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

Differential expression of phosphorylated growth factor receptor tyrosine kinases (RTKs) in PA-SMCs.

<p>Arrays were incubated with 150 µg of lysate prepared from untreated cells or cells treated with suramin (1000 µg/mL, 1 h) and incubated for 15 minutes with 10 ng/mL of PDGF, FGF2, or EGF. RTKs were analyzed with anti-phospho-RTK, while immunoglobulin (IgG) and "reference spots" were used as the negative and positive controls, respectively. n=4.</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 effect of suramin on growth factor-induced pulmonary-artery smooth muscle cell proliferation.

<p>BrdU incorporation and cell count data are shown for quiescent cells incubated with PDGF, FGF2, or EGF (10 ng/mL). Suramin (1000 µg/mL) was added 1 hour before growth factor addition. n=4; *P<0.05; **P<0.001; ***P<0.0001.</p

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

Characterization of under-expressed genes in the CRD signature.

<p>A) Tree analysis of the CRD signature. Identification of the most representative genes by Gominer, PAM, IPA analysis and validation of the candidate genes in the validation cohort by qPCR; B) Network generated by IPA on the most significant GO categories in the CRD signature. Solid lines indicate <i>direct</i> interactions and dashed lines represent <i>indirect</i> interactions. Under-expressed genes in CRD are in gray. C) PAM analysis based on the most representative GO categories of Cluster A and B. Green represents relatively low expression, and red indicates relatively high expression. D) List of 9 genes able to classify correctly CRD and HC. E) The PCA graph of the 9 genes identified by PAM analysis indicated a clear separation between HC and patients with CRD (CF and PAH).</p

Gominer Analysis based for over-expressed genes in CF signature versus HC.

<p>Only GO categories enriched in cluster D (A) and in cluster E (B) with FDR less than 1%, with enrichment superior to 2 and with more than 10 genes are displayed.</p><p>Gominer Analysis based for over-expressed genes in CF signature versus HC.</p

Unsupervised hierarchical clustering analysis.

<p>A) Tree analysis. Clustering analysis based on the 30,146 probes corresponding to 17,163 unique genes expressed in PAH, CF patients and Healthy Controls (HC). 3 signatures were found: 1 common between CF and PAH (named CRD signature), 1 specific to CF and 1 to PAH; B) Principal Component Analysis (PCA) displayed a clear separation between HC and patients with CRD, whereas CF and PAH patients were less distinct; C) 5 groups of genes (or clusters) were selected, A to E, based on a combined approach: selected genes were clustered together and exhibited a t-test p-value below 1% between the CRD group (PAH+CF), CF or PAH versus HC. Green represents relatively low expression, and red indicates relatively high expression.</p

Characterization of over-expressed genes in the PAH signature.

<p>A) Tree analysis of the PAH signature. Identification of the most representative genes by Gominer, PAM, IPA analysis and validation by qPCR of the candidate genes in the validation cohort; B) Network generated by IPA on the most significant GO categories in PAH signature. Over-expressed genes in PAH are in gray. C) PAM analysis based on the cluster, <i>Green</i> represents relatively low expression, and <i>red</i> indicates relatively high expression; D and E) Validation by qPCR of <i>MDK</i> and <i>LGALS3</i> in the validation cohort, respectively.</p