El factor de crecimiento de tejido conectivo: Modulador redox y citoquina proinflamatoria implicada en la génesis del daño vascular

Tesis doctoral inédita leída en la Universidad Autónoma de Madrid, Facultad de Medicina, Departamento de Bioquímica. Fecha de lectura: 22-05-2015El factor de crecimiento de tejido conectivo (CCN2/CTGF), es un gen de desarrollo que se encuentra rexpresado en determinadas condiciones patológicas. El gen de CCN2 codifica para una proteína matricelular compuesta por cuatro módulos, la cual puede ser degradada en diferentes fragmentos que presentan actividad biológica por sí mismos. Entre estos fragmentos, se ha descrito que el módulo IV C-terminal, denominado en este trabajo CCN2(IV), es capaz de regular varias funciones celulares. Tradicionalmente, CCN2 se ha considerado como un mediador de las respuestas fibróticas inducidas por otros factores como TGF-β, Ang II, o ET-1, aunque estudios más recientes sugieren que CCN2 podría tener también un rol importante en la activación de la respuesta inflamatoria. A nivel cardiovascular, se ha descrito un aumento en la expresión tisular de CCN2 en diversas patologías, incluida la aterosclerosis o la fibrosis cardiaca, aunque sus efectos directos sobre la pared vascular no han sido investigados. En el desarrollo de las enfermedades cardiovasculares, se ha descrito la implicación de numerosos procesos moleculares y rutas de señalización, como son la respuesta inflamatoria, el aumento en la producción de ROS o la fibrosis. Como primer objetivo, se quiso determinar si en situaciones de daño vascular existía una asociación entre los niveles de CCN2 y la producción de ROS, utilizando para ello un modelo de infusión de Ang II en ratones. Una vez comprobado que en las aortas de los ratones tratados con Ang II existía un aumento en la producción de O2 .- y que este se localizaba principalmente en las mismas zonas donde se observó un aumento de CCN2, se evaluó la hipótesis de que CCN2 podría estar regulando de forma directa procesos redox a nivel vascular. Por todo esto, el principal objetivo del presente trabajo fue investigar si CCN2(IV) podía inducir, de forma directa, un aumento en la producción de ROS, para lo cual se realizaron estudios en CMLVs y CEs en cultivo, y en aorta de ratón ex vivo e in vivo. Como segundo objetivo, se investigó si CCN2(IV) regulaba la función vascular e inducía procesos inflamatorios y fibróticos. Los resultados obtenidos indicaron que CCN2(IV), tanto en células en cultivo como en aorta de ratón in vivo, producía un aumento en la producción de O2 .- y en la actividad NAD(P)H oxidasa. Además, el tratamiento con CCN2(IV) aumentó la respuesta contráctil vascular, generó disfunción endotelial, e indujo la activación de respuestas proinflamatorias, como son la activación de la ruta NF-κB y la producción de mediadores proinflamatorios. Sin embargo, a los tiempos estudiados, CCN2(IV) no generó un aumento en los niveles de diversos factores profibróticos. Mediante abordajes farmacológicos y silenciamiento génico, demostramos que CCN2(IV), vía Nox1, causó un aumento en la producción de O2 .- en aorta de ratón, el cual dio lugar a disfunción endotelial, a un aumentó en la nitrosilación proteica y a una activación de la respuesta proinflamatoria. Finalmente, se demostró que EGFR era el receptor a través del cual CCN2(IV) ejercía sus acciones vasculares. En conjunto, estos resultados muestran que CCN2 puede actuar como una citoquina proinflamatoria y regular procesos redox mediante la activación del EGFR a nivel vascular, ampliando la visión tradicional de CCN2 y remarcando su importancia en las patologías cardiovasculares.Connective tissue growth factor (CTGF/CCN2) is a developmental gene upregulated in pathological conditions including cardiovascular diseases. CCN2 is a gene that encodes a matricellular protein that can be degraded to biologically active fragments. Among them, the Cterminal module IV (named here CCN2(IV)) regulates many cellular functions. Traditionally, CCN2 has been considered as profibrotic mediator of several factors, including TGF-β, Ang II, or ET-1. Recently, emerging data suggest that CCN2 could participate in the regulation of the inflammatory response. CCN2 overexpression has been found in many cardiovascular diseases, including atherosclerosis and cardiac fibrosis. Among the molecular mechanisms and intracellular signalling systems involved in the pathogenesis of cardiovascular diseases, inflammation, reactive oxygen species (ROS) production and fibrosis have special relevance. The first aim was to determine whether in cardiovascular pathological conditions there was an association between CCN2 levels and ROS production, using a model of systemic infusion of Ang II into mice. In Ang II-treated mice aorta increased ROS production was found mainly located in areas of CCN2 overexpression. Therefore, we evaluated the hypothesis that CCN2 could directly regulate redox processes at the vascular level. The main aim of this thesis was to investigate whether CCN2(IV) could directly increase ROS production, by performing experiments in cultured murine vascular smooth muscle cells (VSMCs), endothelial cells (EC), and in mice aorta ex vivo and in vivo. The second aim was to investigate whether CCN2(IV) could regulate vascular functions, as well as inflammatory and fibrotic processes. In cultured vascular cells and in mice aorta CCN2(IV) increased O2 .- production and NAD(P)H oxidase activity. CCN2(IV) also induced an inflammatory response, characterized by endothelial dysfunction, increased protein nitrosylation, activation of the NF-B transcription factor and upregulation of proinflammatory markers (cytokines, adhesion molecules and chemokines). However, CCN2(IV) did not modulate profibrotic factors, at least at the time point evaluated. By pharmacological and gene silencing approaches we found that CCN2(IV), vía Nox1, increased O2 .- production in murine aorta, linked to endothelial dysfunction, increased protein nitrosylation and the upregulation of proinflammatory factors. Finally, we demonstrated that CCN2(IV) by activation of EGFR pathway, induced vascular inflammation by a mechanism mediated by NAD(P)H oxidase activation and O2 .-production. Thus, these findings expand the current view of CCN2 and remark its important role in inflammatory cardiovascular diseases

Statins Inhibit Angiotensin II/Smad Pathway and Related Vascular Fibrosis, by a TGF-β-Independent Process

We have recently described that in an experimental model of atherosclerosis and in vascular smooth muscle cells (VSMCs) statins increased the activation of the Smad pathway by transforming growth factor-β (TGF-β), leading to an increase in TGF-β-dependent matrix accumulation and plaque stabilization. Angiotensin II (AngII) activates the Smad pathway and contributes to vascular fibrosis, although the in vivo contribution of TGF-β has not been completely elucidated. Our aim was to further investigate the mechanisms involved in AngII-induced Smad activation in the vasculature, and to clarify the beneficial effects of statins on AngII-induced vascular fibrosis. Infusion of AngII into rats for 3 days activates the Smad pathway and increases fibrotic-related factors, independently of TGF-β, in rat aorta. Treatment with atorvastatin or simvastatin inhibited AngII-induced Smad activation and related-fibrosis. In cultured rat VSMCs, direct AngII/Smad pathway activation was mediated by p38 MAPK and ROCK activation. Preincubation of VSMCs with statins inhibited AngII-induced Smad activation at all time points studied (from 20 minutes to 24 hours). All these data show that statins inhibited several AngII-activated intracellular signaling systems, including p38-MAPK and ROCK, which regulates the AngII/Smad pathway and related profibrotic factors and matrix proteins, independently of TGF-β responses. The inhibitory effect of statins on the AngII/Smad pathway could explain, at least in part, their beneficial effects on hypertension-induced vascular damage

Calcineurin inhibitors cyclosporine A and tacrolimus induce vascular inflammation and endothelial activation through TLR4 signaling

The introduction of the calcineurin inhibitors (CNIs) cyclosporine and tacrolimus greatly reduced the rate of allograft rejection, although their chronic use is marred by a range of side effects, among them vascular toxicity. In transplant patients, it is proved that innate immunity promotes vascular injury triggered by ischemia-reperfusion damage, atherosclerosis and hypertension. We hypothesized that activation of the innate immunity and inflammation may contribute to CNI toxicity, therefore we investigated whether TLR4 mediates toxic responses of CNIs in the vasculature. Cyclosporine and tacrolimus increased the production of proinflammatory cytokines and endothelial activation markers in cultured murine endothelial and vascular smooth muscle cells as well as in ex vivo cultures of murine aortas. CNI-induced proinflammatory events were prevented by pharmacological inhibition of TLR4. Moreover, CNIs were unable to induce inflammation and endothelial activation in aortas from TLR4−/− mice. CNI-induced cytokine and adhesion molecules synthesis in endothelial cells occurred even in the absence of calcineurin, although its expression was required for maximal effect through upregulation of TLR4 signaling. CNI-induced TLR4 activity increased O2 −/ROS production and NF-κB-regulated synthesis of proinflammatory factors in cultured as well as aortic endothelial and VSMCs. These data provide new insight into the mechanisms associated with CNI vascular inflammationThis work was supported by grants from the Instituto de Salud Carlos III (Ministerio de Economía Competitividad, Gobierno de España): FEDER funds ISCIII RETIC REDINREN RD12/0021, PI11/02242, PI13/00047, PI14/0041, PI14/00386, PI15/01460; Comunidad de Madrid (CIFRA S2010/BMD-2378); Sociedad Española de Nefrología. Salary support: RR-D: CIFRA; CO-S: Fundación Conchita Rábago de Jiménez Díaz; CG-G and RRR-D: REDINREN; AO: Programa Intensificación Actividad Investigadora (ISCIII/Agencia Laín-Entralgo/CM); JE and MRO: Universidad Autónoma de Madrid; AMR: Contrato Miguel Serve (ISCIII

Gremlin activates the smad pathway linked to epithelial mesenchymal transdifferentiation in cultured tubular epithelial cells

Gremlin is a developmental gene upregulated in human chronic kidney disease and in renal cells in response to transforming growth factor-(TGF-β). Epithelial mesenchymal transition (EMT) is one process involved in renal fibrosis. In tubular epithelial cells we have recently described that Gremlin induces EMT and acts as a downstream TGF-β mediator. Our aim was to investigate whether Gremlin participates in EMT by the regulation of the Smad pathway. Stimulation of human tubular epithelial cells (HK2) with Gremlin caused an early activation of the Smad signaling pathway (Smad 2/3 phosphorylation, nuclear translocation, and Smad-dependent gene transcription). The blockade of TGF-β, by a neutralizing antibody against active TGF-β, did not modify Gremlin-induced early Smad activation.These data showthatGremlin directly, by a TGF-β independent process, activates the Smad pathway. In tubular epithelial cells long-term incubation with Gremlin increased TGF-β production and caused a sustained Smad activation and a phenotype conversion into myofibroblasts-like cells. Smad 7 overexpression, which blocks Smad 2/3 activation, diminished EMT changes observed in Gremlin-transfected tubuloepithelial cells. TGF-β neutralization also diminished Gremlininduced EMT changes. In conclusion, we propose that Gremlin could participate in renal fibrosis by inducing EMT in tubular epithelial cells through activation of Smad pathway and induction of TGF-βThis work was supported by grants from the Instituto de Salud Carlos III (PI11/01854 and REDINREN ISCIIIRETIC RD12/0021/0002 and 0001), Sociedad Española de Nefrología, PCI Iberoamerica (A/9571/07), CYTED IBERERC, FONDECYT Chile 1080083 and 1120480, Comunidad de Madrid (Fibroteam S2010/BMD-2321, S2010/BMD- 2378), Programa Intensificación Actividad Investigadora (ISCIII/Agencia Laín Entralgo/CM) to A.O. Fundación para el fomento en Asturias de la investigaciónn cientíica aplicada y la tecnología (FICYT)

Gremlin regulates tubular epithelial to mesenchymal transition via VEGFR2: Potential role in renal fibrosis

Chronic kidney disease (CKD) is emerging as an important health problem due to the increase number of CKD patients and the absence of an effective curative treatment. Gremlin has been proposed as a novel therapeutic target for renal inflammatory diseases, acting via Vascular Endothelial Growth Factor Receptor-2 (VEGFR2). Although many evidences suggest that Gremlin could regulate renal fibrosis, the receptor involved has not been yet clarified. Gremlin, as other TGF-β superfamily members, regulates tubular epithelial to mesenchymal transition (EMT) and, therefore, could contribute to renal fibrosis. In cultured tubular epithelial cells Gremlin binding to VEGFR2 is linked to proinflammatory responses. Now, we have found out that in these cells VEGFR2 is also involved in the profibrotic actions of Gremlin. VEGFR2 blockade by a pharmacological kinase inhibitor or gene silencing diminished Gremlin-mediated gene upregulation of profibrotic factors and restored changes in EMT-related genes. Moreover, VEGFR2 inhibition blocked EMT phenotypic changes and dampened the rate of wound healing in response to Gremlin. The role of VEGFR2 in experimental fibrosis was evaluated in experimental unilateral ureteral obstruction. VEFGR2 inhibition diminished the upregulation of profibrotic genes and EMT changes, as well as the accumulation of extracellular matrix proteins, such as fibronectin and collagens in the obstructed kidneys. Notch pathway activation participates in renal damage progression by regulating cell growth/proliferation, regeneration and inflammation. In cultured tubular epithelial cells, Notch inhibition markedly downregulated Gremlin-induced EMT changes and wound healing speed. These results show that Gremlin regulates the EMT process via VEGFR2 and Notch pathway activation, suggesting that the Gremlin/VEGFR2 axis could be a potential therapeutic target for CKD.This work was supported by grants from the Instituto de Salud Carlos III (ISCIII) and Fondos FEDER European Union (PI16/02057, PI17/00119, PI17/01495, and Red de Investigación Renal REDINREN: RD16/0009), Sociedad Española de Nefrologia, “NOVELREN-CM: Enfermedad renal crónica: nuevas Estrategias para la prevención, Diagnóstico y tratamiento”; B2017/BMD-3751, B2017/BMD-3686 CIFRA2-CM, PAI 82140017, and FONDECYT 1160465 (Chile) and Bayer HealthCare AG (Grants4Targets initiative, Berlin, Germany)

CCN2 Binds to Tubular Epithelial Cells in the Kidney

Cellular communication network-2 (CCN2), also called connective tissue growth factor (CTGF), is considered a fibrotic biomarker and has been suggested as a potential therapeutic target for kidney pathologies. CCN2 is a matricellular protein with four distinct structural modules that can exert a dual function as a matricellular protein and as a growth factor. Previous experiments using surface plasmon resonance and cultured renal cells have demonstrated that the C-terminal module of CCN2 (CCN2(IV)) interacts with the epidermal growth factor receptor (EGFR). Moreover, CCN2(IV) activates proinflammatory and profibrotic responses in the mouse kidney. The aim of this paper was to locate the in vivo cellular CCN2/EGFR binding sites in the kidney. To this aim, the C-terminal module CCN2(IV) was labeled with a fluorophore (Cy5), and two different administration routes were employed. Both intraperitoneal and direct intra-renal injection of Cy5-CCN2(IV) in mice demonstrated that CCN2(IV) preferentially binds to the tubular epithelial cells, while no signal was detected in glomeruli. Moreover, co-localization of Cy5-CCN2(IV) binding and activated EGFR was found in tubules. In cultured tubular epithelial cells, live-cell confocal microscopy experiments showed that EGFR gene silencing blocked Cy5-CCN2(IV) binding to tubuloepithelial cells. These data clearly show the existence of CCN2/EGFR binding sites in the kidney, mainly in tubular epithelial cells. In conclusion, these studies show that circulating CCN2(IV) can directly bind and activate tubular cells, supporting the role of CCN2 as a growth factor involved in kidney damage progression

Connective tissue growth factor induces renal fibrosis via epidermal growth factor receptor activation

Connective tissue growth factor (CCN2/CTGF) is a matricellular protein that is overexpressed in progressive human renal diseases, mainly in fibrotic areas. In vitro studies have demonstrated that CCN2 regulates the production of extracellular matrix (ECM) proteins and epithelial–mesenchymal transition (EMT), and could therefore contribute to renal fibrosis. CCN2 blockade ameliorates experimental renal damage, including diminution of ECM accumulation. We have reported that CCN2 and its C-terminal degradation product CCN2(IV) bind to epidermal growth factor receptor (EGFR) to modulate renal inflammation. However, the receptor involved in CCN2 profibrotic actions has not been described so far. Using a murine model of systemic administration of CCN2(IV), we have unveiled a fibrotic response in the kidney that was diminished by EGFR blockade. Additionally, in conditional CCN2 knockout mice, renal fibrosis elicited by folic acid-induced renal damage was prevented, and this was linked to inhibition of EGFR pathway activation. Our in vitro studies demonstrated a direct effect of CCN2 via the EGFR pathway on ECM production by fibroblasts and the induction of EMT in tubular epithelial cells. Our studies clearly show that the EGFR regulates CCN2 fibrotic signalling in the kidney, and suggest that EGFR pathway blockade could be a potential therapeutic option to block CCN2-mediated profibrotic effects in renal diseases.Instituto de Salud Carlos IIIUnión EuropeaRed de Investigación RenalComunidad de MadridFundación Renal Iñigo Alvarez de Toledo (FRIAT)Sociedad Española de NefrologíaDepto. de Biología CelularFac. de MedicinaTRUEpu

CTGF Promotes inflammatory cell infiltration of the renal interstitium by activating

Connective tissue growth factor (CTGF) is an important profibrotic factor in kidney diseases. Blockade of endogenous CTGF ameliorates experimental renal damage and inhibits synthesis of extracellular matrix in cultured renal cells. CTGF regulates several cellular responses, including adhesion, migration, proliferation, and synthesis of proinflammatory factors. Here, we investigated whether CTGF participates in the inflammatory process in the kidney by evaluating the nuclear factor-kappa B (NF-κB) pathway, a key signaling system that controls inflammation and immune responses. Systemic administration of CTGF to mice for 24 h induced marked infiltration of inflammatory cells in the renal interstitium (T lymphocytes and monocytes/macrophages) and led to elevated renal NF-κB activity. Administration of CTGF increased renal expression of chemokines (MCP-1 and RANTES) and cytokines (INF-γ, IL-6, and IL-4) that recruit immune cells and promote inflammation. Treatment with a NF-κB inhibitor, parthenolide, inhibited CTGF-induced renal inflammatory responses, including the up-regulation of chemokines and cytokines. In cultured murine tubuloepithelial cells, CTGF rapidly activated the NF-κB pathway and the cascade of mitogen-activated protein kinases, demonstrating crosstalk between these signaling pathways. CTGF, via mitogen-activated protein kinase and NF-κB activation, increased proinflammatory gene expression. These data show that in addition to its profibrotic properties, CTGF contributes to the recruitment of inflammatory cells in the kidney by activating the NF-κB pathwayThis work has been supported by grants from Ministerio de Educación y Ciencia (SAF 2005–03378), Sociedad Española de Nefrología, FIS (PI020822 and PI081564), Red tema´ tica de Investigación Renal REDINREN (ISCIII-RETIC RD06/0016) from the Instituto de Salud Carlos III from Ministerio de Sanidad y Consumo, the EU project DIALOK: LSHB-CT-2007–036644, PCI Iberoamerica (A/9571/07), and FONDECYT, Chile (1080083). Programa Intensificación Actividad Investigadora (ISCIII/Agencia Laín-Entralgo/CM) to A.O. We want to thank Ma Mar Gonzalez Garcia-Parreñoo for her technical hel

Anti-inflammatory, antioxidant and renoprotective effects of SOCS1 mimetic peptide in the BTBR ob/ob mouse model of type 2 diabetes

Introduction Diabetic nephropathy (DN) is the leading cause of chronic kidney disease worldwide. The Janus kinase/signal transducers and activators of transcription (JAK/STAT) pathway participates in the development and progression of DN. Among the different mechanisms involved in JAK/STAT negative regulation, the family of suppressor of cytokine signaling (SOCS) proteins has been proposed as a new target for DN. Our aim was to evaluate the effect of SOCS1 mimetic peptide in a mouse model of obesity and type 2 diabetes (T2D) with progressive DN.Research design and methods Six-week-old BTBR (black and tan brachyuric) mice with the ob/ob (obese/obese) leptin-deficiency mutation were treated for 7 weeks with two different doses of active SOCS1 peptide (MiS1 2 and 4 µg/g body weight), using inactive mutant peptide (Mut 4 µg) and vehicle as control groups. At the end of the study, the animals were sacrificed to obtain blood, urine and kidney tissue for further analysis.Results Treatment of diabetic mice with active peptide significantly decreased urine albumin to creatinine ratio by up to 50%, reduced renal weight, glomerular and tubulointerstitial damage, and restored podocyte numbers. Kidneys from treated mice exhibited lower inflammatory infiltrate, proinflammatory gene expression and STAT activation. Concomitantly, active peptide administration modulated redox balance markers and reduced lipid peroxidation and cholesterol transporter gene expression in diabetic kidneys.Conclusion Targeting SOCS proteins by mimetic peptides to control JAK/STAT signaling pathway ameliorates albuminuria, morphological renal lesions, inflammation, oxidative stress and lipotoxicity, and could be a therapeutic approach to T2D kidney disease