Fig 2: Astrocytic CEBPD and miR-135a regulate the extension of primary cortical neurons. a CEBPD in astrocytes reduces neuronal neurite length. Conditioned medium from primary Cebpd +/+ or Cebpd -/- astrocytes with or without IL-1ß treatment were subjected to exam Thbs1 by Western blot and added to primary neurons for 48 h. b Attenuated miR-135a in astrocytes restore the neuronal neurite outgrowth. Conditioned medium from primary astrocytes infected with IPTG-inducible A-135a expression system with or without IL-1ß treatment were subjected to exam Thbs1 by Western blot and added to primary neurons for 48 h. Neurons were stained with mouse anti-MAP2 Abs for morphological examination. Scale bar = 100 µm. ImageJ software was used to quantification micrometers of neurite length /neuron. Quantification of relative of neurite length/neuron. (*P < 0.05, **P < 0.01, Student’s t test)

Fig 3: miR-135a suppresses Thbs1/THBS1 transcription through its 3'UTR region. a miR-135a attenuates the expression of Thbs1. Induced miR135a in primary astrocytes with DOX-inducible miR-135a expression system; qRT-PCR and western blot analysis confirmed that miR-135a, Thbs1 mRNAs, and proteins levels, respectively. b Two positions of the Thbs1 3'-untranslated region are predicted to be targets of miR-135a. The seed regions were indicated by the open box (upper panel). Luciferase activity of reporter constructs was measured after co-transfected with pre-miR-135a. c Antisense of miR135a (A-135a) antagonize the effects of Cebpd. Induced A-135a in primary astrocytes with IPTG-inducible A-135a expression system and treated with or without IL-1ß treatment. d Induced miR-135a expression repress the THBS1 expression. In stable U373MG cells with DOX-inducible miR-135a expression system, the expression of miR-135a and the level of THBS1 mRNAs and proteins were examined by qRT-PCR, RT-PCR, and Western blot, respectively. e IL-1ß and CEBPD suppresses THBS1 transcription via miR-135a binding motif. The seed region was indicated by the open box (upper panel). Luciferase activity of reporter constructs was measured after co-transfected with pre-miR-135a, CEBPD expression vectors, or treated IL-1ß. f Antagomir of miR135a (AM135a) dose-dependently antagonized the effects of CEBPD. AM135a or scramble antagomir were transfected into the U373MG stable cells with zinc-inducible CEBPD expression system and then incubated in the presence or absence of 100 µM ZnSO4 for 6 h. RT-PCR and Western blot analysis were performed to examine the expressions of THBS1 mRNA and protein. The data represent the mean ± standard error of three independent experiments, each performed in triplicate. (*P < 0.05, **P < 0.01, Student’s t test)

Fig 4: THBS1 transcription is repressed by CEBPD in astrocytes. a CEBPD contribute to THBS1 mRNA and protein degradation. U373MG cells stably transfected with the zinc-inducible CEBPD expression vector and then incubated in the presence or absence of 100 µM ZnSO4 for 6 h. RT-PCR and Western blot analysis were performed to examine the expressions of THBS1 mRNA and protein. b THBS1 expression is suppressed after IL-1ß treatment. U373MG cells were treated with 5 ng/mL IL-1ß for 3 h and then RT-PCR assay and Western blot analysis were performed to examine the expressions of THBS1 mRNA and protein, respectively. c CEBPD participates in IL-1ß repressed THBS1 transcription. U373MG cells were infected with lentivirus of sh-galactosidase (shLacZ) or shCEBPD (shD1, shD2) and then treated with IL-1ß for 3 h. Total RNA or cell lysates were harvested for RT-PCR or Western blot analysis. d Loss of CEBPD in primary astrocytes reverses IL-1ß-attenuated Thbs1 expression. Primary Cebpd +/+ (WT) and Cebpd -/- (CdKO) astrocytes were treated with 20 ng/mL TNFa, or 5 ng/mL IL-1ß for 3 h, then the RT-PCR and Western blot were performed to examine the expression of Thbs1 mRNA and protein, respectively

Fig 5: Validation of THBS1 impacting mLVs function after SAH. SAH modeling and behavioral tests were performed 4 weeks after the AAV injection. A) Flow cytometric analysis of mLECs percentage in Sham, AAV-control, and AAV-THBS1 group at 72 h post-SAH, n = 4 per group, **p < 0.01, ***p < 0.001 by paired two-tailed Student's t-test. B) Representative images and quantification of beads accumulation in dCLNs of Sham, AAV-control, and AAV-THBS1 group at 72 h post-SAH, each data point represents an average of the 2 dCLNs from one individual mouse, n = 8 per group, **p < 0.01, ***p < 0.001 by paired two-tailed Student's t-test. Scale bar: 200 µm. C) Representative confocal images of mLVs region of Sham, AAV-control, and AAV-THBS1 group at 72 h post-SAH. An enlarged view of TS and SSS is shown on the right in each group. Lyve1 (blue), Beads (green), and CD31 (red). Scale bar: 1000 µm. D) Modified Garcia test, E) time turn, F) time total, and G) wire hanging test at 72 h after SAH revealed AAV-THBS1 delivery aggravated short-term neurological function compared with Sham or AAV-control group, n = 10–12 per group. *p < 0.05; **p < 0.01, ***p < 0.001 by paired two-tailed Student's t-test. H) Flow cytometric analysis of mLECs percentage in Sham, SAH-WT, and THBS1-KO group at 24 h post-SAH, n = 4 per group, **p < 0.01, ***p < 0.001 by paired two-tailed Student's t-test. I) Representative images and quantification of beads accumulation in dCLNs in sham, SAH-WT, and THBS-KO group at 24 h post-SAH, each data point represents an average of the 2 dCLNs from one individual mouse. n = 8 per group, **p < 0.01 by paired two-tailed Student's t-test. Scale bar: 200 µm. J) Representative confocal images of mLVs region of Sham, SAH-WT, and THBS1-KO group at 24 h post-SAH. An enlarged view of TS and SSS is shown on the right in each group. Lyve1 (blue), Beads (green), and CD31 (red). Scale bar: 1000 µm. K) Modified Garcia test, L) time turn, M) time total, and N) wire hanging test at 24 h after SAH revealed THBS1 knockout improved short-term neurological function compared with Sham or WT-SAH group. n = 10–12 per group. *p < 0.05; **p < 0.01, ***p < 0.001 by paired two-tailed Student's t-test.