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

Macrophages direct cancer cells through a LOXL2-mediated metastatic cascade in pancreatic ductal adenocarcinoma

[Objective]: The lysyl oxidase-like protein 2 (LOXL2) contributes to tumour progression and metastasis in different tumour entities, but its role in pancreatic ductal adenocarcinoma (PDAC) has not been evaluated in immunocompetent in vivo PDAC models.[Design]: Towards this end, we used PDAC patient data sets, patient-derived xenograft in vivo and in vitro models, and four conditional genetically-engineered mouse models (GEMMS) to dissect the role of LOXL2 in PDAC. For GEMM-based studies, K-Ras +/LSL-G12D;Trp53 LSL-R172H;Pdx1-Cre mice (KPC) and the K-Ras +/LSL-G12D;Pdx1-Cre mice (KC) were crossed with Loxl2 allele floxed mice (Loxl2Exon2 fl/fl) or conditional Loxl2 overexpressing mice (R26Loxl2 KI/KI) to generate KPCL2KO or KCL2KO and KPCL2KI or KCL2KI mice, which were used to study overall survival; tumour incidence, burden and differentiation; metastases; epithelial to mesenchymal transition (EMT); stemness and extracellular collagen matrix (ECM) organisation.[Results]: Using these PDAC mouse models, we show that while Loxl2 ablation had little effect on primary tumour development and growth, its loss significantly decreased metastasis and increased overall survival. We attribute this effect to non-cell autonomous factors, primarily ECM remodelling. Loxl2 overexpression, on the other hand, promoted primary and metastatic tumour growth and decreased overall survival, which could be linked to increased EMT and stemness. We also identified tumour-associated macrophage-secreted oncostatin M (OSM) as an inducer of LOXL2 expression, and show that targeting macrophages in vivo affects Osm and Loxl2 expression and collagen fibre alignment.[Conclusion]: Taken together, our findings establish novel pathophysiological roles and functions for LOXL2 in PDAC, which could be potentially exploited to treat metastatic disease.JCL-G received support from a 'la Caixa' Foundation (ID 100010434) fellowship (LCF/BQ/DR21/11880011). This study was supported by ISCIII FIS grants PI18/00757 and PI21/01110 (BSJ) and PI18/00267 (LG-B), and grants from the Spanish Ministry of Economy and Innovation SAF2016-76504-R (ACan and FP), PID2019-111052RB-I00 (FP), PID2019-104644RB-I00 (GM-B), a Ramón y Cajal Merit Award RYC-2012–12104 (BSJ) and ISCIII, CIBERONC, CB16/12/00446 (ACar) and CB16/12/00295 (ACan and GM-B), all of them co-financed through Fondo Europeo de Desarrollo Regional (FEDER) 'Una manera de hacer Europa'; a Fero Foundation Grant (BSJ); a Coordinated grant (GC16173694BARB) from the Fundación Científica Asociación Española Contra el Cáncer (FC-AECC) (BSJ); a Miguel Servet award (CP16/00121) (PS); a DFG, German Research Foundation Grant—Project no: 492 436 553 (KG); and a Max Eder Fellowship of the German Cancer Aid (111746) (PCH

Comprehensive description of clinical characteristics of a large systemic Lupus Erythematosus Cohort from the Spanish Rheumatology Society Lupus Registry (RELESSER) with emphasis on complete versus incomplete lupus differences

Systemic lupus erythematosus (SLE) is an autoimmune disease characterized by multiple organ involvement and pronounced racial and ethnic heterogeneity. The aims of the present work were (1) to describe the cumulative clinical characteristics of those patients included in the Spanish Rheumatology Society SLE Registry (RELESSER), focusing on the differences between patients who fulfilled the 1997 ACR-SLE criteria versus those with less than 4 criteria (hereafter designated as incomplete SLE (iSLE)) and (2) to compare SLE patient characteristics with those documented in other multicentric SLE registries. RELESSER is a multicenter hospital-based registry, with a collection of data from a large, representative sample of adult patients with SLE (1997 ACR criteria) seen at Spanish rheumatology departments. The registry includes demographic data, comprehensive descriptions of clinical manifestations, as well as information about disease activity and severity, cumulative damage, comorbidities, treatments and mortality, using variables with highly standardized definitions. A total of 4.024 SLE patients (91% with ≥4 ACR criteria) were included. Ninety percent were women with a mean age at diagnosis of 35.4 years and a median duration of disease of 11.0 years. As expected, most SLE manifestations were more frequent in SLE patients than in iSLE ones and every one of the ACR criteria was also associated with SLE condition; this was particularly true of malar rash, oral ulcers and renal disorder. The analysis-adjusted by gender, age at diagnosis, and disease duration-revealed that higher disease activity, damage and SLE severity index are associated with SLE [OR: 1.14; 95% CI: 1.08-1.20 (P < 0.001); 1.29; 95% CI: 1.15-1.44 (P < 0.001); and 2.10; 95% CI: 1.83-2.42 (P < 0.001), respectively]. These results support the hypothesis that iSLE behaves as a relative stable and mild disease. SLE patients from the RELESSER register do not appear to differ substantially from other Caucasian populations and although activity [median SELENA-SLEDA: 2 (IQ: 0-4)], damage [median SLICC/ACR/DI: 1 (IQ: 0-2)], and severity [median KATZ index: 2 (IQ: 1-3)] scores were low, 1 of every 4 deaths was due to SLE activity. RELESSER represents the largest European SLE registry established to date, providing comprehensive, reliable and updated information on SLE in the southern European population

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 VEGF. © 2013 Pérez-Lozano et al.SAF2010-21249 from the Ministerio de Economıa y Competitividad; S2010/BMD-2321 from Comunidad Autonoma de Madrid; PI 09/0776 from Fondo de Investigaciones Sanitarias; RETICS 06/0016 (REDinREN, Fondos FEDER, EU)Peer Reviewe

Asymmetric cellulose triacetate is a safe and effective alternative for online haemodiafiltration

Background: In post-dilution haemodiafiltration only synthetic membranes have been used to date. Asymmetric cellulose triacetate (ATA™) is now available, whose characteristics are suitable for this technique. Objectives: To describe the in vivo performance and behaviour of this membrane, to identify its depurative effectiveness, use in clinical practice and its biocompatibility, both acute and after one month of treatment. Methods: Observational prospective study of 23 patients who were dialysed for 4 weeks using an ATA™ membrane and who maintained their prior regimen. Results: A total of 287 sessions were performed and 264 complete sessions were collected. With an effective time of 243.7 (17.6) min and a mean blood flow of 371.7 (23) ml/min, an average Kt of 56.3 (5.3) l was observed, as well as a convection volume of 27.1 (4.2) l, a filtration fraction of 29.9 (3.7) %, a urea reduction ratio (RR) of 81 (5.2) %, a creatinine RR of 74.7 (4.6) %, a β2-microglobulin RR of 76.5 (4.8) % and a retinol binding protein RR of 18.6 (7.6) %. There were no technical problems or alarms. Changing the heparin dosage was not necessary. No increases in C3a or C5a concentrations or leukopenia were observed in the first 30 min of the session. Neither the monocyte subpopulations nor IL-β1 or IL-6 were significantly altered after one month of treatment. Conclusions: The new ATA™ membrane achieves adequate Kt and convection volume, without technical problems and with good biocompatibility and inflammatory profiles. It is therefore a valid option for post-dilution haemodiafiltration, particularly in patients allergic to synthetic membranes. Resumen: Antecedentes: En la hemodiafiltración posdilucional se han usado solo membranas sintéticas. Ahora contamos con un triacetato de celulosa asimétrico (ATA®) cuyas características lo hacen apto para esta técnica. Objetivos: Describir las prestaciones y el comportamiento in vivo de esta membrana estudiando la eficacia depurativa y el uso clínico, además de su biocompatibilidad aguda tras un mes de tratamiento. Métodos: Estudio prospectivo observacional en el que se incluyeron 23 pacientes que se dializaron durante 4 semanas con ATA® manteniendo su pauta previa. Resultados: Se realizaron 287 sesiones y se recogieron 264 sesiones completas. Con un tiempo efectivo de 243,7 (17,6) min y un flujo medio de sangre de 371,7 (23) ml/min, se obtuvo un Kt medio de 56,3 (5,3) l, un volumen convectivo de 27,1 (4,2) l, con una fracción de filtración del 29,9 (3,7) %, un porcentaje de reducción (RR) de urea de 81 (5,2) %, un RR de creatinina de 74,7 (4,6) %, un RR de β2-microglobulina de 76,5 (4,8) % y un RR de proteína transportadora de retinol de 18,6 (7,6) %. No se produjeron problemas técnicos ni alarmas. No fue preciso cambiar la dosificación de heparina. A los 30 min de la sesión no se produjo ningún aumento de C3a, C5a ni leucopenia. Tampoco se modificaron de forma significativa las poblaciones monocitarias ni la IL-β1 ni IL-6 tras un mes de tratamiento. Conclusiones: ATA® logra un Kt y un volumen convectivo adecuados, sin problemas técnicos y con buen perfil de biocompatibilidad e inflamatorio, lo que lo convierte en una posibilidad más de tratamiento para hemodiafiltración posdilucional, máxime en pacientes alérgicos a membranas sintéticas. Keywords: Online haemodiafiltration, Cellulose triacetate, Suitability, Biocompatibility, Inflammation, Palabras clave: Hemodiafiltración en línea, Triacetato de celulosa, Adecuación, Biocompatibilidad, Inflamació

El triacetato de celulosa asimétrico es una alternativa segura y eficaz para la hemodiafiltración en línea

Resumen: Antecedentes: En la hemodiafiltración posdilucional se han usado solo membranas sintéticas. Ahora contamos con un triacetato de celulosa asimétrico (ATA®) cuyas características lo hacen apto para esta técnica. Objetivos: Describir las prestaciones y el comportamiento in vivo de esta membrana estudiando la eficacia depurativa y el uso clínico, además de su biocompatibilidad aguda tras un mes de tratamiento. Métodos: Estudio prospectivo observacional en el que se incluyeron 23 pacientes que se dializaron durante 4 semanas con ATA® manteniendo su pauta previa. Resultados: Se realizaron 287 sesiones y se recogieron 264 sesiones completas. Con un tiempo efectivo de 243,7 (17,6) min y un flujo medio de sangre de 371,7 (23) ml/min, se obtuvo un Kt medio de 56,3 (5,3) l, un volumen convectivo de 27,1 (4,2) l, con una fracción de filtración del 29,9 (3,7) %, un porcentaje de reducción (RR) de urea de 81 (5,2) %, un RR de creatinina de 74,7 (4,6) %, un RR de β2-microglobulina de 76,5 (4,8) % y un RR de proteína transportadora de retinol de 18,6 (7,6) %. No se produjeron problemas técnicos ni alarmas. No fue preciso cambiar la dosificación de heparina. A los 30 min de la sesión no se produjo ningún aumento de C3a, C5a ni leucopenia. Tampoco se modificaron de forma significativa las poblaciones monocitarias ni la IL-β1 ni IL-6 tras un mes de tratamiento. Conclusiones: ATA® logra un Kt y un volumen convectivo adecuados, sin problemas técnicos y con buen perfil de biocompatibilidad e inflamatorio, lo que lo convierte en una posibilidad más de tratamiento para hemodiafiltración posdilucional, máxime en pacientes alérgicos a membranas sintéticas. Abstract: Background: In post-dilution haemodiafiltration only synthetic membranes have been used to date. Asymmetric cellulose triacetate (ATA™) is now available, whose characteristics are suitable for this technique. Objectives: To describe the in vivo performance and behaviour of this membrane, to identify its depurative effectiveness, use in clinical practice and its biocompatibility, both acute and after one month of treatment. Methods: Observational prospective study of 23 patients who were dialysed for 4 weeks using an ATA™ membrane and who maintained their prior regimen. Results: A total of 287 sessions were performed and 264 complete sessions were collected. With an effective time of 243.7 (17.6) min and a mean blood flow of 371.7 (23) ml/min, an average Kt of 56.3 (5.3) l was observed, as well as a convection volume of 27.1 (4.2) l, a filtration fraction of 29.9 (3.7) %, a urea reduction ratio (RR) of 81 (5.2) %, a creatinine RR of 74.7 (4.6) %, a β2-microglobulin RR of 76.5 (4.8) % and a retinol binding protein RR of 18.6 (7.6) %. There were no technical problems or alarms. Changing the heparin dosage was not necessary. No increases in C3a or C5a concentrations or leukopenia were observed in the first 30 min of the session. Neither the monocyte subpopulations nor IL-β1 or IL-6 were significantly altered after one month of treatment. Conclusions: The new ATA™ membrane achieves adequate Kt and convection volume, without technical problems and with good biocompatibility and inflammatory profiles. It is therefore a valid option for post-dilution haemodiafiltration, particularly in patients allergic to synthetic membranes. Palabras clave: Hemodiafiltración en línea, Triacetato de celulosa, Adecuación, Biocompatibilidad, Inflamación, Keywords: Online haemodiafiltration, Cellulose triacetate, Suitability, Biocompatibility, Inflammatio

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

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

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

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