Transcript levels of VEGF receptors/co-receptors during <i>ex vivo</i> MMT.

<p>The histograms represent the relative mRNA expression of VEGF receptors/co-receptors in non-epithelioid effluent-derived MCs (Non-E, n = 21) compared with epithelioid effluent-derived MCs (E, n = 30). The data are depicted as mean value ± SE of effluent samples from 51 PD patients. (<b>A, B and D</b>) Similar results to those of <i>in vitro</i> MMT: a significant down-regulated expression of receptors VEGFR-1 (p = 0.0009) and VEGFR-2 (p<0.0001) and up-regulation of co-receptor Nrp-1 (p = 0.001). (<b>C and E</b>) The expression of VEGFR-3 and Nrp-2 did not show variation.</p

Expression levels and cellular distribution of Nrp-1 and VEGFR2 proteins during <i>in vitro</i> and <i>ex vivo</i> MMT.

<p>(<b>A</b>) Western blots show the expression levels of Nrp-1 and VEGFR-2 in total cell lysates during <i>in vitro</i> and <i>ex vivo</i> MMT. Expression of α-tubulin is employed as a loading control. Human umbilical vein endothelial cells (HUVEC) are used as a positive control. The histograms depict the quantification of Nrp-1 and VEGFR2 levels compared with α-tubulin. Data are representative of 5 samples for each condition from PD patients and omentum samples included in the study. (<b>B</b>) The expression of Nrp-1, VEGFR-2, and VEGF was analyzed by immunofluorescence microscopy in omentum and effluent-derived MCs. MCs were double stained for Nrp-1 (green) and VEGFR-2 (red), and single stained for VEGF (green). Nuclei were stained with DAPI. Nrp-1 and VEGF show a membrane distribution in omentum and epithelioid MCs (<b>b, c, </b><b>h, i</b>)<b>.</b> During <i>in vitro</i> (<b>e, f</b>) and <i>ex vivo</i> (<b>k, l</b>) MMT both proteins change their localization and are internalized. The expression of VEGFR-2 is down-regulated but it does not show differences in localization during <i>in vitro</i> (<b>a, d</b>) and <i>ex vivo</i> (<b>g, j</b>) MMT.</p

Analysis of the expression levels of VEGF and Sema3A during the MMT process.

<p>(<b>A and B</b>) The expression of VEGF in MC culture supernatants during <i>in vitro</i> (n = 11, p = 0.006) and <i>ex</i> vivo (n = 51, p<0.0001) MMT was analyzed by ELISA. (<b>C</b>) VEGF secretion in effluents of PD patients draining MCs with different phenotypes was also analyzed by ELISA (E, n = 16 and Non-E, n = 11; p = 0.04). (<b>D</b>) Correlation between VEGF levels <i>ex vivo</i> and effluents of PD patients (r = 0.578, p<0.0001) (<b>E and F</b>) Sema-3A mRNA expression, in both <i>in vitro</i> (p = 0.006) and <i>ex vivo</i> (p = 0.0002) MMT, was analyzed by quantitative RT-PCR. (<b>G</b>) Sema-3A secretion in PD effluents (n = 16 E and 11 Non-E; p = 0.02). Box Plots represent 25% and 75% percentiles, median, minimum and maximum values. Numbers above boxes and histograms depict mean ± SE.</p

Baseline characteristics of PD patients with different phenotypes of mesothelial cells.

<p>CCr: creatinine clearance. MTC: mass transfer coefficient. UF: ultrafiltration after a 4-hour dwell with glucose 3.86%. VEGF: vascular endothelial growth factor. Values represent mean ± SD for median clinical parameters or mean ± SE for supernatant VEGF.</p

Nrp-1 immunohistochemical analysis in peritoneal human biopsies.

<p>The expression of Nrp-1 and the mesothelial marker cytokeratin was analyzed in human peritoneal specimens by immunohistochemistry. Positive cells for antibodies used (Nrp-1 and Cytokeratin) show brown staining. Nuclei are counterstained in blue. (<b>a, b</b>) Control peritoneal tissue, with a conserved mesothelial cell monolayer showing an epithelioid morphology (with a 20X objective). These cells show weak expression of Nrp-1 and a marked staining for cytokeratin (arrows). No expression of these proteins was observed in the submesothelial area (region under mesothelial monolayer) (<b>c, d</b>) Fibrotic tissue sample from PD patient showing the loss of mesothelial monolayer and invading spindle-like mesothelial cells in submesothelial area (with a 40X objective). These cells present a strong staining for Nrp-1 (<b>c</b>), and are also positive for cytokeratin (<b>d</b>) (arrows). Pictures are representative of 5 cases of PD patient samples and 4 of control samples.</p

Characterization of MMT <i>in vitro</i> and <i>ex vivo</i>.

<p>(<b>A</b>) Representative pictures of omentum-derived MCs, either untreated or treated with TGF-β1 and IL-1β (MMT <i>in vitro</i>), and the two morphologies observed in confluent cultures of effluent-derived MCs: epithelioid and non-epithelioid. (<b>B</b>) Transcript levels of mesenchymal markers were analyzed by quantitative RT-PCR (n = 11 Omentum, 11 Omentum T+I, 30 E and 21 Non-E). Results show down-regulation of E-cadherin and up-regulation of snail expression during both <i>in vitro</i> and <i>ex vivo</i> MMT. The histograms also show a significant up-regulation of N-cadherin and fibronectin expression in mesenchymal MCs compared to omentum and epithelioid MCs. Data are depicted as mean value ± SE. Symbols show statistical differences between groups.</p

Transcript levels of VEGF receptors/co-receptors during <i>in vitro</i> MMT.

<p>mRNA levels of VEGF receptors/co-receptors were analyzed by quantitative RT-PCR. The results represent the relative mRNAs expression of VEGF receptors in omentum-derived MCs treated with TGF-β1 plus IL-1 β (Omentum T+I) compared with untreated MCs (Omentum NT). The data are depicted as mean value ± SE of omentum samples from 11 healthy donors. (<b>A, B and D</b>) The histograms show a down-regulation in expression of receptors VEGFR-1 (p = 0.005) and VEGFR-2 (p = 0.0005) and up-regulation of co-receptor Nrp-1 (p = 0.004). (<b>C and E</b>) The expression of VEGFR-3 and Nrp-2 did not show significant differences.</p

Proposed model of the role of VEGF receptors and co-receptors during MMT induced by peritoneal dialysis.

<p>(<b>A</b>) General scheme of VEGF receptors and co-receptors and the processes in which they are involved. (<b>B</b>) A normal peritoneum shows baseline VEGF receptors and co-receptors expression, and normal VEGF and Sema-3A levels are secreted at the peritoneal cavity. During continuous peritoneal dialysis, MMT takes place in the peritoneum. Denudation of MC monolayer, submesothelial fibrosis, and augmented vessel number begin to appear as visible signs as a result of the MMT. In addition, MCs change the expression pattern of VEGFRs and co-receptors. At an initial stage, a higher proliferation rate of MCs takes place, perhaps in order to repair the damage in the monolayer. However, MCs could fail to repair the peritoneal damage and MMT progress. As a result of MMT progression, MCs increase secreted VEGF levels while its receptors are down-regulated and Nrp-1 co-receptor increases at late stages of the MMT. The binding of VEGF to Nrp-1 would induce MC invasion to the submesothelial stroma. Therefore, MMT would determine MC behaviour in terms of proliferation and invasion in response to VEGF.</p

The expression of FSP-1 is up-regulated during <i>in vitro</i> and <i>ex vivo</i> MMT.

<p>The expression of FSP-1 and cytokeratin was analyzed in omentum and effluent-derived MCs by immunofluorescence using confocal microscopy. MCs were double stained for cytokeratin (green) and FSP-1 (red). Nuclei were stained with DAPI. Pictures show <i>in vitro</i> a Cyt+++/FSP1- staining for normal MCs (<b>a–c</b>), and Cyt+/FSP1++ for MCs treated with TGF-β1 and IL-1β (<b>d–f</b>). <i>Ex vivo</i>, pictures show Cyt++/FSP1+ staining for epithelioid MCs (<b>g–i</b>) and Cyt+/FSP1++ staining for non-epithelioid MCs (<b>j–l)</b> proving the mesenchymal conversion of MCs. Data are representative of 5 samples for each condition from PD patients and omentum samples included in the study.</p

Treatment with anti-VEGF and anti-Np1b blocking antibodies interferes with the proliferation of mesothelial cells.

<p>(<b>A</b>) The proliferation capacity of MCs throughout <i>in vitro</i> and <i>ex vivo</i> MMT was analyzed by the incorporation of [3H]-thymidine (n = 20). Bar graphic depicts the radioactivity emitted (c.p.m.) in each condition. Data are depicted as mean value ± SE. Symbols show statistical differences between groups. (<b>B and E</b>) Effect of anti-VEGF (n = 10) and anti-Np1b antibodies (n = 8), or IgG isotype control antibody on the proliferation capacity of omentum-derived MCs, either untreated (<b>B</b>) or treated with TGF-β1 plus IL-1β (<b>C</b>); and on the proliferation capacity of effluent-derived MCs with epithelioid (<b>D</b>) or non-epithelioid phenotype (<b>E</b>). Bar graphics represent proliferation percentage of treatments over control cells. Data are depicted as mean value ± SE. Symbols show statistical differences between groups.</p