Fig 2: Intraventricular delivery of recombinant STC2 protein in rodents.a Schematic of the brain slice view (left) and top view (right) in the skull for STC2 protein delivery via mini-osmotic pump. The cannula of the osmotic pump is implanted at 0.8 mm posterior (Y) and 1.5 mm contralateral (X) to the bregma at a depth of 3.5 mm at 1-week after stroke. b Behavioral testing. Vibrissae-forepaw (WP) behavioral testing (left) and overall neurological score (right). Based on WP, STC2 group exhibited statistically significantly greater recovery beginning at 4 weeks post-stroke compared to other groups. c Endogenous neuroblasts. Representative images of DCX + /BrdU+ cells (left) and the cell count of number co-positive cells (right) at peri-infarct region. b Analyzed using a log-transform two-way repeated measures ANOVA which revealed a statistically significant interaction between the effects of treatment group and WP Score (F [12, 162] = 7.058, P < 0.0001); followed by Dunnett’s multiple comparisons test. Neurological scores (NS) were analyzed using a Kruskal–Wallis test (P = 0.018), followed by Dunn’s multiple comparisons test. For both, *P < 0.05, **P < 0.01, data shown as mean ± SEM, n = 10 per group. c Analyzed using one-way ANOVA, followed by Tukey’s HSD post-hoc test with *P < 0.05. Data shown as mean ± SEM, n = 20 images/4 rats.

Fig 3: Transcriptome changes between electrically stimulated and unstimulated NPCs.a Heatmap of transcriptome changes in NPCs from electrical stimulation (NPC – unstimulated, NPCStim – electrically stimulated; blue-downregulated, orange-upregulated). b Volcano plot demonstrating changes in genes with electrical stimulation (blue-downregulated, orange-upregulated). c Top Gene set enrichment pathways with STC2 as the leading edge. d qRT-PCR analysis indicated that STC2 in NPCs was upregulated by electrical stimulation. e ELISA study indicated that the level of STC2 protein after electrical stimulation (NPCStim) is much higher than that in non-stimulated NPCs (NPC). d, e Analyzed using a one-way ANOVA, followed by Tukey’s HSD post-hoc test with **P < 0.01, ****P < 0.0001, data shown as mean ± SEM, n = 4.

Fig 4: Histologic Localization of Selected Genes in PEA Tissue Microarrays(A) Flowchart showing consecutive steps to histologically localize selected genes to specific areas in CTEPH PEA specimens. Laser microdissection and nCounter analysis of mRNA expression levels of (B) TGFBI, (C) FSTL3, (D) STC2, and (E) TAGLN in PEA specimens isolated from 4 CTEPH patients. Quantitative data are presented as mean ± SEM, and P values were determined by comparing findings in vessels area with all other areas (3 comparisons) by means of 1-way ANOVA followed by Bonferroni’s multiple comparisons test for normally distributed values (B, D, E). Values that did not pass the normality test are presented as median with IQR, and P values were determined by means of Kruskal-Wallis test (C). ∗P < 0.05; ∗∗∗P < 0.001; ns = nonsignificant. (F) Representative images after immunohistochemical staining of TGFBI, FSTL3, STC2, and TAGLN on tissue microarrays from patients with CTEPH (PEA material: TMA1 and TMA2), pulmonary arterial hypertension (PAH; lung biopsies) or idiopathic pulmonary fibrosis (IPF; lung biopsies). Insets show higher magnification of selected areas of interest. Scale bars represent 100 μm; scale bars in insets represent 10 μm. Semiquantitative analysis of (G) TGFBI and (H) STC2 expression in tissue. Quantitative data are presented as mean ± SEM, and P values were determined by comparing the mean of each group with the mean of every other group (3 comparisons) by means of 1-way ANOVA followed by Bonferroni’s multiple comparisons test. ∗∗∗P < 0.001; ns = nonsignificant. (I) Representative confocal images of (left) CD31-positive cells (green) expressing TGFBI (red) and (right) STC2 (red) on cryopreserved CTEPH PEA tissue. Scale bars represent 50 μm. Abbreviations as in Figure 1.

Fig 5: Plasma Levels of TGFBI, STC2, FSTL3, and TAGLN in Patients With CTEPH, Patients With PAH, and Healthy Control Subjects(A) Flowchart showing steps of blood analyses in patients with CTEPH enrolled in the CTEPH Registry Bad Nauheim, before and at 12-month follow-up after PEA and in patients with PAH enrolled in the Pulmonary Hypertension Registry Mainz. Results of the quantitative analysis using specific enzyme-linked immunosorbent assay (ELISA) of plasma levels of (B) TGFBI, (C) STC2, (D) FSTL3, and (E) TAGLN in patients with CTEPH (n = 27) compared with patients with PAH (n = 23) or healthy control individuals (n = 6). Quantitative data are presented as violin plot (showing all points). P values were determined by comparing the mean of each group with the mean of every other group (3 comparisons) by means of 1-way ANOVA followed by Bonferroni’s multiple comparisons test for normally distributed values (E) or Kruskal-Wallis test followed by Dunn’s multiple comparisons test for values which did not pass the normality test (B, C, D). ∗P < 0.05; ∗∗P < 0.01; ∗∗∗P < 0.001; ns = nonsignificant. Results of the quantitative analysis using ELISA of plasma levels of (F) TGFBI (n = 18), (G) FSTL3 (n = 10), and (H) TAGLN (n = 10) in patients with CTEPH, before (CTEPH pre) and at 12-month follow-up after PEA surgery (CTEPH post). Quantitative data are presented as symbols and lines. P values were determined with the use of paired Student’s t-test (F) or Wilcoxon matched-pairs signed rank test (G, H). ∗∗∗P < 0.001; ns = nonsignificant. Data points in blue are for male patients, in green for female.