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

Effect of the MDS BM microenvironment on BMEC-1 and HMVEC-L tube formation.

<p>BMEC-1 (A) and HMVEC-L (B) were seeded at a concentration of 8,000 cells per well of 96-well plate and incubated for 7 h at 37°C in 5% CO₂. The endothelial tube formation was photographed at 5 h using a phase contrast inverted microscope. Each experiment was performed in duplicate. The pictures show the appearance of endothelial cell tubes on Matrigel® precoated plates in culture medium (i) and BM supernatant fluid from healthy control (ii), RA (iii), RARS (iv), 5q syndrome (v), RAEB (high-risk MDS) (vi) and RCMD (vii-viii) patients at 1∶10 dilution in culture medium. As the arrows show in the figure, the tube morphology was strikingly influenced by BM supernatant fluid from MDS (iii-viii) with respect to the controls (ii). The tubes originated after the incubation of BMEC-1 or HMVEC-L with the BM supernatant fluid from RCMD patients (vii-viii) were almost completely disrupted and formed closed capillary networks. MDS: myelodysplastic syndrome; BM: bone marrow; BMEC-1: bone marrow endothelial cells; HMVEC-L: lung-derived normal human microvascular endothelial cells; RA: refractory anemia; RARS: refractory anemia with ring sideroblast; RAEB refractory anemia with excess of blasts; RCMD: refractory cytopenia with multilineage dysplasia. (Controls n = 13; RA n = 5; RARS n = 6; 5q syndrome n = 2; RAEB n = 4; RCMD n = 7).</p

Effect of MDS BM microenvironment on BMEC-1 proliferation.

<p>(A) BMEC-1 proliferation curve. To analyze the effect of the BM supernatant fluid from MDS patients and controls on BMEC-1 proliferation, the cell line was incubated with BM supernatant fluid. The cell number was estimated by MTT at two, four or six days. The measurement of absorbance is indicative of the rate of cell proliferation and each value of each patient is the mean of four independent experiments. Each point is the mean of these values ± SEM. The graphics show the increase of proliferation in MDS patients. ANOVA test was used to analyze the overall MDS results at sixth day. The proliferation was 2.4 times higher in MDS than controls (<i>p</i><0.005). (B) The box plot compares median levels of BMEC-1 proliferation at sixth day in the different subtypes of MDS. Whiskers represent the range. Significant differences between RCMD and the control group (<i>p</i><0.01), the other low-risk MDS and the control group (<i>p</i><0.05) and high-risk MDS patients and the controls (<i>p</i><0.05) were observed by Mann-Whitney test. MDS: myelodysplastic syndrome; BM: bone marrow; BMEC-1: bone marrow endothelial cells; MTT: Thiazolyl Blue Tetrazolium Bromide; SEM: standard error of the mean; RCMD: refractory cytopenia with multilineage dysplasia. (Controls n = 8; MDS n = 14; Low-Risk MDS excluding RCMD n = 6; RCMD n = 4; High-Risk MDS n = 4).</p

Circulating soluble endoglin modifies the inflammatory response in mice

<div><p>Inflammation is associated with every health condition, and is an important component of many pathologies such as cardiovascular diseases. Circulating levels of soluble endoglin have been shown to be higher in the serum of patients with cardiovascular diseases with a significant inflammatory component. The aim of this study was to evaluate the implication of circulating soluble endoglin in the inflammatory response. For this purpose, a transgenic mouse expressing human soluble endoglin (sEng+) was employed, and three different inflammatory approaches were used to mimic inflammatory conditions in different tissues. This study shows that control sEng+ mice have a normal inflammatory state. The lung and kidney injury induced by the inflammatory agents was reduced in sEng+ mice, especially the intra-alveolar and kidney infiltrates, suggesting a possible reduction in inflammation induced by soluble endoglin. To deepen into this possible effect, the leukocyte number in the bronchoalveolar lavage and <i>air pouch</i> lavage was evaluated and a significant reduction of neutrophil infiltration in LPS-treated lungs and ischemic kidneys from sEng+ with respect to WT mice was observed. Additionally, the mechanisms through which soluble endoglin prevents inflammation were studied. We found that in sEng+ animals the increment of proinflammatory cytokines, TNFα, IL1β and IL6, induced by the inflammatory stimulus was reduced. Soluble endoglin also prevents the augmented adhesion molecules, ICAM, VCAM and E-selectin induced by the inflammatory stimulus. In addition, vascular permeability increased by inflammatory agents was also reduced by soluble endoglin. These results suggest that soluble endoglin modulates inflammatory-related diseases and open new perspectives leading to the development of novel and targeted approaches for the prevention and treatment of cardiovascular diseases.</p></div

Inflammatory cytokines in plasma.

<p>Quantitative analysis of inflammatory cytokines (TNFα, IL1β and IL6) in plasma was performed by ELISA. Data are expressed as mean ± SEM. n = 5 in each group of mice.</p

Morphological kidney changes after ischemia-reperfusion.

<p>(A) Representative images of hematoxylin and eosin-stained kidney sections from five animals in each experimental group. Kidneys were fixed with 4% paraformaldehyde, embedded in paraffin, and then cut into 5 μm thick sections before being stained. Photomicrographs were obtained with a Nikon Eclipse E800 microscope. Both WT and sEng+ mice kidneys show cortical and medullary hyperemia with areas of tubular necrosis found in the deep and superficial cortex, tubular cast and a significant expansion of the tubular structure with destruction of the epithelium (arrow) and inflammatory infiltrates (•). Magnification x200 and x400. (B) Severity of kidney injury was scored by a pathologist using a semiquantitative histopathology score system which evaluates kidney injury in three categories: glomerular fibrosis, tubular obstruction and dilation and neutrophil infiltration. Data are expressed as mean ± SEM. n = 5 in each group of mice, +p<0,0001 <i>vs</i> control, two-way ANOVA. (C) Evaluation score of neutrophil infiltration. n = 5 in each group of mice, *p<0,01 <i>vs</i> ischemic WT, T test.</p

Inflammatory cytokines in lung tissue.

<p>Quantitative analysis of inflammatory cytokines (TNFα, IL1β and IL6) in lung tissue was performed by RT-PCR (A-B) and ELISA (C-E). Data are expressed as mean ± SEM. n = 5 in each group of mice. (A) IL1β, +p<0,0005 LPS <i>vs</i> control, two-way ANOVA; (B) IL6, +p<0,0001 LPS <i>vs</i> control, two-way ANOVA; (C) TNFα, +p<0,005 LPS <i>vs</i> control, two-way ANOVA; (D) IL1β, +p<0,005 LPS <i>vs</i> control, two-way ANOVA; (E) IL6, +p<0,05 LPS <i>vs</i> control, two-way ANOVA.</p

Inflammatory cytokines in BAL and air-pouch lavage.

<p>Quantitative analysis of proinflammatory cytokines (TNFα, IL1β and IL6) in BAL and air pouch lavage was performed by ELISA and presented as picograms per milliliter of lavage. Data are expressed as mean ± SEM. n = 5 in each group of mice. (A) TNFα concentration in BAL, +p<0,0001 LPS <i>vs</i> control, two-way ANOVA; (B) TNFα concentration in air pouch lavage, +p<0,05 carrageenan <i>vs</i> control, two-way ANOVA; (C) IL1β concentration in BAL, *p<0,05 <i>vs</i> LPS WT, T test; (D) IL1β concentration in air pouch lavage, +p<0,01 carrageenan <i>vs</i> control, two-way ANOVA; (E) IL6 concentration in BAL, #p<0,001 <i>vs</i> control sEng+, T test; *p<0,05 <i>vs</i> LPS, T test; (F) IL6 concentration in air pouch lavage, *p<0,05 <i>vs</i> carrageenan WT, T test.</p

Morphological lung changes after LPS treatment.

<p>(A) Representative images of hematoxylin and eosin stained lung sections of five animals from each experimental group. Lungs were fixed with 4% paraformaldehyde, embedded in paraffin, and cut into 5 μm thick sections before being stained. Photomicrographs were obtained with a Nikon Eclipse E800 microscope. Both WT and sEng+ mice lungs show marked inflammatory infiltrates (arrow) after LPS treatment, inter-alveolar septal thickening (arrow head), and interstitial edema (•). Magnification x200 and x400. (B) Severity of lung injury was scored by a pathologist using a semiquantitative histopathology score system which evaluates lung injury in four categories: alveolar septae, alveolar hemorrhage, intra-alveolar fibrin, and intra-alveolar infiltrates. Data are expressed as mean ± SEM. n = 5 in each group of mice, +p<0,0001 <i>vs</i> control, two-way ANOVA. (C) Evaluation score of intra-alveolar infiltrates. n = 5 in each group of mice.</p