Functional relevance of the switch of VEGF receptors/co-receptors during peritoneal dialysis-induced mesothelial to mesenchymal transition

Vascular endothelial growth factor (VEGF) is up-regulated during mesothelial to mesenchymal transition (MMT) and has been associated with peritoneal membrane dysfunction in peritoneal dialysis (PD) patients. It has been shown that normal and malignant mesothelial cells (MCs) express VEGF receptors (VEGFRs) and co-receptors and that VEGF is an autocrine growth factor for mesothelioma. Hence, we evaluated the expression patterns and the functional relevance of the VEGF/VEGFRs/co-receptors axis during the mesenchymal conversion of MCs induced by peritoneal dialysis. Omentum-derived MCs treated with TGF-β1 plus IL-1β (in vitro MMT) and PD effluent-derived MCs with non-epithelioid phenotype (ex vivo MMT) showed down-regulated expression of the two main receptors Flt-1/VEGFR-1 and KDR/VEGFR-2, whereas the co-receptor neuropilin-1 (Nrp-1) was up-regulated. The expression of the Nrp-1 ligand semaphorin-3A (Sema-3A), a functional VEGF competitor, was repressed throughout the MMT process. These expression pattern changes were accompanied by a reduction of the proliferation capacity and by a parallel induction of the invasive capacity of MCs that had undergone an in vitro or ex vivo MMT. Treatment with neutralizing anti-VEGF or anti-Nrp-1 antibodies showed that these molecules played a relevant role in cellular proliferation only in naïve omentum-derived MCs. Conversely, treatment with these blocking antibodies, as well as with recombinant Sema-3A, indicated that the switched VEGF/VEGFRs/co-receptors axis drove the enhanced invasion capacity of MCs undergoing MMT. In conclusion, the expression patterns of VEGFRs and co-receptors change in MCs during MMT, which in turn would determine their behaviour in terms of proliferation and invasion in response to VEGFThis work was supported by grant SAF2010-21249 from the ‘‘Ministerio de Economía y Competitividad’’ to M.L.C. and by grant S2010/BMD-2321 from ‘‘Comunidad Autónoma de Madrid’’ to M.L.C. and R.S. This work was also partially supported by grants PI 09/0776 from ‘‘Fondo de Investigaciones Sanitarias’’ to A.A., and RETICS 06/0016 (REDinREN, Fondos FEDER, EU) to R.S

Tamoxifen ameliorates peritoneal membrane damage by blocking mesothelial to mesenchymal transition in peritoneal dialysis

Mesothelial-to-mesenchymal transition (MMT) is an auto-regulated physiological process of tissue repair that in uncontrolled conditions such as peritoneal dialysis (PD) can lead to peritoneal fibrosis. The maximum expression of peritoneal fibrosis induced by PD fluids and other peritoneal processes is the encapsulating peritoneal sclerosis (EPS) for which no specific treatment exists. Tamoxifen, a synthetic estrogen, has successfully been used to treat retroperitoneal fibrosis and EPS associated with PD. Hence, we used in vitro and animal model approaches to evaluate the efficacy of Tamoxifen to inhibit the MMT as a trigger of peritoneal fibrosis. In vitro studies were carried out using omentum-derived mesothelial cells (MCs) and effluent-derived MCs. Tamoxifen blocked the MMT induced by transforming growth factor (TGF)-β1, as it preserved the expression of E-cadherin and reduced the expression of mesenchymal-associated molecules such as snail, fibronectin, collagen-I, α-smooth muscle actin, and matrix metalloproteinse-2. Tamoxifen-treatment preserved the fibrinolytic capacity of MCs treated with TGF-β1 and decreased their migration capacity. Tamoxifen did not reverse the MMT of non-epitheliod MCs from effluents, but it reduced the expression of some mesenchymal molecules. In mice PD model, we demonstrated that MMT progressed in parallel with peritoneal membrane thickness. In addition, we observed that Tamoxifen significantly reduced peritoneal thickness, angiogenesis, invasion of the compact zone by mesenchymal MCs and improved peritoneal function. Tamoxifen also reduced the effluent levels of vascular endothelial growth factor and leptin. These results demonstrate that Tamoxifen is a therapeutic option to treat peritoneal fibrosis, and that its protective effect is mediated via modulation of the MMT processThis work was supported by grant SAF2010-21249 from the ‘‘Ministerio de Economia y Competitividad’’ to MLC and by grant S2010/BMD-2321 from ‘‘Comunidad Autónoma de Madrid’’ to MLC and RS. This work was also partially supported by grants PI 09/0776 from ‘‘Fondo de Investigaciones Sanitarias’’ to AA, and RETICS 06/0016 (REDinREN, Fondos FEDER, EU) to R

Analysis of early mesothelial cell responses to Staphylococcus epidermidis isolated from patients with peritoneal dialysis-associated peritonitis

The major complication of peritoneal dialysis (PD) is the development of peritonitis, an infection within the abdominal cavity, primarily caused by bacteria. PD peritonitis is associated with significant morbidity, mortality and health care costs. Staphylococcus epidermidis is the most frequently isolated cause of PD-associated peritonitis. Mesothelial cells are integral to the host response to peritonitis, and subsequent clinical outcomes, yet the effects of infection on mesothelial cells are not well characterised. We systematically investigated the early mesothelial cell response to clinical and reference isolates of S. epidermidis using primary mesothelial cells and the mesothelial cell line Met-5A. Using an unbiased whole genome microarray, followed by a targeted panel of genes known to be involved in the human antibacterial response, we identified 38 differentially regulated genes (adj. p-value < 0.05) representing 35 canonical pathways after 1 hour exposure to S. epidermidis. The top 3 canonical pathways were TNFR2 signaling, IL-17A signaling, and TNFR1 signaling (adj. pvalues of 0.0012, 0.0012 and 0.0019, respectively). Subsequent qPCR validation confirmed significant differences in gene expression in a number of genes not previously described in mesothelial cell responses to infection, with heterogeneity observed between clinical isolates of S. epidermidis, and between Met-5A and primary mesothelial cells. Heterogeneity between different S. epidermidis isolates suggests that specific virulence factors may play critical roles in influencing outcomes from peritonitis. This study provides new insights into early mesothelial cell responses to infection with S. epidermidis, and confirms the importance of validating findings in primary mesothelial cells

Measuring universal health coverage based on an index of effective coverage of health services in 204 countries and territories, 1990–2019: a systematic analysis for the Global Burden of Disease Study 2019

Background Achieving universal health coverage (UHC) involves all people receiving the health services they need, of high quality, without experiencing financial hardship. Making progress towards UHC is a policy priority for both countries and global institutions, as highlighted by the agenda of the UN Sustainable Development Goals (SDGs) and WHO's Thirteenth General Programme of Work (GPW13). Measuring effective coverage at the health-system level is important for understanding whether health services are aligned with countries' health profiles and are of sufficient quality to produce health gains for populations of all ages. Methods Based on the Global Burden of Diseases, Injuries, and Risk Factors Study (GBD) 2019, we assessed UHC effective coverage for 204 countries and territories from 1990 to 2019. Drawing from a measurement framework developed through WHO's GPW13 consultation, we mapped 23 effective coverage indicators to a matrix representing health service types (eg, promotion, prevention, and treatment) and five population-age groups spanning from reproductive and newborn to older adults (≥65 years). Effective coverage indicators were based on intervention coverage or outcome-based measures such as mortality-to-incidence ratios to approximate access to quality care; outcome-based measures were transformed to values on a scale of 0–100 based on the 2·5th and 97·5th percentile of location-year values. We constructed the UHC effective coverage index by weighting each effective coverage indicator relative to its associated potential health gains, as measured by disability-adjusted life-years for each location-year and population-age group. For three tests of validity (content, known-groups, and convergent), UHC effective coverage index performance was generally better than that of other UHC service coverage indices from WHO (ie, the current metric for SDG indicator 3.8.1 on UHC service coverage), the World Bank, and GBD 2017. We quantified frontiers of UHC effective coverage performance on the basis of pooled health spending per capita, representing UHC effective coverage index levels achieved in 2019 relative to country-level government health spending, prepaid private expenditures, and development assistance for health. To assess current trajectories towards the GPW13 UHC billion target—1 billion more people benefiting from UHC by 2023—we estimated additional population equivalents with UHC effective coverage from 2018 to 2023. Findings Globally, performance on the UHC effective coverage index improved from 45·8 (95% uncertainty interval 44·2–47·5) in 1990 to 60·3 (58·7–61·9) in 2019, yet country-level UHC effective coverage in 2019 still spanned from 95 or higher in Japan and Iceland to lower than 25 in Somalia and the Central African Republic. Since 2010, sub-Saharan Africa showed accelerated gains on the UHC effective coverage index (at an average increase of 2·6% [1·9–3·3] per year up to 2019); by contrast, most other GBD super-regions had slowed rates of progress in 2010–2019 relative to 1990–2010. Many countries showed lagging performance on effective coverage indicators for non-communicable diseases relative to those for communicable diseases and maternal and child health, despite non-communicable diseases accounting for a greater proportion of potential health gains in 2019, suggesting that many health systems are not keeping pace with the rising non-communicable disease burden and associated population health needs. In 2019, the UHC effective coverage index was associated with pooled health spending per capita (r=0·79), although countries across the development spectrum had much lower UHC effective coverage than is potentially achievable relative to their health spending. Under maximum efficiency of translating health spending into UHC effective coverage performance, countries would need to reach $1398 pooled health spending per capita (US$ adjusted for purchasing power parity) in order to achieve 80 on the UHC effective coverage index. From 2018 to 2023, an estimated 388·9 million (358·6–421·3) more population equivalents would have UHC effective coverage, falling well short of the GPW13 target of 1 billion more people benefiting from UHC during this time. Current projections point to an estimated 3·1 billion (3·0–3·2) population equivalents still lacking UHC effective coverage in 2023, with nearly a third (968·1 million [903·5–1040·3]) residing in south Asia. Interpretation The present study demonstrates the utility of measuring effective coverage and its role in supporting improved health outcomes for all people—the ultimate goal of UHC and its achievement. Global ambitions to accelerate progress on UHC service coverage are increasingly unlikely unless concerted action on non-communicable diseases occurs and countries can better translate health spending into improved performance. Focusing on effective coverage and accounting for the world's evolving health needs lays the groundwork for better understanding how close—or how far—all populations are in benefiting from UHC

Tamoxifen blocks TGF-β1-induced MMT of MCs.

<p>Omentum-derived MCs were treated or not with 1 ng/mL TGF-β1 for 24 or 48 hours, in the presence of different doses of Tamoxifen (0, 3 and 6 µM). (<b>A</b>) Western blot analyses show that Tamoxifen treatment prevents TGF-β1-induced E-cadherin down-regulation as well as α-SMA, collagen I, fibronectin and MMP-2 up-regulation. A representative experiment is shown. (<b>B to F</b>) The experiments were repeated at least five times and results are depicted as means ± SE. The expressions of E-cadherin (<b>B</b>) and MMP-2 (<b>F</b>) were analyzed at 24 hours, whereas the expressions of α-SMA (<b>C</b>), collagen I (<b>D</b>) and fibronectin (<b>E</b>), were analyzed at 48 hours of treatments. (<b>G</b>) Analysis of the migration capacity in transwell units demonstrates that Tamoxifen (6 µM) reduces the TGF-β1-indued migratory capacity of MCs to basal levels. The experiments, made in triplicates, were repeated at least four times. Box plots represent the median, minimum and maximum values, as well as the 25th and 75th percentiles.</p

Tamoxifen reverts the MMT induced by TGF-β1 <i>in vitro</i>.

<p>Omentum-MCs were stimulated with TGF-β1 during 48 hours and then the cells were either left untreated, treated with TGF-β1 or treated with Tamoxifen (6 or 10 µM) during additional 48 hours. <b>(A)</b> Phase-contrast microscopy shows that Tamoxifen reverts partially the non-epithelioid morphology of omentum-derived MC treated with TGF-β1. <b>(B)</b> Quantitative RT-PCR analysis demonstrates that administration of Tamoxifen (6 µM) after TGF-β1 withdrawal restores partially E-cadherin expression. Bar graphic depicts the expression of E-cadherin-encoding mRNA in relative units (R.U.)</p

Parallelism between MMT, PM thickness and time on PD.

<p><b>(A)</b> Immunofluorescence microscopy images of parietal peritoneal sections stained for cytokeratin (green) and FSP-1 (red), with DAPI counterstaining, show accumulation of trans-differentiated mesothelial cells in the submesothelial space at 7, 15 and 30 days of PD mice. Progressive time-dependent increases of MMT and PM thickness is observed during PD fluid exposure. Representative slides are presented. Magnification ×200. <b>(B)</b> Quantification of the submesothelial MMT (cytokeratin/FSP-1 double positive cells per field) at different time points. <b>(C)</b> Quantification of peritoneal thickness ( µm) at different time points. Box Plots represent 25% and 75% percentiles, median, minimum and maximum values. Numbers above boxes depict means ± SE. Symbols show statistical differences between groups. <b>(D)</b> Correlation between both MMT and peritoneal thickness was determined by Spearman regression analysis.</p

Tamoxifen preserves the fibrinolytic capacity of TGF-β1-treated MCs.

<p>Omentum-derived MCs were treated or not with 1 ng/mL TGF-β1 during 48 hours, in the presence of different doses of Tamoxifen (0, 3 and 6 µM). <b>(A to C)</b> Stimulation of MCs with TGF-β1 inhibits the expression of the fibrinolytic factors uPA <b>(A)</b>, uPAR <b>(B)</b> and tPA <b>(C)</b>, and treatments with different doses of Tamoxifen restore the basal levels of these factors or increase their synthesis above basal levels. <b>(D)</b>. TGF-β1 treatment increases the expression of PAI-1, and its expression is not affected by Tamoxifen. The levels of these factors were measured in culture media supernatants by ELISA and results are depicted as nanograms per milligrams of total cellular proteins <b>(E)</b>. The PAI/tPA-ratio, an important marker of fibrinolytic capacity decline, increases in response to TGF-β1 and returns to basal levels when Tamoxifen is added at 6 µM. Box plots show the 25th and 75th percentiles, median, minimum and maximum values of five independent experiments. The symbols represent the statistical differences between the groups.</p

Tamoxifen reverts partially the mesenchymal phenotype of effluent-derived MCs.

<p>Effluent-derived MCs with mesenchymal phenotype (as determined by non-epitheliod morphology, low expression of E-cadherin and up-regulated expression of mesenchymal markers) were treated with different doses of Tamoxifen (0, 3, 6, and 10 µM) and analyzed at 48 hours. Omentum-derived MCs were employed as control. (<b>A</b>) Western blot analyses show that Tamoxifen treatments do not re-induce E-cadherin expression but inhibit the expression of the mesenchymal molecules α-SMA, collagen I, fibronectin and MMP-2; being the effects of Tamoxifen more evident at high doses (6 and 10 µM). A representative experiment is shown. (<b>B to F</b>) The experiments were repeated with five different samples of effluent-derived MCs and results of the expression of E-cadherin <b>(B)</b>, α-SMA <b>(C)</b>, collagen I <b>(D)</b>, MMP-2 <b>(E)</b> and fibronectin <b>(F)</b> are depicted as means ± SE. Quantitative RT-PCR demonstrates that the expression of Snail mRNA is not inhibited by any dose of Tamoxifen tested <b>(G)</b>. Bars depict means ± SE of five independent experiments.</p

Effects of Tamoxifen on the number of fibroblasts derived from MCs.

<p><b>(A)</b> Immunofluorescence microscopy analysis of parietal peritoneal sections stained for cytokeratin (green) and FSP-1 (red) with DAPI counterstaining show accumulation of trans-differentiated mesothelial cells in the submesothelial space (cytokeratin positive cells) in the PDF group, some of which co-express FSP-1 (yellow cells in the Merge panel). The administration of Tamoxifen reduces the number of cytokeratin/FSP-1 double positive cells per field. Representative slides are presented. Magnification ×200. <b>(B)</b> Reductions of the number of cytokeratin/FSP-1 positive fibroblasts by Tamoxifen are significant. Box Plots represent 25% and 75% percentiles, median, minimum and maximum values. Numbers above boxes depict means ± SE. Symbols show statistical differences between groups.</p