Fig 2: Overexpression of PGC-1a in pulmonary artery endothelial cells ameliorated hypoxia-induced PAH and vascular remodeling in lungs. Nanoparticles containing control vector or PGC1-a overexpression plasmid (OE:PGC-1a) was injected to mice via tail vein. Mice were exposed to normoxia or hypoxia (10% O2) for 4 weeks. A-B, Assessment of mean pulmonary aorta pressure (mPAP). *P < 0.01 vs. Ctrl; #P < 0.01 vs. Vector group; n = 6. C, Ratio of right ventricle to left ventricle + septum weight [RV/(LV + S)]. *P < 0.01 vs. Ctrl; #P < 0.01 vs. Vector group; n = 6. D-E, H &E (D) and elastic van Gieson staining (E) of the pulmonary arteries. Artery wall thickening of pulmonary arteries with different diameters are shown. Scale Bar: 100 µm. F, Quantification of pulmonary arterial muscularization in arteries with diameters 20–70 µm. N = non-muscularized vessels; P = partially muscularized vessels. F = fully muscularized vessels. *P < 0.01 vs. Ctrl; #P < 0.01 vs. the vector group for each vessel type, respectively; n = 6. G, Quantitative assessment of the wall thickness of the pulmonary arteries as described in Methods. *P < 0.01 vs. Ctrl; #P < 0.01 vs. Vector group; n = 6.

Fig 3: A proposed model of a5 protective actions on mitochondrial damage induced by doxorubicin (DOX) in cardiomyocytes. Plus (+) and minus (-) symbol in red colour indicate stimulation or inhibition induced by DOX and, potentially, the a5 target sites. DOX reduces mitochondrial biogenesis and function, (1) binding to topoisomerase IIß (TOP2B), which complexes to the promoters of Ppargc1a and Ppargc1b and blocks transcription of mitochondrial genes, including ROS defense genes, (2) reducing eNOS expression and mTORC1 activity—which are important regulators of mitochondrial physiology, (3) limiting KLF15 gene expression and presumably BCAA oxidation, with a-ketoacid and BCAA accumulation—which are toxic at high levels—beyond reduced TCA cycle intermediates and mitochondrial energy production. Mitochondrial accumulation of DOX is accompanied by increased ROS.

Fig 4: Overexpression of PGC-1a improved PAEC function and inhibits calcium release in smooth muscle cells (SMCs). Nanoparticles containing control vector or PGC1-a expression plasmid (OE:PGC1-a) were injected to mice via tail vein. Mice were exposed to normoxia (Ctrl) or hypoxia (10% O2) for 4 weeks. A-B, Endothelial nitric oxide synthase (eNOS) expression in lung tissues, as detected by Western blotting (A) and normalized to GAPDH. *P < 0.01 vs. Ctrl; #P < 0.01 vs. Vector group; n = 6. C-D, eNOS protein expression. Human PAECs were transfected with empty vector (Ctrl) or PGC-1a expression plasmid and then treated with vehicle or TGF-ß (5 ng/ml) for 3 days. eNOS expression was detected by Western blotting (C) and normalized to GAPDH (D). *P < 0.05 vs. vehicle-treated cells; #P < 0.01 vs. Ctrl vector-transfected cells with TGF-ß; n = 6. E, Nitric oxide (NO) release assay. PAECs were treated with nanoparticles containing empty Vector (Ctrl) or PGC-1a expression plasmid (PGC-1a). One day later, cells were treated with vehicle or TGF-ß for 20 min. NO generated by PAECs was measured using a Nitric Oxide Assay Kit (Abcam, ab65327). The relative NO levels were quantified by normalizing to the vehicle-treated cells, which was set as 1. *P < 0.01 vs. vehicle-treated cells; #P < 0.01 vs. Ctrl vector nanoparticle-treated cells with TGF-ß; n = 6. F-G, Calcium flux assay. PAECs were transfected with empty vector (Ctrl) or PGC-1a expression plasmid and cocultured with SMCs in a transwell plate. PACEs in the upper chamber were treated with vehicle (Ctrl) or TGF-ß (5 ng/ml) for 3 days. Then SMCs in the lower chamber were incubated with Fluo-4 AM at 37 °C and room temperature each for 45 min. Calcium flux signal (green) was detected with a Nikon fluorescence microscope using FITC channel (F) and normalized to control (G). *P < 0.05 vs. vehicle-treated cells; #P < 0.01 vs. Ctrl vector-transfected cells with TGF-ß; n = 6. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

Fig 5: Expression of Akt, CREB, FoxO1, PGC-1, PEPCK, and G-6-Pase in the liver tissues of rats. (A, B, C, D) The mRNA expression of FoxO1, PGC-1a, PEPCK, G-6-Pasea in the HFD + spexin group was significantly decreased as compared with that in the HFD group. (E) The spexin treatment increased the p-FoxO1 Ser256/FoxO1 ratio. (F) The spexin treatment increased the p-Akt /Akt ratio. (G) The intervention of spexin decreased the p-CREB /CREB ratio. (H) The protein expression of p-Akt, Akt, p-CREB, CREB, FoxO1, PGC-1, PEPCK, and G-6-Pase. * P < 0.05, # P > 0.05.