatures: Reference and marker genes with cellular composition.

Publications

Comprehensive evaluation of blood-brain barrier-forming micro-vasculatures: Reference and marker genes with cellular composition.

Similar Datasets

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