Contribution of fatty acid oxidation to the pathogenesis of pulmonary hypertension

Abstract

Dysregulated metabolism characterizes both animal and human forms of pulmonary hypertension (PH). Enzymes involved in fatty acid metabolism have previously not been assessed in human pulmonary arteries affected by pulmonary arterial hypertension (PAH), and how inhibition of fatty acid oxidation (FAO) may attenuate PH remains unclear. Fatty acid metabolism gene transcription was quantified in laser-dissected pulmonary arteries from 10 explanted lungs with advanced PAH (5 idiopathic, 5 associated with systemic sclerosis), and 5 donors without lung diseases. Effects of oxfenicine, a FAO inhibitor, on female Sugen 5416-chronic hypoxia (SuHx) rats were studied in vivo using right heart catheterization, and ex vivo using perfused lungs and pulmonary artery ring segments. The impact of pharmacologic (oxfenicine) and genetic (carnitine palmitoyltransferase 1a heterozygosity) FAO suppression was additionally probed in mouse models of Schistosoma and hypoxia-induced PH. Potential mechanisms underlying FAO-induced PH pathogenesis were examined by quantifying ATP and mitochondrial mass in oxfenicine-treated SuHx pulmonary arterial cells, and by assessing pulmonary arterial macrophage infiltration with immunohistochemistry. We found upregulated pulmonary arterial transcription of 26 and 13 FAO genes in idiopathic and systemic sclerosis-associated PAH, respectively. In addition to promoting de-remodeling of pulmonary arteries in SuHx rats, oxfenicine attenuated endothelin-1-induced vasoconstriction. FAO inhibition also conferred modest benefit in the two mouse models of PH. Oxfenicine increased mitochondrial mass in cultured rat pulmonary arterial cells, and decreased the density of perivascular macrophage infiltration in pulmonary arteries of treated SuHx rats. In summary, FAO inhibition attenuated experimental PH, and may be beneficial in human PAH.

Keywords: fatty acid oxidation; metabolism; pulmonary arterial hypertension; pulmonary hypertension.

Figure 1.

Translation of fatty acid oxidation enzymes is increased in the pulmonary artery of patients with pulmonary arterial hypertension (PAH). Immunohistochemical staining of human lung tissues [left: control; middle: systemic sclerosis-associated PAH (APAH), right: idiopathic PAH (IPAH)] with antibodies against fatty acid binding protein 4 (FABP4; top row) and carnitine palmitoyltransferase 1 (CPT1; bottom row). Scale bars indicate 100 µm in the top left (FABP4-Control), 300 µm in the bottom right (CPT1-IPAH), and 200 µm in the other four panels. The arrows and the arrowhead indicate pulmonary arteries and macrophage, respectively.

Figure 2.

Oxfenicine attenuates right ventricular systolic pressure (RVSP) elevation and reverses pulmonary vascular remodeling in Sugen-chronic hypoxia rats. A and B: pulmonary artery intimal (A) and medial (B) carnitine palmitoyltransferase 1 (CPT1) expression, measured by immunohistochemistry, is increased in SuHx rats compared with the three control groups. n = 4 or 5/group. C: cartoon representations of SuHx rat oxfenicine treatment (top) and prevention (bottom) protocols. D and E: right ventricular systolic pressure (RVSP) in oxfenicine-fed female SuHx rats. Tx and Prev refer to the oxfinicine treatment (D) and prevention (E) protocols, respectively. n = 6 (Oxf), 13 (SuHx). F: representative hematoxylin-eosin (H&E) stained lung histopathological sections of normoxia (A and B) or hypoxia-exposed (C–F) female rats, treated with the vehicle carboxymethylcellulose (CMC; A and C), Sugen 5416 (B and D), or Sugen 5416 + oxfenicine per the treatment protocol (E and F). Small muscular and prealveolar pulmonary arteries are highlighted by arrows. CMC-normoxia (A) and Sugen 5416-normoxia (B) show normal pulmonary arterial thickness. Lungs from Sugen 5416-treated rats show mild airspace enlargement as previously reported. CMC-hypoxia (C) resulted in media thickening; SuHx (D) showed marked endothelial cell proliferation and vascular obliterations. SuHx rats fed oxfenicine per the treatment protocol (E and F) demonstrated a marked decrease in pulmonary arterial intima lesions (arrows), with some residual media thickening (short arrow in E) (b: bronchiole) (25 µm magnification bar shown in A). G: thickness of individual small pulmonary artery (PA), sized 50–100 µm in diameter, is expressed as the difference between the outer and the inner diameters, divided by the outer diameter [(O − I)/O]. The increased PA thickness in Sugen-chronic hypoxia rats was partially attenuated with oxfenicine (Oxf) administration according to the prevention protocol (Prev). n = 5 or 6 rats/group. Statistical analyses were performed with Kruskal–Wallis test with Dunn’s multiple comparisons test (A, B, and G), and Mann–Whitney test (D and E). ****P < 0.0001, **P < 0.01, *P < 0.05. AU, arbitrary unit; CMC-Hx, carboxymethylcellulose-containing diluent with hypoxia; CMC-Nx, carboxymethylcellulose-containing diluent with normoxia; Oxf, oxfenicine; SuHx, Sugen 5416-chronic hypoxia; Su-Nx, Sugen 5416-normoxia.

Figure 3.

Elevated pulmonary vascular resistance in Sugen 5416-chronic hypoxia (SuHx) rats is attenuated by oxfenicine (Oxf). Pulmonary artery pressure measured in isolated lungs taken from control (Ctrl) and SuHx rats fed either water or oxfenicine (Oxf) for 3 days. n = 3 or 6/group. Statistical analysis was performed with ordinary two-way ANOVA with Tukey’s multiple comparisons test, and interaction P value is reported. ****P < 0.0001, ***P < 0.001, *P < 0.05.

Figure 4.

Oxfenicine (Oxf) attenuates endothelin-1-induced vasoconstriction of Sugen 5416-chronic hypoxia (SuHx) pulmonary artery. A: representative isometric tension tracings of pulmonary artery (PA) ring segments from a control rat (black) and a SuHx rat (red), first exposed to phenylephrine (PE) then subsequently to acetylcholine (ACh). B: percent relaxation of PA ring segments of control (Ctrl) and SuHx rats in response to acetylcholine. n = 3 or 4/group. C and D: representative isometric tension tracings (C) and change in tension (D) of PA ring segments, taken from SuHx rats fed either water or oxfenicine (Oxf) for 3 days, in response to endothelin-1 (ET-1) treatment. n = 4/group. E and F: representative isometric tension tracings (E) and change in tension (F) of PA ring segments, taken from SuHx rats fed either water or oxfenicine for 3 days, in response to KCl. n = 6/group. Each data point in both D and F represents an individual PA ring segment. Statistical analyses were performed with unpaired t test (B), and Mann–Whitney test (D and F). **P < 0.01, *P < 0.05.

Figure 5.

CPT1a heterozygosity attenuates pulmonary hypertension (PH) in two mouse models. A: cartoon representations of CPT1a^+/− mouse experiment protocols involving chronic hypoxia (top) and Schistosoma mansoni-induced (bottom) pulmonary hypertension. B and C: right ventricular systolic pressure (RVSP) (B) and right ventricular weight (C) expressed as the Fulton index (right ventricular weight divided by the combined weight of the left ventricle and the interventricular septum) in CPT1a^+/+ (WT) and CPT1a^+/− (CPT1a Het) littermate mice exposed to chronic hypoxia for 3 wk (Hx). n = 10–13/group. D and E: RVSP (D) and right ventricular weight (E) in CPT1a^+/+ and CPT1a^+/− littermate mice challenged with S. mansoni eggs (Schisto). n = 11 or 12/group. To minimize the effect of potential inter-litter genetic variability, RVSP and Fulton index of each mouse are displayed and analyzed as the difference between the individual measurement and the litter average value, and littermates with different genotypes are compared against one another. F and G: pulmonary arterial intimal (F) and medial (G) remodeling in CPT1a^+/+ and CPT1a^+/− mice challenged with S. mansoni eggs. n = 11 or 12/group. Statistical analyses were performed with unpaired t test. H: lung tissue and pulmonary artery histopathology of hypoxia-exposed wild type (a) and CPT1a-heterozygous (b) mice, and S. mansoni egg-infected wildtype (c), with prominent perivascular granulomas, and CPT1a-heterozygous (d) mice. Hematoxylin-eosin (H&E) stained, ×10 magnification, scale bars included. All lungs were inflated with agarose as apparent with the eosinophilic material in the alveolar and airway spaces. *P < 0.05. CPT1a, carnitine palmitoyltransferase 1a.

Figure 6.

Oxfenicine (Oxf) attenuates pulmonary hypertension (PH) in two mouse models. A: cartoon representations of oxfenicine mouse experiment protocols involving acute 24-h hypoxia (top) and Schistosoma mansoni-induced (bottom) pulmonary hypertension. Female mice were used for oxfenicine experiments. B and C: right ventricular systolic pressure (RVSP) (B) and right ventricular weight (C) expressed as the Fulton index (right ventricular weight divided by the combined weight of the left ventricle and the interventricular septum) in C57BL/6J wild type mice that were fed water with or without oxfenicine (Oxf) prior to acute hypoxia exposure. n = 6 or 7/group. D and E: RVSP (D) and Fulton index (E) in C57BL/6J mice that received S. mansoni egg injections (Schisto) and were fed water with or without oxfenicine. n = 9 or 10/group. F and G: pulmonary arterial intimal (F) and medial (G) remodeling in C57BL/6J mice that received S. mansoni egg injections and were fed water with or without oxfenicine. n = 9/group. Statistical analyses were performed with Mann–Whitney test (B and C), and unpaired t test (D–G). H: lung tissue and pulmonary artery histopathology of 24-h acute hypoxia-exposed mice fed water (a) or oxfenicine (b), and S. mansoni egg-infected mice fed water (c), with prominent perivascular inflammation and granulomas, or oxfenicine (d). Hematoxylin-eosin (H&E) stained, ×10 magnification, scale bars included. All lungs were inflated with agarose as apparent with the eosinophilic material in the alveolar and airway spaces. **P < 0.01, *P < 0.05.

Figure 7.

Oxfenicine reduces rat pulmonary arterial fatty acid oxidation in an ATP-independent manner. A and B: 9,10-³H-palmitic acid oxidation by rat pulmonary artery (PA) endothelial (A) and PA smooth muscle cells (B) normalized by protein. C and D: 5-³H-glucose oxidation by rat PA endothelial (C) and PA smooth muscle cells (D) normalized by protein. E and F: ATP concentrations (nM) in rat PA endothelial (E) and PA smooth muscle cells (F). n = 6/group. For oxfenicine treatment (SuHx + Oxf), cells isolated from SuHx rats were first incubated for 24 h in 1 mM oxfenicine in enriched media prior to adding ³H-labeled compounds or measuring ATP. G: ATP normalized by protein in rat pulmonary artery ring segments, in which SuHx + Oxf represents oxfenicine administration per the treatment protocol. N = 3–5/group. Statistical analyses were performed with Kruskal–Wallis test with Dunn’s multiple comparisons test. **P < 0.01, *P < 0.05 ATP, adenosine triphosphate; CMC + Nx, carboxymethylcellulose-containing diluent with normoxia; CPM, counts per minute; Oxf oxfenicine; PAEC, pulmonary artery endothelial cell; PASMC pulmonary artery smooth muscle cell; SuHx Sugen 5416-chronic hypoxia.