Hypoxia-Induced Inflammation in In Vitro Model of Human Blood-Brain Barrier: Modulatory Effects of the Olfactory Ensheathing Cell-Conditioned Medium

Abstract

Hypoxia compromises the integrity of the blood-brain barrier (BBB) and increases its permeability, thereby inducing inflammation. Olfactory ensheathing cells (OECs) garnered considerable interest due to their neuroregenerative and anti-inflammatory properties. Here, we aimed to investigate the potential modulatory effects of OEC-conditioned medium (OEC-CM) on the response of human brain microvascular endothelial cells (HBMECs), constituting the BBB, when exposed to hypoxia. HBMECs were utilized to establish the in vitro BBB model. OECs were isolated from mouse olfactory bulbs, and OEC-CM was collected after 48 h of culture. The effect of OEC-CM treatment on the HBMEC viability was evaluated under both normoxic and hypoxic conditions at 6 h, 24 h, and 30 h. Western blot and immunostaining techniques were employed to assess NF-κB/phospho-NF-κB expression. HIF-1α, VEGF-A, and cPLA₂ mRNA expression levels were quantified using digital PCR. ELISA assays were performed to measure PGE2, VEGF-A, IL-8 secretion, and cPLA₂ specific activity. The in vitro formation of HBMEC capillary-like structures was examined using a three-dimensional matrix system. OEC-CM attenuated pro-inflammatory responses and mitigated the HIF-1α/VEGFA signaling pathway activation in HBMECs under hypoxic condition. Hypoxia-induced damage of the BBB can be mitigated by novel therapeutic strategies harnessing OEC potential.

Keywords: Blood–brain barrier; Human brain microvascular endothelial cells; Hypoxia; Inflammation; Olfactory ensheathing cells.

Fig. 1

Effect of olfactory ensheathing cell conditioned medium (OEC-CM) on HBMEC viability under normoxic (21% O₂, 5% CO₂, and 74% N₂) or hypoxic conditions (HYP) (1% O₂, 5% CO₂, and 94% N₂). The CCK-8 assay was employed to assess the proliferation and viability of HBMECs at different time points (6 h, 24 h, and 30 h) under normoxic and hypoxic conditions. Bars represent means ± SD of three independent experiments, each performed in triplicate (n = 3). Statistically significant differences, determined by one-way ANOVA, are indicated: *p ≤ 0.05 vs CTRL; § p ≤ 0.05 vs HYP + OEC-CM

Fig. 2

Evaluation of the absolute levels of NF-κB and p-NF-κB expression in HBMECs. Western blot analysis was conducted on samples cultured either in basal culture medium or treated with OEC-CM under normoxic and hypoxic (HYP) conditions for 6 h and 30 h. The analyses were performed on the relevant lysates using specific antibodies against NF-κB and p-NF-κB. Densitometric analysis was carried out using ImageJ software. Bars in the graph represent means ± SD of three independent experiments, each performed in triplicate (n = 3). Statistically significant differences, determined by one-way ANOVA followed by Tukey’s multiple comparisons test, are indicated: *p ≤ 0.05 vs respective CTRL at 6 h and 30 h; # p ≤ 0.05 vs respective HYP at 6 h and 30 h; ǂ p ≤ 0.05 vs respective OEC-CM at 6 h and 30 h; § p ≤ 0.05 30 h vs 6 h

Fig. 3

NF-κB and p-NF-κB immunostaining of HBMECs cultured in basal culture medium or treated with OEC-CM under normoxic and hypoxic (HYP) conditions for 6 h and 30 h. Scale bar: 20 µm. Fluorescence intensity quantification was carried out using ImageJ software. Bars in the graph represent means ± SD of three independent experiments, each performed in triplicate (n = 3). Statistically significant differences, determined by one-way ANOVA followed by Tukey’s multiple comparisons test are indicated: * p ≤ 0.05 vs respective CTRL at 6 h and 30 h; # p ≤ 0.05 vs respective HYP at 6 h and 30 h; § p ≤ 0.05 30 h vs 6 h

Fig. 4

Comparison of a HIF-1α, b VEGF-A, and c cPLA₂ mRNA levels in the presence of OEC-CM under normoxic or 6-h hypoxic condition in HBMECs. Data were normalized to the GAPDH expression level. All data points are means ± SD (n = 3). Statistically significant differences, determined by two-way ANOVA followed by Šídák’s multiple comparisons test, are indicated: ****p < 0.0001

Fig. 5

PGE2 (a), VEGF-A (b) and IL-8 (c) levels in media from HBMECs cultured in basal culture medium (CTRL) or treated with OEC-CM under normoxic and hypoxic (HYP) conditions for 6h and 30h. All data points are means ± S.E.M. from three independent experiments (n = 3). Statistically significant differences, determined by one-way ANOVA followed by Dunnett’s multiple comparison test, are indicated as follows: * p < 0.05 vs respective 6h or 30h CTRL; § p < 0.05 vs respective 6h or 30h HYP

Fig. 6

Calcium-dependent cytosolic PLA₂ specific activity in HBMEC lysates from 6 and 30 h normoxic and hypoxic HBMECs, with or without OEC-CM treatment. All data points are means ± S.E.M. from three independent experiments (n = 3). Statistically significant differences, determined by one-way ANOVA, followed by Dunnett’s multiple comparison test, are indicated as follows: * p < 0.05 vs respective 6 h or 30 h CTRL; § p < 0.05 vs respective 6 h or 30 h HYP

Fig. 7

Evaluation of the angiogenic potential of HBMECs in the presence of OEC-CM under normoxic or hypoxic conditions. a Representative microphotographs showing three-dimensional cultures in Matrigel of each sample. Magnification: 40 × . Scale bar: 500 μm. The quantification of main parameters describing the capillary network formation after 6 h: b total master segments length; c number of master segments; d total branches length; e number of branches. Values are expressed as a mean ± SEM of three independent experiments (n = 3). * p < 0.05 vs. CTRL; # p < 0.05 vs. HYP. One-way ANOVA, followed by Tukey’s multiple comparisons test