Figure 5

<p>Mouse lung PECAM-1 immunostaining (<b>A</b>). (<b>B</b>) Number of vessels per 100 alveoli. (<b>C</b>) Mouse lung F4/80 immunostaining. (<b>D</b>) Number of macrophages per 100 alveoli. Values are mean±SEM. *<i>P</i><0.02 compared with wild-type mice exposed to normoxia and ^σ<i>P</i><0.02 compared with wild-type mice exposed to hypoxia.</p

Key Role of the Endothelial TGF-β/ALK1/Endoglin Signaling Pathway in Humans and Rodents Pulmonary Hypertension

<div><p>Mutations affecting transforming growth factor-beta (TGF-β) superfamily receptors, activin receptor-like kinase (ALK)-1, and endoglin (ENG) occur in patients with pulmonary arterial hypertension (PAH). To determine whether the TGF-β/ALK1/ENG pathway was involved in PAH, we investigated pulmonary TGF-β, ALK1, ALK5, and ENG expressions in human lung tissue and cultured pulmonary-artery smooth-muscle-cells (PA-SMCs) and pulmonary endothelial cells (PECs) from 14 patients with idiopathic PAH (iPAH) and 15 controls. Seeing that ENG was highly expressed in PEC, we assessed the effects of TGF-β on Smad1/5/8 and Smad2/3 activation and on growth factor production by the cells. Finally, we studied the consequence of ENG deficiency on the chronic hypoxic-PH development by measuring right ventricular (RV) systolic pressure (RVSP), RV hypertrophy, and pulmonary arteriolar remodeling in ENG-deficient (<i>Eng^+/−</i>) and wild-type (<i>Eng^+/+</i>) mice. We also evaluated the pulmonary blood vessel density, macrophage infiltration, and cytokine expression in the lungs of the animals. Compared to controls, iPAH patients had higher serum and pulmonary TGF-β levels and increased ALK1 and ENG expressions in lung tissue, predominantly in PECs. Incubation of the cells with TGF-β led to Smad1/5/8 phosphorylation and to a production of FGF2, PDGFb and endothelin-inducing PA-SMC growth. Endoglin deficiency protected mice from hypoxic PH. As compared to wild-type, <i>Eng^+/−</i> mice had a lower pulmonary vessel density, and no change in macrophage infiltration after exposure to chronic hypoxia despite the higher pulmonary expressions of interleukin-6 and monocyte chemoattractant protein-1. The TGF-β/ALK1/ENG signaling pathway plays a key role in iPAH and experimental hypoxic PH via a direct effect on PECs leading to production of growth factors and inflammatory cytokines involved in the pathogenesis of PAH.</p></div

Effect of pulmonary endothelial cells (PECs) from controls incubated with TGF-β with or without anti-ENG antibody (ENG Ab) on mRNA expression of A) <i>preproET-1</i>, (B) <i>PDGFa</i>, (C) <i>PDGFb</i>, (D) <i>FGF2</i>, (E) <i>EGF</i>, (F) <i>MCP-1</i>, and (G) <i>IL-6</i>.

<p>Values are mean±SEM.*<i>P</i><0.05 compared to PECs without TGF-β treatment.</p

Figure 2

<p>Effect of increasing TGF-β doses on Smad 1,5,8 and Smad 2,3 phosphorylation, respectively, in pulmonary endothelial cells (PECs) from controls and from patients with idiopathic pulmonary hypertension (iPAH) (<b>A</b> and <b>B</b>). Protein levels were normalized for β-actin. Values are means±SEM normalized for results without TGF-β. *<i>P</i><0.05 compared to relevant controls without TGF-β, ^§<i>P</i><0.05 compared to control PECs under the same conditions. (<b>C</b>) Growth of pulmonary-artery smooth-muscle cells (PA-SMCs) from controls in response to serum-free media derived from cultured PECs from controls and stimulated by TGF-β with or without anti-ENG antibody (ENG Ab).Values are mean±SEM. *<i>P</i><0.05 compared to basal condition, ^§<i>P</i><0.05 compared to PA-SMCs stimulated with PEC medium.</p

Figure 5

<p>Mouse lung PECAM-1 immunostaining (<b>A</b>). (<b>B</b>) Number of vessels per 100 alveoli. (<b>C</b>) Mouse lung F4/80 immunostaining. (<b>D</b>) Number of macrophages per 100 alveoli. Values are mean±SEM. *<i>P</i><0.02 compared with wild-type mice exposed to normoxia and ^σ<i>P</i><0.02 compared with wild-type mice exposed to hypoxia.</p

The open bars indicate the results in controls and the closed bars in patients with idiopathic pulmonary hypertension (iPAH).

<p>TGF-β was assayed using an ELISA in serum and lung homogenates (controls, n = 15; iPAH patients, n = 14) (<b>A</b> and <b>B</b>). (<b>C</b>) TGF-β mRNA measured in pulmonary endothelial cells (PECs) and pulmonary-artery smooth-muscle cells (PA-SMCs; controls, n = 7; iPAH patients, n = 7). (<b>D</b>) TGF-β receptor expression: <i>ALK1, ALK5</i>, and <i>ENG</i> mRNA measured in PECs and PA-SMCs (controls, n = 7; iPAH patients, n = 7). (<b>E</b>): ENG, ALK1, and ALK5 protein expression in LUNG, PECs and PA-SMCs. Protein levels were normalized for β-actin (controls, n = 7; iPAH patients, n = 7). Values are mean±SEM. *<i>P</i><0.05 and **<i>P</i><0.01 compared with controls.</p

BrdU incorporation and changes in cell number in human pulmonary-artery smooth muscle cells in response to 10% fetal calf serum in the presence or absence of a range of concentrations of suramin (100 to 1000 µg/mL).

<p>n=4; *P<0.05; **P<0.001; ***P<0.0001.</p

The effect of suramin on PDGF, FGF2, and EGF-induced ERK1/2 phosphorylation in PA-SMCs.

<p>Western blotting was used to assess ERK1/2 phosphorylation in PA-SMCs incubated with PDGF, FGF2, or EGF (10 ng/mL, 10 min) with or without suramin pretreatment (1000 µg/mL, 1 h). The results shown are typical findings from five independent experiments. The level of phosphorylated ERK1/2 was normalized against β-actin. n=4. *P<0.05 versus control; **P<0.001 versus control. The phospho/total ratio was expressed as the mean±SEM from five PA-SMC samples. </p

The preventive effect of suramin on the development of monocrotaline-induced pulmonary hypertension.

<div><p>Compared with vehicle administered alone, suramin treatment significantly prevented the development of pulmonary hypertension and right ventricular hypertrophy. <b>A</b>) Pulmonary arterial pressure measurements. <b>B</b>) Right ventricular hypertrophy as assessed by the RV/(LV+S) weight ratio. </p> <p><b>C</b>) Percentages of nonmuscular (NM), partially muscular (PM), fully muscular (FM), and completely obliterated (FM+) intra-acinar vessels. All values are the mean±SEM from at least 5 animals per group. </p> <p><b>D</b>-<b>O</b>) Histochemical and immunohistochemical analysis of rat lung tissue at 21 days after monocrotaline injection. Medial hypertrophy was associated with an increased number of proliferating vascular cells shown by immunohistochemistry for PCNA; PCNA-positive cells have dark nuclei. Suramin prevented the development of medial hypertrophy and the deposition of collagen around the pulmonary arteries. <b>D</b>-<b>F</b>) Hematoxylin-phloxine-saffron staining. <b>G</b>-<b>I</b>) Proliferation of pulmonary artery smooth muscle cells<b>. J</b>-<b>L</b>) Collagen deposition. <b>M</b>-<b>O</b>) Macrophages in the lung tissue. All values are the mean±SEM from at least 5 animals in each group. *P<0.05; **P<0.001; ***P<0.0001. The statistical analyses compared MCT-injected rats (no suramin treatment) vs. rats injected with saline instead of MCT or rats treated with suramin. Scale bar=25 µm in all sections.</p></div

The structure and wall thickness of human pulmonary artery segments after 10 days of incubation in culture medium.

<p>The culture medium was either unsupplemented or supplemented with 10% FCS, suramin (1000 µg/mL) or masitinib (10^-5 M). (<b>A</b>) Hematoxylin-phloxine-saffron stain Scale bars: 25 µm (<b>B</b>) Masson trichrome stain Scale bars: 25 µm. (<b>C</b>) Double immunofluorescence staining performed with an anti-proliferating cell nuclear antigen (PCNA) antibody (red signal) and an anti-α-smooth muscle actin (α-SMA) antibody (green signal). DAPI nuclear staining is also shown (blue signal). Scale bars: 50 µm.</p