Histological analysis of activated fibroblasts presence during Eng^+/− wound healing.

<p>(<b>A</b> and <b>B</b>) Immunofluorescence staining of alpha-smooth muscle actin (α-SMA) on Formalin-fixed Parafin-embedded wounds from <i>Eng^+/+</i> (<b>A</b>) and <i>Eng^+/−</i> (<b>B</b>) mice at day 6. (<b>C</b> and <b>D</b>) Granulation tissue on wounds edges from <i>Eng^+/+</i> (<b>C</b>) and <i>Eng^+/−</i> (<b>D</b>) mice in more details. (<b>E</b> and <b>F</b>) Immunofluorescence staining of α-SMA on Formalin-fixed Parafin-embedded scars from <i>Eng^+/+</i> (<b>E</b>) and <i>Eng^+/−</i> (<b>F</b>) mice at day 12. (<b>G</b> and <b>H</b>) <i>Eng^+/+</i> (<b>G</b>) and <i>Eng^+/−</i> (<b>H</b>) day-12 scars magnification. Bars = 500 mm. Arrow = Activated fibroblasts accumulation. A representative image from four independent experiments is shown.</p

Sequences of primers.

<p>Sequences of primers.</p

Effect of PI3K inhibition on endoglin-mediated different cell proliferation.

<p>(<b>A</b>) Cell number of MDF was analyzed by crystal violet assay after 10 μM LY294002 treatment for 4 days. Inhibition of Akt activation after LY294002 treatment was analyzed by western blot (<b>A</b>, upper panel). (<b>B</b>) BrdU incorporation of <i>Eng^+/+</i> and <i>Eng^+/−</i> MDF after LY294002 treatment was also analyzed. (<b>C</b>) Endo and Mock fibroblasts proliferation was assessed by MTT after 10 μM LY294002 treatment. Inhibition of Akt activation after LY294002 treatment was analyzed by western blot (<b>C</b>, upper panel). Mean+SEM is represented (n = 3). * p<0.05-significance of the difference between cells in control conditions, ** p<0.05-significance of the difference between LY294002 treatment and control conditions, Two-way ANOVA. Tubulin was used as loading control. A representative blot from three independent experiments is shown.</p

Effect of endoglin haploinsufficiency on extracellular matrix formation.

<p>(<b>A</b>) Synthesis of collagen Iα and fibronectin by <i>Eng^+/+</i> and <i>Eng^+/−</i> MDF were analyzed by RT-PCR. GAPDH was used as housekeeping (<b>B</b>) Synthesis of collagen Iα and fibronectin were also evaluated by western blot in total protein extracts from <i>Eng^+/+</i> and <i>Eng^+/−</i> MDF using Tubulin as loading control. A representative blot from three independent experiments is shown.</p

Effect of endoglin expression on fibroblast migration.

<p>(<b>A</b> and <b>B</b>) <i>Eng^+/+</i> and <i>Eng^+/−</i> MDF <i>in vitro</i> migration was analyzed by wound healing assay (<b>A</b>) and 8-mm pore transwell migration (<b>B</b>). (<b>C</b>) Migration of NIH3T3 fibroblasts overexpressing human endoglin (Endo) and infected with empty vector (Mock) was assessed by transwell assay. * p<0.05-significance of the difference between cells, Student's <i>t</i>-test.</p

Histological analysis of Akt phosphorylation after wound healing.

<p>Immunofluorescence staining of alpha-smooth muscle actin (α-SMA) and phospho-Akt (pAkt) on Formalin-fixed Parafin-embedded wounds from <i>Eng^+/+</i> and <i>Eng^+/−</i> mice at day 12. Bars = 50 mm. Arrow = Colocalization of α-SMA and pAkt. A representative image from four independent experiments is shown.</p

Effect of endoglin expression on Akt phosphorylation.

<p>(<b>A</b>) Ser⁴⁷³ and Thr³⁰⁸ Akt phosphorilation and Akt expression were analyzed by western blot in total protein extracts from <i>Eng^+/+</i> and <i>Eng^+/−</i> MDF. (<b>B</b>–<b>D</b>) Similar analysis were carried on in <i>Eng^+/−</i> MDF transiently transfected with ENG or empty vector (<b>B</b>); NIH3T3 overexpressing by retroviral infection endoglin (Endo) and its controls (Mock) (<b>C</b>); and Endo fibroblasts transfected with anti-Eng siRNA (<b>D</b>). Tubulin was used as loading control. A representative blot from three independent experiments is shown.</p

Effect of endoglin haploinsufficiency on TGFβ1 signaling pathways.

<p>(<b>A</b>) <i>Eng^+/+</i> and <i>Eng^+/−</i> MDF were stimulated with 1 ng/ml TGFβ1 for 30 minutes. Total protein extracts were analyzed by western blot with anti-phospho-Smad1/3, anti-phospho-Smad2, anti-Smad1 and anti-Smad2/3 (<b>B</b>). Id1 expression was assessed by western blot after 1 hour TGFβ1 treatment. (<b>C</b>) TGFβ1-induced PAI-1 expression increment was analyzed by quantitative RT-PCR. A representative experiment of three independent experiments using triplicate samples is shown. (<b>D</b>–<b>F</b>) MAPKs and PI3K/Akt pathways were analyzed after stimulation with 1 ng/ml TGFβ1 for 5, 15 and 30 minutes. Total proteins extracts were resolved by western blot and incubated with anti-phospho-Erk1/2 and anti-Erk1/2 (<b>D</b>), anti-phospho-JNK1 and anti-JNK1 (<b>E</b>) and anti-phospho-Akt and anti-Akt (<b>F</b>) respectively. Tubulin was used as loading control. Mean+SEM is represented (n = 3). * p<0.05-significance of the difference between TGFβ1 treatment and control conditions, Two-way ANOVA. A representative blot from three independent experiments is shown.</p

Soluble angiogenic factors in MDS BM microenvironment.

<p>The box plot compares median levels of sENG, sFLT-1 and sVEGF in BM supernatant fluid of various types of MDS. To measure the levels of angiogenic factors present in the BM supernatant fluid in the different MDS groups, ELISA assays were carried out in the BM supernatant fluid from MDS patients and controls. Whiskers represent the range. Mann-Whitney test showed that sENG concentrations in BM supernatants was higher in RCMD with respect to the healthy cases (<i>p</i><0.005), the remaining low-risk MDS (<i>p</i><0.05) and high-risk patients (<i>p</i> = 0.05) (A). RCDM displayed higher levels of sFLT-1 with respect to the controls (<i>p</i> = 0.001), the remaining low-risk MDS (<i>p</i><0.005) and the high-risk MDS patients (<i>p</i><0.005) (B). No significant differences in sVEGF concentration of MDS groups were found (C). MDS: myelodysplastic syndrome; BM: bone marrow; ENG: endoglin; sFLT-1: fms-like tyrosine kinase 1; VEGF: vascular endothelial grow factor; RCMD: refractory cytopenia with multilineage dysplasia. (Controls n = 24; Low-Risk MDS excluding RCMD n = 15; RCMD n = 15; High-Risk MDS n = 6).</p

<i>ENG</i> and <i>VEGF</i> RNA expression in mononuclear BM cells of MDS subtypes.

<p>The box plot compares median of <i>ENG</i> and <i>VEGF</i> expression levels in BM mononuclear cells between the different MDS groups and controls. The gene expression levels were analyzed by RT-PCR. Each sample was performed in triplicate. Value of each patient is the mean of these three experiments. Mann-Whitney test was used to analyze the results. The box plot compares the RNA expression in BM mononuclear cells of subtypes of MDS. Whiskers represent the range. A down-regulation of <i>ENG</i> was showed in RCMD cases (<i>p</i><0.05). By contrast, <i>ENG</i> expression in high-risk MDS patients was higher than in controls or in the other MDS (<i>p</i><0.05). No significant differences in low-risk MDS excluding RCMD patients in <i>ENG</i> expression with respect to the healthy controls were found (A). The low-risk MDS groups showed over-expression of <i>VEGF</i> with respect to the control group (<i>p</i><0.05). Moreover, patients with RCMD showed the highest values in the expression of this gene with respect to the other low-risk MDS. No significant differences in high-risk MDS patients in <i>VEGF</i> expression with respect to the healthy controls were found (B). ENG: endoglin; VEGF: vascular endothelial grow factor; BM: bone marrow; MDS: myelodysplastic syndrome; RCMD: refractory cytopenia with multilineage dysplasia; RAEB: refractory anaemia with excess of blasts. (Controls n = 13; Low-Risk MDS excluding RCMD n = 22; RCMD n = 12; High-Risk MDS n = 16).</p