Caracterización inmunofenotípica de los fibroblastos peritoneales durante el tratamiento con diálisis peritoneal

Premio Extraordinario de Doctorado 2012En esta tesis doctoral se recogen varios trabajos relacionados con fibrogénesis peritoneal, transición epitelio-mesenquimal de la célula mesotelial y correlación morfo-funcional entre datos obtenidos de biopsias peritoneales y transporte peritoneal. Durante el tratamiento crónico con diálisis peritoneal se produce una progresiva fibrosis del peritoneo así como cambios vasculares y disminución de la capacidad dialítica de la membrana peritoneal, que en muchas ocasiones supone un abandono de la técnica. Durante los últimos años se han producido importantes avances en el origen y función de los miofibroblastos, que no han sido estudiados en este modelo de fibrosis. Objetivo: Estudiar in vivo la población fibroblástica del peritoneo normal y durante la fibrogénesis asociada al tratamiento dialítico, en especial en relación al posible origen y función miofibroblástica. Establecer una posible correlación entre estos mecanismos etiopatogénicos y las anomalías en fisiopatología peritoneal observadas en estos pacientes. Pacientes y métodos: Pare ello se han empleado biopsias peritoneales tanto de pacientes en tratamiento con diálisis peritoneal como controles urémicos y sanos. El estudio inmunofenotípco ha sido realizado mediante inmunohistoquímica, analizándose con especial atención la expresión de alfa-actina, CD34 y marcadores de transición epitelio-mesenquimal (citoqueratinas AE1/AE3, calretinina, ICAM-1, cadherina-E y VEGF). Estos trabajos inmunohistoquímicos son el complemento in vivo a experimentos realizados in vitro y ex vivo sobre población celular mesotelial. De todos estos pacientes se han obtenido datos clínicos y de función peritoneal. Resultados: En primer lugar, nuestra serie de biopsias peritoneales mostró las anomalías estructurales descritas en asociación al tratamiento con diálisis peritoneal (pérdida mesotelial, fibrosis y vasculopatía). En los controles sanos y urémicos (sin diálisis peritoneal) los fibroblastos submesoteliales expresaron CD34 y no alfa-actina, ni marcadores de transición epitelio-mesenquimal. Durante el tratamiento con diálisis peritoneal se invierte este patrón, con aparición de subpoblaciones sin CD34 y con expresión de alfa-actina (miofibroblastos). Parte de estos miofibroblastos expresan marcadores mesoteliales apoyando la hipótesis de que tienen un origen mesotelial. Se observó una distribución submesotelial de los miofibroblastos. En el análisis de correlación morfo-funcional se observó relación entre la presencia de marcadores de transición epitelio-mesenquimal y alto transporte peritoneal. En las biopsias existe correlación entre la presencia de marcadores de transición epitelio-mesenquimal y expresión fibroblástica de VEGF, siendo dichos marcadores mas frecuentes en los pacientes con alto transporte peritoneal. Conclusiones: El tratamiento con diálisis peritoneal induce alteraciones estructurales del peritoneo, entre las que destaca fibrosis. Dicha fibrosis es producida principalmente por el fibroblasto peritoneal que en situaciones de daño peritoneal se convierte en miofibroblasto, en parte mediante un mecanismo de transición epitelio-mesenquimal de la célula mesotelial. Dicho miofibroblasto resultante de transición produce elevados niveles de VEGF que es, en parte, responsable del estado de alto transporte peritoneal. En este sentido los estudios de correlación morfo-funcional establecen una relación entre la presencia de transición epitelio-mesenquimal y la situación de alto transporte peritoneal

Caracterización inmunofenotípica de los fibroblastos peritoneales durante el tratamiento con diálisis peritoneal

Premio Extraordinario de Doctorado 2012En esta tesis doctoral se recogen varios trabajos relacionados con fibrogénesis peritoneal, transición epitelio-mesenquimal de la célula mesotelial y correlación morfo-funcional entre datos obtenidos de biopsias peritoneales y transporte peritoneal. Durante el tratamiento crónico con diálisis peritoneal se produce una progresiva fibrosis del peritoneo así como cambios vasculares y disminución de la capacidad dialítica de la membrana peritoneal, que en muchas ocasiones supone un abandono de la técnica. Durante los últimos años se han producido importantes avances en el origen y función de los miofibroblastos, que no han sido estudiados en este modelo de fibrosis. Objetivo: Estudiar in vivo la población fibroblástica del peritoneo normal y durante la fibrogénesis asociada al tratamiento dialítico, en especial en relación al posible origen y función miofibroblástica. Establecer una posible correlación entre estos mecanismos etiopatogénicos y las anomalías en fisiopatología peritoneal observadas en estos pacientes. Pacientes y métodos: Pare ello se han empleado biopsias peritoneales tanto de pacientes en tratamiento con diálisis peritoneal como controles urémicos y sanos. El estudio inmunofenotípco ha sido realizado mediante inmunohistoquímica, analizándose con especial atención la expresión de alfa-actina, CD34 y marcadores de transición epitelio-mesenquimal (citoqueratinas AE1/AE3, calretinina, ICAM-1, cadherina-E y VEGF). Estos trabajos inmunohistoquímicos son el complemento in vivo a experimentos realizados in vitro y ex vivo sobre población celular mesotelial. De todos estos pacientes se han obtenido datos clínicos y de función peritoneal. Resultados: En primer lugar, nuestra serie de biopsias peritoneales mostró las anomalías estructurales descritas en asociación al tratamiento con diálisis peritoneal (pérdida mesotelial, fibrosis y vasculopatía). En los controles sanos y urémicos (sin diálisis peritoneal) los fibroblastos submesoteliales expresaron CD34 y no alfa-actina, ni marcadores de transición epitelio-mesenquimal. Durante el tratamiento con diálisis peritoneal se invierte este patrón, con aparición de subpoblaciones sin CD34 y con expresión de alfa-actina (miofibroblastos). Parte de estos miofibroblastos expresan marcadores mesoteliales apoyando la hipótesis de que tienen un origen mesotelial. Se observó una distribución submesotelial de los miofibroblastos. En el análisis de correlación morfo-funcional se observó relación entre la presencia de marcadores de transición epitelio-mesenquimal y alto transporte peritoneal. En las biopsias existe correlación entre la presencia de marcadores de transición epitelio-mesenquimal y expresión fibroblástica de VEGF, siendo dichos marcadores mas frecuentes en los pacientes con alto transporte peritoneal. Conclusiones: El tratamiento con diálisis peritoneal induce alteraciones estructurales del peritoneo, entre las que destaca fibrosis. Dicha fibrosis es producida principalmente por el fibroblasto peritoneal que en situaciones de daño peritoneal se convierte en miofibroblasto, en parte mediante un mecanismo de transición epitelio-mesenquimal de la célula mesotelial. Dicho miofibroblasto resultante de transición produce elevados niveles de VEGF que es, en parte, responsable del estado de alto transporte peritoneal. En este sentido los estudios de correlación morfo-funcional establecen una relación entre la presencia de transición epitelio-mesenquimal y la situación de alto transporte peritoneal

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

Impact of COVID-19 on cardiovascular testing in the United States versus the rest of the world

Objectives: This study sought to quantify and compare the decline in volumes of cardiovascular procedures between the United States and non-US institutions during the early phase of the coronavirus disease-2019 (COVID-19) pandemic. Background: The COVID-19 pandemic has disrupted the care of many non-COVID-19 illnesses. Reductions in diagnostic cardiovascular testing around the world have led to concerns over the implications of reduced testing for cardiovascular disease (CVD) morbidity and mortality. Methods: Data were submitted to the INCAPS-COVID (International Atomic Energy Agency Non-Invasive Cardiology Protocols Study of COVID-19), a multinational registry comprising 909 institutions in 108 countries (including 155 facilities in 40 U.S. states), assessing the impact of the COVID-19 pandemic on volumes of diagnostic cardiovascular procedures. Data were obtained for April 2020 and compared with volumes of baseline procedures from March 2019. We compared laboratory characteristics, practices, and procedure volumes between U.S. and non-U.S. facilities and between U.S. geographic regions and identified factors associated with volume reduction in the United States. Results: Reductions in the volumes of procedures in the United States were similar to those in non-U.S. facilities (68% vs. 63%, respectively; p = 0.237), although U.S. facilities reported greater reductions in invasive coronary angiography (69% vs. 53%, respectively; p < 0.001). Significantly more U.S. facilities reported increased use of telehealth and patient screening measures than non-U.S. facilities, such as temperature checks, symptom screenings, and COVID-19 testing. Reductions in volumes of procedures differed between U.S. regions, with larger declines observed in the Northeast (76%) and Midwest (74%) than in the South (62%) and West (44%). Prevalence of COVID-19, staff redeployments, outpatient centers, and urban centers were associated with greater reductions in volume in U.S. facilities in a multivariable analysis. Conclusions: We observed marked reductions in U.S. cardiovascular testing in the early phase of the pandemic and significant variability between U.S. regions. The association between reductions of volumes and COVID-19 prevalence in the United States highlighted the need for proactive efforts to maintain access to cardiovascular testing in areas most affected by outbreaks of COVID-19 infection

La herencia de Beckett

En 2006 se cumplieron cien años del nacimiento de Samuel Beckett, aniversario que el CBA quiso conmemorar con los ciclos del teatro y conferencias "El eco de sus pasos". Minerva recoge las intervenciones de la mesa redonda que puso punto final al ciclo de conferencias, en la que Julián Jiménez Heffernan, profesor de literatura inglesa en la Universidad de Córdoba y coordinador de estas jornadas, Derek Attridge, profesor de literatura inglesa en la Universidad de York, José Antonio Sanchez, catedrático de teoría literaria en la Universidad de Valencia, intentaron rastrear las diversas influencias de la producción beckettiana en el campo cultural y artístico en un animado debate. Terry Eagleton, nos ofrece un perfil poco habitual del autor irlandés

Are the mesothelial-to-mesenchymal transition, sclerotic peritonitis syndromes, and encapsulating peritoneal sclerosis part of the same process?

Mesothelial-to-mesenchymal transition (MMT) is an autoregulated physiological process of tissue repair that in uncontrolled conditions, such as peritoneal dialysis (PD), can lead to peritoneal fibrosis. The maximumexpression of sclerotic peritoneal syndromes (SPS) is the encapsulating peritoneal sclerosis (EPS) for which no specific treatment exists. The SPS includes a wide range of peritoneal fibrosis that appears progressively and is considered as a reversible process, while EPS does not. EPS is a serious complication of PD characterized by a progressive intra-abdominal inflammatory process that results in bridles and severe fibrous tissue formation which cover and constrict the viscera. Recent studies show that transdifferentiated mesothelial cells isolated from the PD effluent correlate very well with the clinical events such as the number of hemoperitoneum and peritonitis, as well as with PD function (lower ultrafiltration and high Cr-MTC). In addition, in peritoneal biopsies from PD patients, the MMT correlates very well with anatomical changes (fibrosis and angiogenesis). However, the pathway to reach EPS from SPS has not been fully and completely established. Herein, we present important evidence pointing to the MMT that is present in the initial peritoneal fibrosis stages and it is perpetual over time, with at least theoretical possibility that MMT initiated the fibrosing process to reach EPSThis work was supported by Grant SAF2010-21249 from the Ministerio de Economía y Competitividad to M. López- Cabrera and by Grant S2010/BMD-2321 from Comunidad Autónoma de Madrid to M. López-Cabrera and R. Selgas This work was also partially supported by Grants PI 09/0776 from Fondo de Investigaciones Sanitarias to A. A. Peralta and RETICS 06/0016 (REDinREN, Fondos FEDER, EU) to R. Selga

Biocompatible Dialysis Solutions Preserve Peritoneal Mesothelial Cell and Vessel Wall Integrity. A Case-Control Study on Human Biopsies.

♦ Chronic exposure to conventional peritoneal dialysis (PD) solutions has been related to peritoneal function alterations in PD patients, and associated with mesothelial cell loss, submesothelial fibrosis, vasculopathy, and angiogenesis. In vitro and ex vivo analyses, as well as studies with animal models, have demonstrated that biocompatible PD solutions attenuate these morphological alterations. Our aim was to confirm the morphological benefits of biocompatible solutions in PD patients. ♦ We analyzed biopsies from 23 patients treated with biocompatible solutions (study group, SG), and compared them with a control group (n = 23) treated with conventional solutions (CG), matched for time on PD. ♦ A total of 56.5% of SG patients showed total or partial preservation of mesothelial cells monolayer, in contrast with 26.1% of patients in CG (p = 0.036). Peritoneal fibrosis was not significantly less frequent in SG patients (47.8% SG vs 69.6% CG; p = 0.13). In patients without previous peritonitis, a significantly lower prevalence of fibrosis was present in SG patients (41.7% SG vs 77.8% CG; p = 0.04). Hyalinizing vasculopathy (HV) was significantly lower in SG (4.3% SG vs 30.4% CG; p = 0.02). Cytokeratin-positive fibroblast-like cells were detected in 10 patients (22%), but the prevalence was not significantly lower in SG. In the univariate regression analysis, the use of biocompatible solutions was associated with mesothelial monolayer integrity (p = 0.04) and an absence of vasculopathy (p = 0.04). ♦ The present study demonstrates in vivo in human biopsies that biocompatible solutions are better tolerated by the peritoneum in the medium and long term than conventional solutions

Rapamycin Protects from Type-I Peritoneal Membrane Failure Inhibiting the Angiogenesis, Lymphangiogenesis, and Endo-MT

Preservation of peritoneal membrane (PM) is essential for long-term survival in peritoneal dialysis (PD). Continuous presence of PD fluids (PDF) in the peritoneal cavity generates chronic inflammation and promotes changes of the PM, such as fibrosis, angiogenesis, and lymphangiogenesis. Mesothelial-to-mesenchymal transition (MMT) and endothelial-to-mesenchymal transition (Endo-MT) seem to play a central role in this pathogenesis. We speculated that Rapamycin, a potent immunosuppressor, could be beneficial by regulating blood and lymphatic vessels proliferation. We demonstrate that mice undergoing a combined PD and Rapamycin treatment (PDF + Rapa group) presented a reduced PM thickness and lower number of submesothelial blood and lymphatic vessels, as well as decreased MMT and Endo-MT, comparing with their counterparts exposed to PD alone (PDF group). Peritoneal water transport in the PDF + Rapa group remained at control level, whereas PD effluent levels of VEGF, TGF-β, and TNF-α were lower than in the PDF group. Moreover, the treatment of mesothelial cells with Rapamycin in vitro significantly decreased VEGF synthesis and selectively inhibited the VEGF-C and VEGF-D release when compared with control cells. Thus, Rapamycin has a protective effect on PM in PD through an antifibrotic and antiproliferative effect on blood and lymphatic vessels. Moreover, it inhibits Endo-MT and, at least partially, MMT

Caveolin1 and YAP drive mechanically induced mesothelial to mesenchymal transition and fibrosis

Despite their emerging relevance to fully understand disease pathogenesis, we have as yet a poor understanding as to how biomechanical signals are integrated with specific biochemical pathways to determine cell behaviour. Mesothelial-to-mesenchymal transition (MMT) markers colocalized with TGF-β1-dependent signaling and yes-associated protein (YAP) activation across biopsies from different pathologies exhibiting peritoneal fibrosis, supporting mechanotransduction as a central driving component of these class of fibrotic lesions and its crosstalk with specific signaling pathways. Transcriptome and proteome profiling of the response of mesothelial cells (MCs) to linear cyclic stretch revealed molecular changes compatible with bona fide MMT, which (i) overlapped with established YAP target gene subsets, and were largely dependent on endogenous TGF-β1 signaling. Importantly, TGF-β1 blockade blunts the transcriptional upregulation of these gene signatures, but not the mechanical activation and nuclear translocation of YAP per se. We studied the role therein of caveolin-1 (CAV1), a plasma membrane mechanotransducer. Exposure of CAV1-deficient MCs to cyclic stretch led to a robust upregulation of MMT-related gene programs, which was blunted upon TGF-β1 inhibition. Conversely, CAV1 depletion enhanced both TGF-β1 and TGFBRI expression, whereas its re-expression blunted mechanical stretching-induced MMT. CAV1 genetic deficiency exacerbated MMT and adhesion formation in an experimental murine model of peritoneal ischaemic buttons. Taken together, these results support that CAV1-YAP/TAZ fine-tune the fibrotic response through the modulation of MMT, onto which TGF-β1-dependent signaling coordinately converges. Our findings reveal a cooperation between biomechanical and biochemical signals in the triggering of MMT, representing a novel potential opportunity to intervene mechanically induced disorders coursing with peritoneal fibrosis, such as post-surgical adhesions

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