Fig 1.
Generation of a mouse model of hypoxia-induced PH.
(A) Scheme for generating the mouse model of hypoxia-induced PH. Eight-week-old mice were housed in a hypoxic chamber (8% O2) for 4 weeks before experiments were performed. (B) Right ventricular systolic pressure (RVSP) in mice exposed to normoxia (Con) or hypoxia (PH) (n = 9, 8). (C, D) Right ventricular wall thickness (RVWT) (n = 8, 10) (C) and right ventricular diameter (RVD) (n = 8, 10) (D) in the indicated mice. (E) Hematoxylin and eosin (HE) staining of tissues harvested from the indicated mice. Scale bar = 50μm. (F) Western blot analysis of p53 in lungs from the indicated mice. The right panel displays quantification of p53 relative to the actin loading control (n = 5, 5). Data are shown as the mean ± s.e.m. *P<0.05, **P<0.01 by the 2-tailed Student’s t-test (B-D, F).
Fig 2.
Glycolysis in p53-depleted pulmonary arterial smooth muscle cells (PASMCs).
(A) Western blot analysis of p53 in PASMCs exposed to normoxia (Con) or hypoxia (Hypoxia). The right panel shows quantification of p53 relative to the actin loading control (n = 4,4). (B) Western blot analysis of PASMCs treated with siRNA for p53 (si-p53) or control siRNA (Mock). The right panel displays quantification of p53 relative to the actin loading control (n = 3,3). (C) Glucose-6-phosphate (n = 3,4), fructose-1,6-bisphosphate (n = 5,4), pyruvate (n = 5,5), and lactate (n = 5,5) levels determined by metabolomic analyses in PASMCs treated with siRNA for p53 (si-p53) or control siRNA (Mock). (D) PASMCs were treated with siRNA for p53 (si-p53) or control siRNA (Mock), followed by Seahorse XF extracellular flux analysis of glycolytic function, including the extracellular acidification rate (ECAR), glycolysis (n = 5,5), glycolytic capacity (n = 3,5), and glycolytic reserve (n = 5,5). Results represent the mean ± s.e.m. *P<0.05, **P<0.01 by the 2-tailed Student’s t-test (A–D). In Fig 2C, PASMCs treated with siRNA for p53 (si-p53) or control siRNA (Mock) were analyzed in 5 groups each, but glucose-6-phosphate became undetectable in 2 Mock groups and 1 si-p53 group. Boxplot analysis of fructose-1,6-bisphosphate data identified 1 abnormal value in the si-p53 group, and this was excluded from the figure and from analysis. In Fig 2D, glycolytic function was analyzed in 5 groups each of PASMCs treated with siRNA for p53 (si-p53) or control-siRNA (Mock), but boxplot analysis identified 2 abnormal values for glycolytic capacity in the Mock group and these were excluded from analysis.
Fig 3.
Mitochondrial respiration in p53-depleted pulmonary arterial smooth muscle cells (PASMCs).
(A) Levels of acetyl-CoA (n = 5,5), citrate (n = 4,5), cis-aconitate (n = 5,5), isocitrate (n = 4,4), fumarate (n = 5,5) and malate (n = 5,5) determined by metabolomic analysis in PASMCs treated with siRNA for p53 (si-p53) or control siRNA (Mock). (B) PASMCs were treated with siRNA for p53 (si-p53) or control siRNA (Mock), followed by Seahorse XF extracellular flux analysis of mitochondrial respiration, including the oxygen consumption rate (OCR), basal respiration (n = 5,5), maximal respiration (n = 5,4), ATP production (n = 5,4) and spare capacity (n = 5,4). Results represent the mean ± s.e.m. *P<0.05, **P<0.01 by the 2-tailed Student’s t-test (A, B). In Fig 3A, PASMCs treated with siRNA for p53 (si-p53) or control siRNA (Mock) were analyzed in 5 groups each. However, boxplot analysis identified 1 abnormal value for citrate in the Mock group, 1 for isocitrate in the Mock group, and 1 for isocitrate in the si-p53 group, and these values were excluded from analyses. In Fig 3B, mitochondrial respiration was analyzed in 5 groups each of PASMCs treated with siRNA for p53 (si-p53) or control siRNA (Mock). Boxplot analysis identified 1 abnormal value each for maximal respiration, ATP production, and Spare capacity in the si-p53 group, and these values were excluded from analyses.
Fig 4.
Mouse models of PH with SMC-specific gain or loss of p53 function.
Scheme for generation of mice with hypoxia-induced PH. (B) Western blot analysis of p53 expression in mice with SMC-specific gain of p53 function (Myh11-Cre/ERT2; Mdm4fl/fl (SMC-Mdm4KO)) or loss of p53 function (Myh11-Cre/ERT2; Trp53fl/fl (SMC-p53KO)). The right panel shows quantification of p53 relative to the actin loading control in the indicated groups (n = 3,3,3). (C) Right ventricular systolic pressure (RVSP) of the indicated mice housed under normoxia (Con) or hypoxia (PH) (n = 9,4,11). (D, E) Right ventricular wall thickness (RVWT) (n = 9,4,11) (D) and right ventricular diameter (RVD) (n = 9,4,11) (E) of the indicated mice housed under hypoxia (PH). (F) Hematoxylin and eosin (HE) staining of tissues from the indicated mice. Scale bar = 50 μm. Data represent the mean ± s.e.m. *P<0.05, **P<0.01 by the 2-tailed Student’s t-test (B), or 2-way ANOVA followed by Tukey’s multiple comparison test (C–E).