Figure 9.

In vivo treatment of hypoxia-exposed mice with the CtBP1 inhibitor, MTOB, inhibited lung interstitial/perivascular macrophage activation. (A) Immunofluorescent double staining (green: CtBP1, red: CD68, blue: DAPI, scale bar = 50μm for mice and scale bar = 100μm for bovine or human. “m”-media, “adv”-adventitia, “ni”-neointima) demonstrated that CtBP1 protein is overexpressed in the adventitia of 4-day hypoxia-exposed mice, neonatal calves with chronic hypoxia-induced pulmonary hypertension (PH), and human PH patients, but not in their respective controls. Single arrows showed the cells with positive staining for both CtBP1 and CD68. Double arrows showed cells with positive staining for CtBP1 only. (B) Immunofluorescent staining of mouse lung sections obtained from sea level (SL) control, 4-day hypoxic (4d-Hx), or 4d-Hx mice treated with MTOB (4d-Hx + MTOB) showed that MTOB decreased inflammatory cell recruitment (CD68) and vascular remodeling (TNC). (C) Quantification of CD68 and TNC staining on lung sections from SL, 4d-Hx and 4d-Hx+MTOB mice. **p < 0.01, ****p < 0.0001. (D) Lung interstitial/perivascular macrophages were isolated by flow cytometry from mice exposed to hypoxia (HX-IM) and treated with MTOB or PBS. Experiments were repeated three times. qPCR showed MTOB decreased the gene expression of Arg1, Glut1, Ldha, Pkm2, S100A4, Tnc, and Ctss in interstitial/perivascular macrophages exposed to hypoxia. Data is represented as fold change relative to PBS treated hypoxic mice and is displayed as mean ± sem. *P < 0.05, **P < 0.01.