Fig 2: NEO1 is decreased in the SAH cortex and resulted in EC proliferation increasing and EC apoptosis reducing.A Total RNA of the cortex was extracted and the expression of NEO1 was quantitated by RT-qPCR (n = 6 mice/group). The expression level of NEO1 was normalized against β-actin. B, C Representative immunoblotting (B) and quantification (C) of NEO1 of cortex from mice subjected to SAH for different time points and sham surgery (n = 6 mice/group). β-actin was used as a loading control. D, E Representative images of coimmunostained NEO1 and Neun (D) or GFAP (E) in sham and SAH 7 days cortex (n = 10 mice/group). F, G Quantification of NEO1 in neuron (F) and astrocytes (G). H–J MBMEC cocultured with Hb-treated neurons (n = 6 experiments with Hb-treated neurons and its control group neurons). K–M Reduced proliferation in MBMEC cocultured with Hb-treated astrocytes (n = 6 experiments with Hb-treated astrocytes and its control group astrocytes). N Representative immunoblotting of cleaved-caspase3 from MBMEC cocultured with CM of NEO1 knockdown astrocyte. O Quantitative analysis of data in (G). β-actin was used as a loading control (n = 6 experiments with NEO1 knockdown astrocytes and its control group astrocytes). P–R MBMEC cocultured with NEO1 knockdown neurons (n = 6 experiments with NEO1 knockdown neurons and its control group neurons). S–U MBMEC cocultured with NEO1 knockdown astrocytes (n = 6 experiments with NEO1 knockdown astrocytes and its control group astrocytes). Scale bars: 20 μm. Data are presented as mean ± SD. *P < 0.05; **P < 0.01, ***P < 0.01. 1-way ANOVA with Tukey’s correction for multiple comparisons (A, C). Student’s t-test.

Fig 3: Hepcidin alleviation of EC iron storage decreases, proliferation, migration, and leakage induced by CM of astrocytes from NEO1GFAP-Cre.A–C Representative immunoblotting (A) and quantification (B, C) of hepcidin effect on FTH and FTL of MBMEC in the presence of CM of astrocytes. Data are presented as mean ± SD (n = 6 experiments/group). D–F Hepcidin (800 nM) inhibition of MBMEC proliferation. D Schematic of hepcidin administration in MBMEC cultures in the presence of CM of NEO1 KO astrocytes. EDU incubated for 4 h. E Representative images of EDU + MBMEC. F Quantitative analysis of data in (E). Data are presented as mean ± SD (n = 6 experiments/group). G–I Hepcidin inhibition of MBMEC migration. G, H Schematic of hepcidin administration in MBMEC cultures in the presence of CM of NEO1 KO astrocytes to access migration by wound-healing assay. I Representative images of MBMEC migration 24 h after wound scratching. J Quantification analysis of data in (H). Data are presented as mean ± SD (n = 6 experiments/group). K Representative images of caveolae of MBMEC. L Hepcidin inhibition of permeability of MBMEC monolayers. Data are presented as mean ± SD (n = 3 experiments/group). *P < 0.05; **P < 0.01; ns not significant. 1-way ANOVA with Tukey’s correction.

Fig 4: Increased EC migration in HUVEC cultures exposed to CM of NEO1 KO astrocytes and hepcidin expression needs NEO1.A–C Increased EC proliferation in HUVEC cocultured with CM of NEO1-KO astrocytes. EDU incubated for 4 h (n = 6 experiments of astrocytes from NEO1 GFAP-Cre and its control group). D–F Increased EC migration in HUVEC cocultured with CM of NEO1-KO astrocytes (n = 6 experiments of astrocytes from NEO1GFAP-Cre and its control group). G The permeability of HUVEC monolayers (n = 3 experiments of astrocytes from NEO1GFAP-Cre and its control group). H–J Transwell assay to access HUVECs migration. K–Q Representative immunoblotting (K–M) and quantification (L, M, O, and Q) of FTH, FTL, FPN1, and hepcidin of cortex endothelial cells from mice subjected to SAH for different time points and sham surgery (n = 6 mice/group). PECAM-1 was used as a loading control. R The Quantification analysis of hepcidin in CM of primary astrocytes and primary neurons by ELISA assay (n = 6 experiments/group). S, T FISH analysis of hepcidin mRNA in primary astrocytes and primary neurons (n = 6 experiments/group). Scale bars: 20 μm. U Quantification analysis of hepcidin in CM of astrocytes from NEO1GFAP-Cre and its control group by ELISA assay (n = 6 experiments/group). V, W Representative immunoblotting (V) and quantification (W) of hepcidin in astrocytes from NEO1GFAP-Cre and its control group (n = 6 experiments of astrocytes from NEO1GFAP-Cre and its control group). Data are presented as mean ± SD. *P < 0.05; **P < 0.01, ***P < 0.01. 1-way ANOVA with Tukey’s correction for multiple comparisons (B, C, E, and G). Student’s t-test.