A double-edged effect of hypoxia on astrocyte-derived exosome releases

Abstract

Exosomes are the smallest extracellular vesicles secreted from cells, carrying different cargos, including nucleic acids, proteins and others which transfer from cells to cells. The properties of exosomes depend on the donor cells. Hypoxia, referring to a sublethal and insufficient oxygen supply, reportedly influences exosome secretion of hypoxic cells. In the present study, we focused on the effects of hypoxia on exosomes obtained from CTX-TNA2 astrocyte cells exposed to different durations of hypoxia followed by normoxia as a model of hypoxic preconditioning. To evaluate the functions of exosomes, primary cultured cortical neurons were treated with hemin, a potent neurotoxin. Our sulforhodamine B assay showed that incubation of hemin (30 μM) consistently induced neuronal death. Co-incubation of exosomes from CTX-TNA2 cells subjected to 2 hr-hypoxia plus 6 hr-renormoxia (2H/6R exosomes), but not 12 hr-hypoxia plus 24 hr-renormoxia (12H/24R exosomes), attenuated hemin-induced cell death and reduction in growth associated protein 43 level (a biomarker of neurite outgrowth). Western blot assay demonstrated that 2H/6R exosomes attenuated hemin-induced elevations in inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) levels (two proinflammatory biomarkers) as well as heme oxygenase-1 (HO-1). In contrast, 12H/24R exosomes did not alter hemin-induced elevation in HO-1 but further augmented hemin-induced increases in iNOS and COX-2. Moreover, 2H/6R exosomes attenuated hemin-induced reduction in glutathione hydroperoxidase 4 (a biomarker of ferroptosis) and elevation in active caspase 3 (a biomarker of apoptosis) while 12H/24R exosomes did not effectively alter hemin-induced programed cell death. In conclusion, our study showed that 2H/6R exosomes possessed neuroprotective activities while 12H/24R exosomes had mild pro-inflammatory activities, suggesting that different hypoxic preconditionings influenced CTX-TNA2 cells which then secreted exosomes with differential biological activities. These findings highlight a double-edged role of hypoxia on exosome functions.

Keywords: CTX-TNA2; double-edged role; exosomes; hemin; hypoxic preconditioning.

FIGURE 1

Characterization of exosomes secreted from CTX-TNA2 cells subjected to different hypoxic preconditionings. (A) CTX-TNA2 cells were treated with hypoxia for 3, 6, and 12 h. Cell viability was measured using SRB assay. Values are the mean ± S.E.M. (n = 3/each group). No statistical signifijcance was observed using one-way ANOVA followed by the LSD test as post hoc method. (B) The diagram illustrated two different hypoxic preconditioings of CTX-TNA2 cells for exosomes collection. One was 2H/6R exosomes which were obtained from CTX-TNA2 cells exposed to 2-h hypoxia followed by 6-h normoxia. The other was 12H/24R exosomes which were obtained from CTX-TNA2 cells exposed to 12-h hypoxia followed by 24-h normoxia (C,D) 2H/6R exosomes, 12H/24R exosomes and control exosomes obtained from 1 × 10⁶ CTX-TNA2 cells were used. Western blot assay was employed to measure exosomal markers, including Alix and CD63 (C). TEM images of control exosomes, 2H/6R exosomes and 12H/24R exosomes were performed (D). Representative exosomes were indicated by arrows in each images. Scale bar = 200 nm.

FIGURE 2

Differential effects of 2H/6R exosomes and 12H/24R exosomes on hemin neurotoxicity in primary cultured cortical neurons. (A) Primary cultured cortical neurons were treated with hemin (30 μM) plus 2H/6R exosomes obtained from 1 × 10⁶ CTX-TNA2 cells (as 1-fold) and 12H/24R exosomes (1 fold, 3 folds) for 16 h. Cell death was measured by SRB assay. (B,C) Primary cultured cortical neurons were treated with hemin (30 μM) plus 2H/6R exosomes (1-fold) (B) and 12H/24R exosomes (1 fold and 3 folds) for 16 h. Western blot assay was employed to measure GAP43. Each lane contained 30 μg protein for all experiments. Graphs show statistic results from relative optical density of bands on the blots. Values are the mean ± S.E.M. (n = 3/each group). *, p < 0.05 statistically significant in the hemin groups compared with the control groups; #, P < 0.05 in hemin plus exosomes compared with hemin alone by one-way ANOVA followed by the LSD test as post hoc method.

FIGURE 3

Differential effects of 2H/6R exosomes and 12H/24R exosomes on hemin-induced impairment of neurite outgrowth in primary cultured cortical neurons. Primary cultured cortical neurons were treated with hemin (30 μM) plus 2H/6R exosomes obtained from 1 × 10⁶ CTX-TNA2 cells for 1 h. The cells were immunostained with Neu N, PHK26 and DAPI. Calibration: 10 μm. The results were duplicated.

FIGURE 4

Differential effects of 2H/6R exosomes and 12H/24R exosomes on hemin-induced neuroinflammation in primary cultured cortical neurons. (A,C) Primary cultured cortical neurons were treated with hemin (30 μM) plus 2H/6R exosomes obtained from 1 × 10⁶ CTX-TNA2 cells (as 1 fold) for 16 h (B,D) Primary cultured cortical neurons were treated with hemin (30 μM) plus 12H/24R exosomes (1 fold and 3 folds) for 16 h. Western blot assay was employed to measure iNOS (A,B) and COX-2 (C,D). Each lane contained 30 μg protein for all experiments. Graphs show statistic results from relative optical density of bands on the blots. Values are the mean ± S.E.M. (n = 3/each group). *, p < 0.05 statistically significant in the hemin groups compared with the control groups; #, P < 0.05 in hemin plus exosomes compared with hemin alone by one-way ANOVA followed by the LSD test as post hoc method.

FIGURE 5

Differential effects of 2H/6R exosomes and 12H/24R exosomes on hemin-induced HO-1 expression in primary cultured cortical neurons. (A) Primary cultured cortical neurons were treated with hemin (30 μM) plus 2H/6R exosomes obtained from 1 × 10⁶ CTX-TNA2 cells (as 1-fold) for 16 h. (B) Primary cultured cortical neurons were treated with hemin (30 μM) plus 12H/24R exosomes (1 fold and 3 folds) for 16 h. Western blot assay was employed to measure HO-1. Each lane contained 30 μg protein for all experiments. Graphs show statistic results from relative optical density of bands on the blots. Values are the mean ± S.E.M. (n = 3/each group). *, p < 0.05 statistically significant in the hemin groups compared with the control groups; #, P < 0.05 in hemin plus exosomes compared with hemin alone by one-way ANOVA followed by the LSD test as post hoc method.

FIGURE 6

Differential effects of 2H/6R exosomes and 12H/24R exosomes on hemin-induced programmed cell death in primary cultured cortical neurons. (A,C) Primary cultured cortical neurons were treated with hemin (30 μM) plus 2H/6R exosomes obtained from 1 × 10⁶ CTX-TNA2 cells (as 1 fold) for 16 h (B,D) Primary cultured cortical neurons were treated with hemin (30 μM) plus 12H/24R exosomes (1 fold and 3 folds) for 16 h. Western blot assay was employed to measure GPX4 (A,B) and active-caspase 3 (C,D). Each lane contained 30 μg protein for all experiments. Graphs show statistic results from relative optical density of bands on the blots. Values are the mean ± S.E.M. (n = 3/each group). *, p < 0.05 statistically significant in the hemin groups compared with the control groups; #, P < 0.05 in hemin plus exosomes compared with hemin alone by one-way ANOVA followed by the LSD test as post hoc method.

FIGURE 7

Cargos in control exosomes, 2H/6R and 12H/24R exosomes by miRNA sequencing analysis. Sequencing was performed with single-end reads (100 bp) at a depth exceeding 10 million reads per sample. miRNA expression levels were analyzed on the Qiagen GeneGlobe platform with UMI-based correction.