Fig 2: Increased lipid synthesis is required for hyper-proliferation and survival of human PAH PAVSMC. (A–E) Early passage distal primary human PAVSMC from non-diseased (CTRL) and PAH subjects were serum-deprived for 48 hours and subjected to immunoblot analysis to detect indicated proteins. n = 5 (CTRL), n = 7 (PAH for P-ACLY, ACLY, see Supplementary Figure 1A for additional immunoblots used for statistical analysis) or n = 5 (PAH for ACC, FASN, CPT1A). Data are means ± SE, fold to control. (F,G) Human PAH PAVSMC were incubated for 48 h in serum deprived media supplemented with regular (+ Lipids) or lipid-deprived (-Lipids) 0.1% BSA and subjected to fluorescent BODIPY 493/503 staining (green) to detect intracellular neutral lipids followed by DAPI (blue) staining to detect nuclei. Representative images with enlarged area (F) and statistical analysis (G) are shown. Bar equals 50 μm. Data are means ± SE from n = 5 subjects/group, fold to cells incubated in regular 0.1% BSA (+ Lipids) group. (H) Equal amount of human PAVSMC from five PAH and five control subjects were seeded to 6 well plates. 48 h later (day 0), media was changed to the serum-free media supplemented with regular (+ Lipids) or lipid-deprived (-Lipids) 0.1% BSA; six days later, cell count assay was performed. Data are means ± SE, n = 5 subjects/group. (I) PAH PAVSMC were treated with ACC inhibitor TOFA (20 μM) for 48 h followed by proliferation analysis (Ki-67). Data are means ± SE, percentage of Ki-67 positive cells/total number of cells, n = 5 subjects/group. (J) PAH PAVSMC were treated with ACC inhibitor TOFA (20 μM) for 48 h followed by apoptosis analysis. Data are means ± SE, percentage of TUNEL-positive cells/total number of cells, n = 5 subjects/group. *p < 0.05, **p < 0.01 by Mann–Whitney U test.

Fig 3: Enhancing Cpt1a increased FAO attenuating CSE-induced apoptosis in vitro. (A) Annexin V+ cells were assessed by flow cytometry. (B) FAO was measured by the Seahorse Analyzer. Data are expressed as mean ± standard error of the mean, n=10, **P<0.01 vs. Control; ##P<0.01 vs. pGLVU/GFP-shRNA control; &&P<0.01 vs. pGLVU/GFP control. Cpt1a, Carnitine Palmitoyl Transferase 1A; FAO, Fatty Acid Oxidation; CSE, Cigarette Smoke Extract; COPD, Chronic Obstructive Pulmonary Disease.

Fig 4: A proposed model showing how Cpt1a suppresses vascular endothelial apoptosis and inflammation induced by COPD. Cpt1a, carnitine palmitoyltransferase 1A; COPD, chronic obstructive pulmonary disease; ECs, endothelial cells.

Fig 5: Enhancing Cpt1a attenuating CSE-induced apoptosis and inflammation in vivo. (A) Haematoxylin and eosin-stained sections (200x) showing the lung injury in vivo. (B) Apoptosis were assessed by TUNEL (Bar=100 µm). (C) ELISA demonstrating the levels of TGF-β, IL-6, TNF-α and IL-1β and in lung tissues. Data are expressed as mean ± standard error of the mean, n=10; **P<0.01 vs. Control; ##P<0.01 vs. pGLVU/GFP-shRNA control; &&P<0.01 vs. pGLVU/GFP control. Cpt1a, Carnitine Palmitoyl Transferase 1A; CSE, cigarette smoke extract; COPD, chronic obstructive pulmonary disease.