Atorvastatin combined with low-dose dexamethasone for vascular endothelial cell dysfunction induced by chronic subdural hematoma

Abstract

Atorvastatin has been shown to be a safe and effective non-surgical treatment option for patients with chronic subdural hematoma. However, treatment with atorvastatin is not effective in some patients, who must undergo further surgical treatment. Dexamethasone has anti-inflammatory and immunomodulatory effects, and low dosages are safe and effective for the treatment of many diseases, such as ankylosing spondylitis and community-acquired pneumonia. However, the effects of atorvastatin and low-dose dexamethasone for the treatment of chronic subdural hematoma remain poorly understood. Hematoma samples of patients with chronic subdural hematoma admitted to the General Hospital of Tianjin Medical University of China were collected and diluted in endothelial cell medium at 1:1 as the hematoma group. Atorvastatin, dexamethasone, or their combination was added to the culture medium. The main results were as follows: hopping probe ion conductance microscopy and permeability detection revealed that the best dosages to improve endothelial cell permeability were 0.1 μM atorvastatin and 0.1 μM dexamethasone. Atorvastatin, dexamethasone, or their combination could markedly improve the recovery of injured endothelial cells. Mice subcutaneously injected with diluted hematoma solution and then treated with atorvastatin, dexamethasone, or their combination exhibited varying levels of rescue of endothelial cell function. Hopping probe ion conductance microscopy, western blot assay, and polymerase chain reaction to evaluate the status of human cerebral endothelial cell status and expression level of tight junction protein indicated that atorvastatin, dexamethasone, or their combination could reduce subcutaneous vascular leakage caused by hematoma fluid. Moreover, the curative effect of the combined treatment was significantly better than that of either single treatment. Expression of Krüppel-like factor 2 protein in human cerebral endothelial cells was significantly increased, as was expression of the tight junction protein and vascular permeability marker vascular endothelial cadherin in each treatment group compared with the hematoma stimulation group. Hematoma fluid in patients with chronic subdural hematoma may damage vascular endothelial cells. However, atorvastatin combined with low-dose dexamethasone could rescue endothelial cell dysfunction by increasing the expression of tight junction proteins after hematoma injury. The effect of combining atorvastatin with low-dose dexamethasone was better than that of atorvastatin alone. Increased expression of Krüppel-like factor 2 may play an important role in the treatment of chronic subdural hematoma. The animal protocols were approved by the Animal Care and Use Committee of Tianjin Medical University of China on July 31, 2016 (approval No. IRB2016-YX-036). The study regarding human hematoma samples was approved by the Ethics Committee of Tianjin Medical University of China on July 31, 2018 (approval No. IRB2018-088-01).

Keywords: brain; brain trauma; cells; central nervous system; inflammation; plasticity; protein; repair.

Figure 1

The optimum time required for induction of cell damage by hematoma fluid, and optimum drug dosage for treatment of cells after hematoma injury. (A) Hopping probe ion conductance microscopy was employed to scan morphological changes of the human cerebral endothelial cell line (hCMEC/D3) following hematoma injury. White arrows indicate intercellular spaces. (B) Relative concentrations of FITC-dextran that passed through hCMEC/D3 monolayers in PBS or atorvastatin treatment groups after hematoma injury. (C) Relative concentrations of FITC-dextran that passed through hCMEC/D3 monolayers in the PBS or dexamethasone treatment groups after hematoma injury. Data are expressed as the mean ± SD (one-way analysis of variance followed by the least significant difference post hoc test). *P < 0.05, vs. hematoma group. (B, C) Independent experiments were conducted twice. FITC: Fluorescein isothiocyanate; PBS: phosphate-buffered saline.

Figure 2

Atorvastatin and low-dose dexamethasone treatment maintains the stability of human cerebral endothelial cell line (hCMEC/D3) following injury. (A) Hopping probe ion conductance microscopy analysis of changes in hCMEC/D3 cells with atorvastatin, dexamethasone, or a combination of atorvastatin and dexamethasone following hematoma-induced injury at various scan times. (B) Changes in cell height. (C) Percentage of altered cells after treatments. Data are expressed as the mean ± SD (n= 3; B, two-way analysis of variance followed by Tukey’s post hoc test; C, one-way analysis of variance followed by the least significant difference post hoc test). **P < 0.01, ***P < 0.001, vs. hematoma (+) atorvastatin (–) dexamethasone (–) group; ##P < 0.01, vs. hematoma (+) atorvastatin (+) dexamethasone (–) group; ††P < 0.01, vs. hematoma (+) atorvastatin (–) dexamethasone (+) group. Experiments were conducted twice.

Figure 3

Atorvastatin and low-dose dexamethasone treatment increases expression of vascular endothelial cadherin (VE-cadherin) and other tight junction proteins, which rescue vascular permeability after hematoma-induced injury. (A) Immunofluorescence photomicrographs of VE-cadherin (red) expressed in the human cerebral endothelial cell line (hCMEC/D3) after treatment. Scale bar: 50 μm. (B) Western blot assay of VE-cadherin in hCMEC/D3 cells after treatment. (C–E) Relative expression of VE-cadherin, claudin-5, and zonula occludens-1 in hCMEC/D3 cells treated with PBS, atorvastatin, dexamethasone, or a combination of atorvastatin and dexamethasone after hematoma injury, as measured by quantitative real-time polymerase chain reaction. Experiments were independently performed in triplicate. (F) Relative concentrations of FITC-dextran that passed through hCMEC/D3 cell monolayers treated with PBS, atorvastatin, dexamethasone, or a combination of atorvastatin and dexamethasone after hematoma injury. Data are expressed as the mean ± SD (n = 3; one-way analysis of variance followed by the least significant difference post hoc test). *P < 0.05; **P < 0.01, vs. hematoma (+) atorvastatin (–) dexamethasone (–) group. #P < 0.05, vs. hematoma (+) atorvastatin (+) dexamethasone (–) group; †P < 0.05, vs. hematoma (+) atorvastatin (–) dexamethasone (+) group. Experiments were conducted twice. FITC: Fluorescein isothiocyanate; PBS: phosphate-buffered saline; VE-cadherin: vascular endothelial cadherin.

Figure 4

KLF2 expression is significantly increased in the combination treatment group after hematoma injury. (A) Heat map representation of gene sequencing data for altered expression levels of the top 50 genes in cell lines subjected to combination treatment after hematoma injury (red indicates high expression, green indicates low expression). (B) Fold change of KLF2 gene expression in different treatment groups (qRT-PCR), compared with the hematoma stimulation group. (C) qRT-PCR analysis of KLF2 mRNA expression in different treatment groups. (D) Western blot assay of changes in KLF2 expression in various treatment groups, compared with the hematoma stimulation group. Data are expressed as the mean ± SD (n = 3; one-way analysis of variance followed by the least significant difference post hoc test). *P < 0.05, **P < 0.01, vs. hematoma (+) atorvastatin (–) dexamethasone (–) group; #P < 0.05, vs. hematoma (+) atorvastatin (+) dexamethasone (–) group. KLF2: Krüppel-like factor 2; qRT-PCR: quantitative real-time polymerase chain reaction.

Figure 5

Atorvastatin and low-dose dexamethasone treatment reduces high permeability in vivo. (A) Evans blue images of hematoma-induced leakage into the skin with PBS, atorvastatin, dexamethasone, or a combination of atorvastatin and dexamethasone. (B) Quantitative analysis of dye extracted from sham-, hematoma-, atorvastatin-, dexamethasone-, or combination-treated skin samples retrieved from experimental animals. Data are expressed as the mean ± SD (n = 10; one-way analysis of variance followed by the least significant difference post hoc test). *P < 0.05; **P < 0.01, vs. hematoma (+) atorvastatin (–) dexamethasone (–) group. ##P < 0.01, vs. hematoma (+) atorvastatin (+) dexamethasone (–) group. ††P < 0.01, vs. hematoma (+) atorvastatin (–) dexamethasone (+) group. PBS: Phosphate-buffered saline.