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Multicenter Study
. 2020 Jan 1;10(1):231-246.
doi: 10.7150/thno.37142. eCollection 2020.

Soluble CD146, a cerebrospinal fluid marker for neuroinflammation, promotes blood-brain barrier dysfunction

Daji Wang  1 , Hongxia Duan  2 , Jing Feng  2 , Jianquan Xiang  3 , Liqun Feng  4 , Dan Liu  2 , Xuehui Chen  2 , Lin Jing  2 , Zheng Liu  2 , Dexi Zhang  2 , Hongjun Hao  5 , Xiyun Yan  1   2
Affiliations
Multicenter Study

Soluble CD146, a cerebrospinal fluid marker for neuroinflammation, promotes blood-brain barrier dysfunction

Daji Wang et al. Theranostics. .

Abstract

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 αvβ1. Blocking integrin αvβ1 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.

Keywords: blood-brain barrier damage and neuroinflammation.; cerebrospinal fluid; sCD146.

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Conflict of interest statement

Competing Interests: The authors have declared that no competing interest exists.

Figures

Figure 1
Figure 1
CSF sCD146 is elevated in neuroinflammatory diseases. (A-B) sCD146 levels in CSF and serum from patients with NIND (n=217), remitting MS (n=44), IIDD (n=136), CNSI (n=210), PNS (n=216) were assayed using an ELISA sandwich system. *p<0.05; **p<0.01; and ***p<0.001. The data are representative of three independent experiments.
Figure 2
Figure 2
The ROC curves of sCD146 and related molecules to predict the BBB damage.
Figure 3
Figure 3
sCD146 promotes BBB permeability in vitro. (A) Analysis of paracellular barrier function by permeability assay. hCMEC/D3 cells were seeded into the upper chambers of a transwell system, and permeability was measured with 0.5 μg/mL HRP after hCMEC/D3 cells were incubated with 5 μg/mL BSA, 0.05-5 μg/mL rhsCD146 for 2 h. *p<0.05; **p<0.01; and ***p<0.001. (B) Immunofluorescence staining of the TJPs (occludin, ZO-1 and JAM-1) and F-actin after hCMEC/D3 cells were treated with 5 μg/mL BSA or rhsCD146 for 4 h. Bar, 10 μm. (C) hCMEC/D3 cells were preincubated with 5 μg/mL BSA, 0.5 μg/mL or 5 μg/mL rhsCD146, TJP expression levels were verified by western blotting. (D) hCMEC/D3 cells were treated with 5 μg/mL BSA or 0.5, 2 or 5 μg/mL rhsCD146 for 12 h, and apoptosis was detected by flow cytometry with Annexin V and 7-AAD. (E) hCMEC/D3 cells were treated with 5 μg/mL BSA, 0.5 rhsCD146 or 5 μg/mL rhsCD146 for 12 h, and cell lysates were used to detect the expression of caspase 9, caspase 3, Bcl-2 and Bax.
Figure 4
Figure 4
sCD146 interacts with integrin αvβ1. (A) Total RNA of hCMEC/D3 cells was extracted to measure the mRNA expression of integrin subunits by Q-PCR. (B) Whole-cell lysates of hCMEC/D3 cells were collected, and the protein expression levels of the integrin subunits were detected by western blot. (C) Co-IP assays show the association between sCD146 and integrin αvβ1 in hCMEC/D3 cells. Protein levels were analyzed with anti-integrin αv and β1 antibodies. (D-E) Cell adhesion assay. The interaction between sCD146 and integrin αvβ1 was blocked by anti-integrin αv and β1 antibodies. Bar, 100 μm. *p<0.05; **p<0.01; and ***p<0.001.
Figure 5
Figure 5
MAPK, Akt and NF-кB signaling pathways are involved in sCD146-integrin αvβ1 induced hyperpermeability of hCMEC/D3 cells. (A-C) Phosphorylation of p38, ERK1/2, JNK, Akt and NF-кB was induced by treatment with 0.5, 2 or 5 μg/mL rhsCD146 for 10 min in hCMEC/D3 cells. At least three independent assays were performed. (D) MAPK, Akt and NF-кB signaling pathways are involved in sCD146-induced hyperpermeability of hCMEC/D3 cells. hCMEC/D3 cells were preincubated with signaling inhibitors 45 min before treatment with 5 μg/mL rhsCD146. The working concentration of signaling inhibitor of p38 (FHPI), JNK (SP600125), and NF-кB (BAY11-7082) is 10 μM, of ERK1/2 (SCH772984) is 2 μM and of Akt (LY294002) is 5 μM. (E-H) rhsCD146-induced phosphorylation of p38, ERK1/2, JNK, Akt and NF-кB was inhibited by anti-integrin αv and β1 antibodies. hCMEC/D3 cells were preincubated with 3 μg/mL IgG, anti-integrin αv, anti-integrinβ1 or anti-integrin αvβ1 antibodies for 30 min, and then, 5 μg/mL BSA or rhsCD146 was added to the culture medium and incubated for another 10 min. The cell lysates were harvested for western blot analysis.
Figure 6
Figure 6
Blocking integrin αvβ1 attenuates sCD146-induced BBB dysfunction. (A) rhsCD146-induced paracellular permeability of hCMEC/D3 cells was blocked by anti-integrin αv and β1 antibodies. hCMEC/D3 cells were seeded to the upper chambers of a transwell system; preincubated with 3 μg/mL IgG, anti-integrin αv or β1 antibody for 30 min; and then cultured with 5 μg/mL BSA or rhsCD146 for another 2 h. Permeability was measured using 0.5 μg/mL HRP. *p<0.05; **p<0.01; and ***p<0.001. (B) hCMEC/D3 cells were preincubated with 3 μg/mL IgG, anti-integrin αv or β1 antibody for 30 min and then treated with 5 μg/mL BSA or rhsCD146. TJP expression was verified by western blotting. (C) Immunofluorescence staining of the TJPs and F-actin in hCMEC/D3 cells pretreated with 3 μg/mL IgG, anti-integrin αv or β1 antibody for 30 min and 5 μg/mL BSA or rhsCD146 for 4 h. Bar, 10 μm. (D) hCMEC/D3 cells were preincubated with 3 μg/mL IgG, anti-integrin αv or β1 antibody for 30 min and treated with 5 μg/mL BSA or rhsCD146 for another 12 h; apoptosis was detected by flow cytometry with Annexin V and 7-AAD. (E-F) hCMEC/D3 cells were preincubated with 3 μg/mL IgG, anti-integrin αv or β1 antibody for 30 min and treated with 5 μg/mL BSA or rhsCD146 for another 12 h; western blotting was performed to detect the expression of caspase 9, caspase 3, Bcl-2 and Bax.
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