Figures
- FIGURE 1
Upregulation of leptin (Ob)/Ob main receptor (ObR-b) axis in human idiopathic pulmonary arterial hypertension (iPAH). a and b) Human pulmonary endothelial cells (P-ECs) as Ob in PAH. a) Representative images of in situ Ob staining (red) from controls and patients with iPAH, in which P-ECs are positive for lectin (green) and pulmonary arterial smooth muscle cells (PA-SMCs) are positive for SM22 (white). b) In vitro quantification of Ob production in conditioned media of primary early (≤5) passage cultures of human P-ECs from controls (n=15) and iPAH patients (n=10). c–e) Human and rodent PA-SMCs overexpress the ObR-b in PAH. c) Representative image of ObR-b (red) staining in PA-SMCs, positive for SM22 (white), in lungs from controls and iPAH patients, in which P-ECs are positive for lectin (green). d) Quantification of ObR-b mRNA level in primary early (≤5) passage cultures of human PA-SMCs from controls (n=10) and iPAH (n=10), and quantification for ObR-b/β-actin ratio in primary early (≤5) passage cultures of human PA-SMCs from controls (n=5) and iPAH (n=5) with representative Western blots. e) Quantification of ObR-b/β-actin ratio in rat lungs, from controls (n=5) and chronic hypoxia-induced pulmonary hypertension (PH) (n=5), with representative Western blot. f–i) Dichloroacetate (DCA) abolishes the excessive endothelial-derived Ob production. f) Schematic representation of the role of dimethyloxaloylglycine (DMOG) and DCA in Ob signalling pathway. PDK: pyruvate dehydrogenase kinase; PDH: pyruvate dehydrogenase; PHD: prolyl hydroxylase domains; HIF: hypoxia-inducible factor. g) Quantification of HIF-1α/β-actin ratio and a representative Western blot in primary early (≤5) passage cultures of human P-ECs from controls in untreated (vehicle, n=3), DMOG-treated (n=3) or DMOG+DCA-treated (n=3) conditions. h and i) Quantification of Ob production in conditioned media of primary early (≤5) passage cultures of human P-ECs from h) controls in untreated (n=3), DMOG-treated (n=3) or DMOG+DCA-treated (n=3) conditions and from i) human P-ECs from iPAH patients in untreated (n=3) or DCA-treated conditions (n=3). DAPI: 4',6-diamidino-2-phenylindole; AU: arbitrary unit. *: p<0.05; **: p<0.01. Scale bar=20 µm.
- FIGURE 2
Leptin (Ob)/Ob main receptor (ObR-b) axis contributes to pulmonary vascular remodelling in idiopathic pulmonary arterial hypertension (iPAH). a–c) The Ob/ObR-b axis induces human and rodent pulmonary arterial smooth muscle cell (PA-SMC) proliferation. a) Quantification of 5-bromo-2-deoxyuridine (BrdU) incorporation in primary early (≤5) passage cultures of PA-SMCs from controls and iPAH treated with increasing doses of recombinant Ob (n=3 for each condition and replicated three times). b) Quantification of mRNA levels of Ki67 and c) cyclin D1 in lungs from chronically hypoxic mice, treated or untreated (vehicle) with recombinant Ob (n=5 in each group). d–k) Ob/ObR-b axis induces monocyte activation in iPAH. d) Representative fluorescence-activated cell sorter (FACS) dot plots of peripheral blood mononuclear cells (PBMCs) withdrawn from controls (n=10) and iPAH patients (n=10), and stained with an anti-CD14 antibody. The red quadrants represent PBMCs positive for CD14 (CD14+ cells). SSC: side-scattered light. e) Quantification of CD14+ PMBCs. f) Representative FACS dot plots of PBMCs withdrawn from controls (n=10) and iPAH patients (n=10), and dual stained with anti-CD11b and CD25 antibodies. The red quadrants in the dot plots represent double-positive cells for CD11b and CD25 (CD11b+ CD25+ cells). g) Quantification of CD11b+ CD25+ PBMCs. h) Quantification of the number of CD14+ cells expressing ObR-b by FACS in controls (n=10) and iPAH patients (n=10). i) Quantification of the number of CD14+ PBMCs from controls (n=3) treated with increasing doses of recombinant Ob (n=3 in each condition, replicated three times). j) Representative FACS dot plots quantification of control PBMCs (n=3) treated with increasing doses of recombinant Ob double stained with anti-CD11b and CD25 antibodies. The red quadrants in the dot plots represent double-positive cells for CD11b and CD25 (CD11b+ CD25+ cells). k) Quantification of CD11b+ CD25+ PBMCs treated with increasing doses of recombinant Ob (n=3 in each condition, replicated three times). l–n) Ob/ObR-b axis induces overexpression of key adhesion molecules in human pulmonary endothelial cells (P-ECs). l) Quantification of mRNA levels of intercellular adhesion molecule (ICAM)-1, m) vascular cell adhesion molecule (VCAM)-1 and n) E-selectin in primary early (≤5) passage cultures of human P-ECs from controls (n=3) treated with increasing doses of recombinant Ob (n=3 in each condition, replicated three times). AU: arbitrary unit; ns: nonsignificant; #: normalised to untreated conditions. *: p<0.05; **: p<0.01; ***: p<0.001
- FIGURE 3
Daily leptin (Ob) injections act on chronic hypoxia-induced pulmonary hypertension (PH). a and b) Murine model of chronic activation of Ob/Ob main receptor (Obr-b) axis. a) Experimental strategy of chronic recombinant Ob intraperitoneal (i.p.) injections in mice exposed to normoxia or hypoxia (n=5 in each condition). b) Body weight. c–f) Chronic Ob injections increase right ventricular systolic pressure (RVSP) in hypoxia-induced-PH. c) RVSP. d) Right ventricular hypertrophy measured by Fulton index normalised by body weight. e) Representative images of haematoxylin and eosin staining. Scale bar=50 μm. f) Quantification of the percentage of muscularised pulmonary arteries (PA) in mice. g–k) Chronic Ob injections enhance pulmonary arterial smooth muscle cell proliferation and perivascular macrophage activation. Representative images of g) proliferating cell nuclear antigen (PCNA), scale bar=50 µm and i) F4/80 markers, scale bar=20 µm. h and j) Quantification of cells expressing PCNA and F4/80 markers, respectively, in mice PA. k) Quantification of double-positive cells for CD206 and F4/80 in mice PA. ns: nonsignificant; *: p<0.05; **: p<0.01; ***: p<0.001.
- FIGURE 4
Leptin (Ob)/Ob main receptor (ObR-b) axis enhances chronic hypoxia-induced pulmonary hypertension (PH) susceptibility. a and b) ObR-deficient Zucker Diabetic Fatty (ZDF) transgenic rats. a) Experimental strategy of ZDF transgenic rats exposed to normoxia or hypoxia (n=5 in each condition). b) Body weight. c–g) ObR-deficiency protects against chronic hypoxia-induced-PH development. c) Mean pulmonary arterial pressure (PAP), d) pulmonary vascular resistance (PVR), and e) right ventricular hypertrophy measured by Fulton index normalised by body weight. f) Representative images of haematoxylin and eosin stain (HE) and α-smooth muscle cell actin (α-SMA) and g) quantification of the percentage of muscularised pulmonary arteries (PA) in rat lungs. h–k) ObR-deficiency decreases pulmonary arterial smooth muscle cell proliferation and perivascular macrophage accumulation. h and j) Representative images of proliferating cell nuclear antigen (PCNA) and CD68 staining, respectively, in rat PA. i and k ) Quantification of cells expressing PCNA and CD68 markers, respectively, in rat PA. ns: nonsignificant; *: p<0.05; **: p<0.01. Scale bar=50 µm.
- FIGURE 5
Targeting leptin (Ob)/Ob main receptor (ObR-b) axis with an Ob neutraliser (ObR:Fc) protects against pulmonary hypertension (PH) progression. a and b) Efficacy of an Ob neutraliser treatment in chronic hypoxia-induced PH. a) Quantification of 5-bromo-2-deoxyuridine (BrdU) incorporation in primary early (≤5) passage cultures of human pulmonary arterial smooth muscle cells (PA-SMCs) from controls exposed to the conditioned media of pulmonary endothelial cells (P-ECs) from idiopathic pulmonary arterial hypertension (iPAH) patients untreated (vehicle) or treated with ObR:Fc (n=3 in each condition). b) Experimental strategy of chronic ObR:Fc intraperitoneal (i.p.) injections in mice exposed to normoxia or hypoxia (n=5 in each condition). c–f) ObR:Fc treatment protects against chronic hypoxia induced PH progression. c) Right ventricular systolic pressure (RVSP), d) right ventricular hypertrophy measured by Fulton index, normalised by body weight, e) representative images of haematoxylin and eosin staining, and f) quantification of the percentage of muscularised pulmonary arteries (PA) in mice. g–k) ObR:Fc treatment increases PA-SMC proliferation and perivascular macrophage activation. Representative images of g) proliferating cell nuclear antigen (PCNA) and i) F4/80 staining. h and j) Quantification of cells expressing PCNA and F4/80 markers, respectively, in mice PA. k) Quantification of double-positive cells for CD206 and F4/80 in mice PA. ns: nonsignificant; *: p<0.05; **: p<0.01; ***: p<0.001. Scale bar=50 µm.
- FIGURE 6
Chronic dichloroacetate (DCA) treatment modulates leptin (Ob)/Ob main receptor (ObR-b) axis in hypoxia-induced pulmonary hypertension (PH). a and b) Chronic DCA treatment abolishes endothelial-derived Ob production in hypoxia. a) Representative images of in situ Ob staining (red) in rat lungs exposed to normoxia or hypoxia, treated with or not treated with DCA at a concentration of 1 g·L-1, in which pulmonary endothelial cells (P-ECs) are positive for Tie2 (green). b) Experimental strategy of chronic DCA treatment in drinking water in rats exposed to normoxia or hypoxia (n=5 in each condition). c–g) DCA treatment protects against chronic hypoxia-induced PH progression. c) Mean pulmonary arterial pressure (PAP), d) pulmonary vascular resistance (PVR), and e) right ventricular hypertrophy measured by Fulton index normalised by body weight. f) Representative images of haematoxylin and eosin staining (HE) and α-smooth muscle cell actin (α-SMA). g) Quantification of the percentage of muscularised pulmonary arteries (PA) in rat lungs. h–k) DCA treatment decreases pulmonary arterial smooth muscle cell proliferation and perivascular macrophage accumulation. Representative images of stained h) proliferating cell nuclear antigen (PCNA) and j) CD68+. i and k) Quantification of cells expressing PCNA and CD68 markers, respectively, in rat PA. DAPI: 4',6-diamidino-2-phenylindole; ns: nonsignificant; *: p<0.05; ***: p<0.001. Scale bar=50 µm.
Tables
- TABLE 1
Characteristics of idiopathic pulmonary arterial hypertension (iPAH) patients and controls
iPAH Controls Patients 10 10 Age years 54.9±3.1 41.9±2.1 Sex male/female (ratio) 3/7 (0.43) 4/6 (0.67) NYHA functional class Class I 2 NA Class II 5 NA Class III 3 NA 6MWD m 465±20 NA Mean PAP mmHg 48.8±3.5 NA Cardiac index Lmin−1·m−2 2.9±0.2 NA PVRI Wood unit·m−2 8.3±0.9 NA PCWP mmHg 8.4±0.7 NA Specific PAH therapy ERA 8 NA PDE5i 6 NA Prostanoids 2 NA No treatment 0 NA -
Data are presented as n or mean±sem, unless otherwise stated. NYHA: New York Heart Association; 6MWD: 6-min walking distance; PAP: pulmonary arterial pressure; PVRI: pulmonary vascular resistance index; PCWP: pulmonary capillary wedge pressure; ERA: endothelin receptor antagonists; PDE5i: phophodiesterase 5 inhibitors; NA: not applicable.
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- TABLE 2
Characteristics of transplanted idiopathic pulmonary arterial hypertension (iPAH) patients and lung resected subjects (controls)
iPAH Controls Patients 7 7 Age years 39.8±2.2 46.4±2.3 Sex male/female (ratio) 2/5 (0.40) 2/5 (0.40) Mutation in BMPR2 gene Carrier 0 NA Non-carrier 7 NA NYHA functional class Class III 2 NA Class IV 5 NA Mean PAP mmHg 69.9±2.1 NA Cardiac index L·min−1·m−2 2.6±0.1 NA PVRI Wood unit·m−2 14.6±0.6 NA PCWP mmHg 8±0.5 NA -
Data are presented as n or mean±sem, unless otherwise stated. NYHA: New York Heart Association; PAP: pulmonary arterial pressure; PVRI: pulmonary vascular resistance index; PCWP: pulmonary capillary wedge pressure; NA: not applicable.
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