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. 2020 Feb 4;17(1):48.
doi: 10.1186/s12974-020-1727-6.

CLEC14A deficiency exacerbates neuronal loss by increasing blood-brain barrier permeability and inflammation

Yeomyeong Kim  1 , Sungwoon Lee  1 , Haiying Zhang  1 , Sunghye Lee  1 , Hyejeong Kim  1 , Yeaji Kim  1 , Moo-Ho Won  2 , Young-Myeong Kim  3 , Young-Guen Kwon  4
Affiliations

CLEC14A deficiency exacerbates neuronal loss by increasing blood-brain barrier permeability and inflammation

Yeomyeong Kim et al. J Neuroinflammation. .

Abstract

Background: Ischemic stroke is a main cause of mortality. Blood-brain barrier (BBB) breakdown appears to play a critical role in inflammation in patients with ischemic stroke and acceleration of brain injury. The BBB has a protective function and is composed of endothelial cells, pericytes, and astrocytes. In ischemic stroke treatments, regulation of vascular endothelial growth factor (VEGF)-A and vascular endothelial growth factor receptor (VEGFR)-2 is a crucial target despite adverse effects. Our previous study found that loss of C-type lectin family 14 member A (CLEC14A) activated VEGF-A/VEGFR-2 signaling in developmental and tumoral angiogenesis. Here, we evaluate the effects of BBB impairment caused by CLEC14A deficiency in ischemia-reperfusion injury.

Methods: In vitro fluorescein isothiocyanate (FITC)-dextran permeability, transendothelial electrical resistance (TEER) assay, and immunostaining were used to evaluate endothelial integrity. BBB permeability was assessed using Evans blue dye and FITC-dextran injection in Clec14a-/- (CLEC14A-KO) mice and wild-type mice. Middle cerebral artery occlusion surgery and behavioral assessments were performed to evaluate the neurologic damage. The change of tight junctional proteins, adhesion molecules, pro-inflammatory cytokines, and microglial were confirmed by immunofluorescence staining, Western blotting, and quantitative reverse transcription polymerase chain reaction of brain samples.

Results: In endothelial cells, knockdown of CLEC14A increased FITC-dextran permeability and decreased transendothelial electrical resistance; the severity of this effect increased with VEGF treatment. Immunofluorescence staining revealed that tight junctional proteins were attenuated in the CLEC14A knockdown endothelial cells. Consistent with the in vitro results, CLEC14A-KO mice that were injected with Evans blue dye had cerebral vascular leakage at postnatal day 8; wild-type mice had no leakage. We used a middle cerebral artery occlusion model and found that CLEC14A-KO mice had severe infarcted brain and neurological deficits with upregulated VEGFR-2 expression. FITC-dextran leakage was present in CLEC14A-KO mice after ischemia-reperfusion, and the numbers of tight junctional molecules were significantly decreased. Loss of CLEC14A increased the pro-inflammatory response through adhesion molecule expression, and glial cells were activated.

Conclusions: These results suggest that activation of VEGFR-2 in CLEC14A-KO mice aggravates ischemic stroke by exacerbating cerebral vascular leakage and increasing neuronal inflammation after ischemia-reperfusion injury.

Keywords: Blood-brain barrier (BBB); C-type lectin family 14 member A (CLEC14A); Ischemic stroke; Vascular endothelial growth factor (VEGF); Vascular endothelial growth factor receptor-2 (VEGFR-2).

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Conflict of interest statement

The authors declare that they have no competing interests.

Figures

Fig. 1
Fig. 1
CLEC14A knockdown decreased junctional integrity in ECs. a, d HUVEC and HBMEC monolayer permeability to FITC-dextran increased after knockdown of CLEC14A using siRNA(50 nM) and treatment with VEGF (50 ng/ml, 30 min). Absorbance of the solution in the lower chamber was measured after FITC-dextran was added to the transwell. b, e TEER was reduced by VEGF stimulation under the same conditions of the permeability assay before FITC-dextran was added. The TEER was measured using a Millicell ERS-2 (Millipore). c Immunofluorescence staining of VE-cadherin, ZO-1, and DAPI in untreated or VEGF-treated (50 ng/ml, 30 min) HUVECs after knockdown of CLEC14A. Each arrow indicates an attenuated junction. Scale bars: 20 μm. *P < 0.05, **P < 0.01, and ***P < 0.001. The results are mean values and the error bars represent the standard error of the mean values
Fig. 2
Fig. 2
Loss of CLEC14A exacerbated cerebral injury with VEGFR2 activation in MCAO stroke model. a, c Images of whole brains and comparison of Evans blue dye (EB) leakage in brains (P8 and adult) after EB injection (intraperitoneal route) in WT and CLEC14A KO mice (n = 6, each group). b, d The quantitative analysis of Evans blue (EB) dye extravasation using spectrophotometer, 24 h after intraperitoneal injection of EB dye. e TTC staining of brain slices in the sham, WT, and CLEC14A KO groups 24-h post ischemia-reperfusion. Severe infarction was present in the CLEC14A KO group. Scale bar = 1 mm. f Percentage of infarct volume in the WT and CLEC14A KO groups 24 h after ischemia-reperfusion. g Neurological scores of WT and CLEC14A KO mice were evaluated. h VEGFR2, pVEGFR2 protein expression in ipsilateral brain lysate after ischemia-reperfusion injury. i, j Quantification data of relative pVEGFR2 and VEGFR2 protein expression. n = 6 per group; *P < 0.05, **P < 0.01, ***P < 0.001. The results are mean values and the error bars represent the mean ± SD
Fig. 3
Fig. 3
CLEC14A deletion increased cerebral vascular permeability after ischemia-reperfusion. a Confocal microscopic images of the cerebral cortex and subcortex. FITC-dextran (70 kD, 30 mg/ml) was injected in left ventricle 24 h after ischemia-reperfusion. n = 6 per group. Scale bars: 50 μm. b, c Quantitative analysis was performed on values for FITC-dextran mean intensity of cerebral cortex and subcortex in WT and CLEC14A KO mice. **P < 0.01, ***P < 0.001. The results are mean values and the error bars represent the mean ± SD
Fig. 4
Fig. 4
CLEC14A deletion attenuated tight junctional proteins in cerebral ischemic regions. a Immunofluorescence staining for Occludin, Claudin-5, ZO-1, and CD31 with DAPI in the ischemic cortex of WT and CLEC14A KO mice 24 h after ischemia-reperfusion. n = 6 per group. Scale bars: 20 μm. b Quantitative analysis of Occludin/CD31 ratio. c Quantitative analysis of Claudin-5/CD31 ratio. d Quantitative analysis of ZO-1/CD31 ratio. e, f Western blot analysis of tight junctional proteins from ischemic brain and quantitative graph. *P < 0.05, **P < 0.01, and ***P < 0.001. The results are mean values and the error bars represent the mean ± SD
Fig. 5
Fig. 5
Loss of CLEC14A upregulated expression of adhesion molecules and pro-inflammatory cytokines after ischemia-reperfusion. a Representative images of results for ICAM-1 and VCAM-1 immunofluorescence staining in the ischemic region of WT and CLEC14A KO mice. Merged images of ICAM-1 or VCAM-1 with CD31 and DAPI staining are also shown. n = 6 per group. Scale bars: 20 μm. b, c Quantification of the relative ICAM-1 or VCAM-1 levels. d–g Relative mRNA expression level of TNF-α, IL-6, IL-1β, and MCP-1. *P < 0.05, **P < 0.01, and ***P < 0.001. The results are mean values and the error bars represent the mean ± SD
Fig. 6
Fig. 6
CLEC14A KO mice exhibited glial activation after ischemia-reperfusion. a Picture of TTC stained coronal brain section indicating immunofluorescence staining regions. The square fields represent the observed regions of GFAP (red) and Iba1 (green). b Immunofluorescence staining of GFAP (red) and Iba1 (green) in the ischemic cortex of the WT and CLEC14A KO groups after ischemia-reperfusion. Increased expression of GFAP and Iba1 was present in the CLEC14A-KO group, compared with the WT group. n = 6 per group. Scale bars: 100 μm. c, d Glial activation was quantified based on the percentage of the GFAP-positive area and Iba1 positive cells per mm2. *P < 0.05, **P < 0.01, and ***P < 0.001, paired comparisons. The results are mean values and the error bars represent the mean ± SD
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