Fig 1: I-BET protects against apoptosis and alters C15 gene expression in cultured PAECs.(A) Schematic representation of clusters and relevant mediator genes demonstrated rewiring across four PH DDNs (clusters 3, 15, 27, and 35) by all three BET inhibitor drugs represented in CTRP (I-BET151, I-BET762, and JQ-1). Black cells: the cluster to which each mediator belongs. Bar graph on y axis: number of BET inhibitor–associated mediators for each cluster. (B) DDN of cluster 15 representing rewiring associated with the collective actions of all three BET inhibitors; red, drug-sensitive interactions; blue, drug resistant; gray, both. Solid lines, known interactions; dotted lines, new statistically determined dependencies; square boxes, critical mediators. (C to E) In PAECs ± IL-1β exposure, I-BET762 (I-BET), when compared with vehicle control (VC), reversed the IL-1β–induced increases of (C) mitochondrial superoxide (O2−) levels as determined by flow cytometery of MitoSOX Red staining (n = 5 per group), (D) apoptosis as assessed by caspase-3/7 activity (n = 6 per group), and (E) expression of cluster 15 (C15) gene galectin-8 (LGALS8) as determined by quantitative reverse transcription polymerase chain reaction (RT-qPCR) (n = 3 per group). (F to H) By representative immunoblot (F) and densitometry of LGALS8-L (G) and LGALS8-M (H) in PAECs (n = 3 per group), I-BET reversed the IL-1β–induced increase of the L isoform, but not the M isoform, of LGALS8, as compared with VC. ACTNB, b-actin. (I and J) Using immunofluorescence staining (I) and respective quantification, expression of LGALS8 was increased in CD31+ pulmonary arteriolar endothelium (J) of human patients with WSPH group 1 (n = 8) and group 3 (n = 8) PH as compared to non-PH controls (n = 6). Scale bars, 50 μm. Data from (C) to (H) are represented as fold change with respect to Un and plotted as means ± SEM. Statistical significance is indicated using one-way analysis of variance (ANOVA) with Bonferroni’s multiple comparisons testing (*P < 0.05, **P < 0.01, and ***P < 0.001). AU, arbitrary units. DAPI, 4′,6-diamidino-2-phenylindole.
Fig 2: I-BET762 reduces LGALS8, endothelial apoptosis, and improves existing PAH in multiple PAH rat models.(A) Sprague-Dawley rats were administered SU5416 intraperitoneally (i.p.) (20 mg/kg) followed by hypoxia for 21 days to promote PAH. Rats were then treated with I-BET762 versus vehicle control by daily intraperitoneal injection (30 mg/kg) at days 21 to 35 in normoxia (n = 3 to 6 per group). (B to E) By immunofluorescence staining and quantification of LGALS8 (B to E) and cleaved caspase-3 (CC-3) expression (D and E) in pulmonary arterioles, I-BET decreased LGALS8 and apoptotic CC-3, notably in CD31+ endothelium. (F to H) I-BET reduced arteriolar muscularization (F), right ventricular systolic pressure (RVSP) (G), and Fulton index [right ventricle (RV)/left ventricle + septum (LV + S) mass ratio; H]. (I) Sprague-Dawley rats were administered MCT intraperitoneally (60 mg/kg) to promote PAH within 3 weeks. Rats were then treated with I-BET762 versus vehicle control by daily intraperitoneal injection (30 mg/kg) at days 12 to 26 after MCT injection (n = 3 to 6 per group). (J to M) By immunofluorescence staining and quantification of LGALS8 (J and K) and cleaved caspase-3 (CC-3) expression (L and M) in pulmonary arterioles, I-BET decreased LGALS8 and apoptotic CC-3, notably in CD31+ endothelium. (N to P) I-BET reduced arteriolar muscularization (N), RVSP (O), and Fulton index (RV/LV + S; P). Data are plotted as means ± SEM. Scale bars, 50 μm. Statistical significance is indicated using one-way ANOVA with Bonferroni’s multiple comparisons testing (*P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001).
Fig 3: I-BET and genetic deficiency of LGALS8 independently protect against hypoxia-induced PH in mice.(A to I) Wild-type (WT) mice were exposed to 1 or 3 weeks of hypoxia and treated with daily I-BET versus VC. VC-treated normoxic mice were used as comparators (n = 3 to 6 per group). With the exception of Fulton index, I-BET reversed the 3-week hypoxia-dependent increases of these indices: muscularization as indicated by α-SMA+ staining (A and B); RVSP (C); Fulton index (RV/[LV + S] mass ratio) (D); LGALS8 expression (E) in CD31+ endothelial cells (F), whole arterioles (G), or α-SMA+ smooth muscle cells (H); and cleaved caspase-3 (CC-3, I). Consistent with the fact that endothelial apoptosis in PH is more readily observed early in disease (66), the reduction of endothelial CC-3 by I-BET was more prominent at the earlier 1-week hypoxia time point. (J to N) In parallel, as compared with hypoxic WT mice, hypoxic Lgals8−/− mice displayed reductions in vascular cleaved caspase-3 (J and K), muscularization (L), RVSP (M), and Fulton index (N) (n = 8 to 9 Lgals8−/− and 8 to 13 WT; black, male; red, female). (O) Cartoon representing effect of I-BET on Lgals8 expression, controlling downstream STAT signaling pathway, endothelial apoptosis, and PH. Data are plotted as means ± SEM. Statistical significance is indicated using one-way ANOVA with Bonferroni’s multiple comparisons testing for (A) to (I) and Student’s t test for (J) to (N) (*P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001). Scale bars, 50 μm.
Fig 4: LGALS8 is a major effector of C15 controlling endothelial cell apoptosis via STAT1 signaling.(A and B) Interaction of LGALS8 and the α3 subunit of integrin α3β1 was demonstrated by proximity ligation assay (PLA) in PAECs. Positive interaction was depicted by Texas Red signal; blue, DAPI. Controls include deletion of either antibody or both (n = 3 per group). Scale bars, 200 μm. Ab, antibody. (C to E) In PAECs, representative immunoblot (C) and densitometry demonstrated increased STAT1 (D) and pSTAT1 (E) levels with IL-1β exposure (1 hour); these levels were attenuated by knockdown of integrin α3 (siITGα3), integrin β1 (siITGβ1), or both (n = 3 per group). (F to H) By representative immunoblots (F) and densitometry of total STAT1 (G) and phosphorylated STAT1 (pSTAT1, H) in PAECs, knockdown of LGALS8 (siLGALS8) attenuated the IL-1β (1 hour)–induced increase of pSTAT1 (n = 3 per group). (I and J) Similarly, siLGALS8 reduced the IL-1β (48 hours)–dependent increases of mitochondrial O2− as assessed by MitoSOX staining and flow cytometry (I) and apoptosis as assessed by caspase-3/7 activity (J) (n = 6 per group). (K and L) In IL-1β–exposed (48 hours) PAECs treated with I-BET and recombinant galectin-8 (rhGal8; 24 hours), rhGal8 reversed the I-BET–induced attenuation of mitochondrial O2− (K) and caspase 3/7 activity (L) (n = 3 to 6 per group). Data plotted as means ± SEM. Statistical significance is indicated using one-way ANOVA with Bonferroni’s multiple comparisons testing (*P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001).
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