Abstract Aims: Connective tissue growth factor (CTGF/CCN2) is a developmental gene upregulated in pathological conditions, including cardiovascular diseases, whose product is a matricellular protein that can be degraded to biologically active fragments. Among them, the C-terminal module IV [CCN2(IV)] regulates many cellular functions, but there are no data about redox process. Therefore, we investigated whether CCN2(IV) through redox signaling regulates vascular responses. Results: CCN2(IV) increased superoxide anion (O2(•-)) production in murine aorta (ex vivo and in vivo) and in cultured vascular smooth muscle cells (VSMCs). In isolated murine aorta, CCN2(IV), via O2(•-), increased phenylephrine-induced vascular contraction. CCN2(IV) in vivo regulated several redox-related processes in mice aorta, including increased nonphagocytic NAD(P)H oxidases (Nox)1 activity, protein nitrosylation, endothelial dysfunction, and activation of the nuclear factor-?B (NF-?B) pathway and its related proinflammatory factors. The role of Nox1 in CCN2(IV)-mediated vascular responses in vivo was investigated by gene silencing. The administration of a Nox1 morpholino diminished aortic O2(•-) production, endothelial dysfunction, NF-?B activation, and overexpression of proinflammatory genes in CCN2(IV)-injected mice. The link CCN2(IV)/Nox1/NF-?B/inflammation was confirmed in cultured VSMCs. Epidermal growth factor receptor (EGFR) is a known CCN2 receptor. In VSMCs, CCN2(IV) activates EGFR signaling. Moreover, EGFR kinase inhibition blocked vascular responses in CCN2(IV)-injected mice. Innovation and Conclusion: CCN2(IV) is a novel prooxidant factor that in VSMCs induces O2(•-) production via EGFR/Nox1 activation. Our in vivo data demonstrate that CCN2(IV) through EGFR/Nox1 signaling pathway induces endothelial dysfunction and activation of the NF-?B inflammatory pathway. Therefore, CCN2(IV) could be considered a potential therapeutic target for redox-related cardiovascular diseases. Antioxid. Redox Signal. 00, 000-000.
Gremlin is a developmental gene upregulated in human chronic kidney disease and in renal cells in response to transforming growth factor-? (TGF-?). 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-? 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-?, by a neutralizing antibody against active TGF-?, did not modify Gremlin-induced early Smad activation. These data show that Gremlin directly, by a TGF-? independent process, activates the Smad pathway. In tubular epithelial cells long-term incubation with Gremlin increased TGF-? 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-? neutralization also diminished Gremlin-induced 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-?.
The classical view of the immune system has changed by the discovery of novel T-helper (Th) subsets, including Th17 (IL-17A-producing cells). IL-17A participates in immune-mediated glomerulonephritis and more recently in inflammatory pathologies, including experimental renal injury. Peritoneal dialysis patients present chronic inflammation and Th1/Th2 imbalance, but the role of the Th17 response in peritoneal membrane damage has not been investigated. In peritoneal biopsies from dialyzed patients, IL-17A immunostaining was found mainly in inflammatory areas and was absent in the healthy peritoneum. IL-17A-expressing cells included lymphocytes (CD4+ and ??), neutrophils, and mast cells. Elevated IL-17A effluent concentrations were found in long-term peritoneal dialysis patients. Studies in mice showed that repeated exposure to recombinant IL-17A caused peritoneal inflammation and fibrosis. Moreover, chronic exposure to dialysis fluids resulted in a peritoneal Th17 response, including elevated IL-17A gene and protein production, submesothelial cell infiltration of IL-17A-expressing cells, and upregulation of Th17 differentiation factors and cytokines. IL-17A neutralization diminished experimental peritoneal inflammation and fibrosis caused by chronic exposure to dialysis fluids in mice. Thus, IL-17A is a key player of peritoneum damage and it may be a good candidate for therapeutic intervention in peritoneal dialysis patients.
Chronic kidney disease is reaching epidemic proportions worldwide and there is no effective treatment. Connective tissue growth factor (CCN2) has been suggested as a risk biomarker and a potential therapeutic target for renal diseases, but its specific receptor has not been identified. Epidermal growth factor receptor (EGFR) participates in kidney damage, but whether CCN2 activates the EGFR pathway is unknown. Here, we show that CCN2 is a novel EGFR ligand. CCN2 binding to EGFR extracellular domain was demonstrated by surface plasmon resonance. CCN2 contains four distinct structural modules. The carboxyl-terminal module (CCN2(IV)) showed a clear interaction with soluble EGFR, suggesting that EGFR-binding site is located in this module. Injection of CCN2(IV) in mice increased EGFR phosphorylation in the kidney, mainly in tubular epithelial cells. EGFR kinase inhibition decreased CCN2(IV)-induced renal changes (ERK activation and inflammation). Studies in cultured tubular epithelial cells showed that CCN2(IV) binds to EGFR leading to ERK activation and proinflammatory factors overexpression. CCN2 interacts with the neurotrophin receptor TrkA, and EGFR/TrkA receptor crosstalk was found in response to CCN2(IV) stimulation. Moreover, endogenous CCN2 blockade inhibited TGF-?-induced EGFR activation. These findings indicate that CCN2 is a novel EGFR ligand that contributes to renal damage through EGFR signalling.
Connective tissue growth factor (CTGF/CCN2) is a matricellular protein susceptible to proteolytic degradation. CCN2 levels have been suggested as a potential risk biomarker in several chronic diseases. In body fluids, CCN2 full-length and its degradation fragments can be found; however, their in vivo effects are far from being elucidated. CCN2 was described as a profibrotic mediator, but this concept is changing to a proinflammatory cytokine. In vitro, CCN2 full-length and its C-terminal module IV (CCN2(IV)) exert proinflammatory properties. Emerging evidence suggest that Th17 cells, and its effector cytokine IL-17A, participate in chronic inflammatory diseases. Our aim was to explore whether CCN2(IV) could regulate the Th17 response. In vitro, stimulation of human naive CD4+ T lymphocytes with CCN2(IV) resulted in differentiation to Th17 phenotype. The in vivo effects of CCN2(IV) were studied in C57BL/6 mice. Intraperitoneal administration of recombinant CCN2(IV) did not change serum IL-17A levels, but caused an activation of the Th17 response in the kidney, characterized by interstitial infiltration of Th17 (IL17A+/CD4+) cells and upregulation of proinflammatory mediators. In CCN2(IV)-injected mice, elevated renal levels of Th17-related factors (IL-17A, IL-6, STAT3 and ROR?t) were found, whereas Th1/Th2 cytokines or Treg-related factors (TGF-? and Foxp-3) were not modified. Treatment with an anti-IL-17A neutralizing antibody diminished CCN2(IV)-induced renal inflammation. Our findings unveil that the C-terminal module of CCN2 induces the Th17 differentiation of human Th17 cells and causes a renal Th17 inflammatory response. Furthermore, these data bear out that IL-17A targeting is a promising tool for chronic inflammatory diseases, including renal pathologies.
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-? (TGF-?), leading to an increase in TGF-?-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-? 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-?, 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-? 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.
Angiotensin II (Ang II) modulates vasomotor tone, cell growth, and extracellular matrix deposition. This study analyzed the effect of atorvastatin in the possible alterations induced by Ang II on structure and mechanics of mesenteric resistance arteries and the signaling mechanisms involved. Wistar rats were infused with Ang II (100 ng/kg per day, SC minipumps, 2 weeks) with or without atorvastatin (5 mg/kg per day). Ang II increased blood pressure and plasmatic malondialdehyde levels. Compared with controls, mesenteric resistance arteries from Ang II-treated rats showed the following: (1) decreased lumen diameter; (2) increased wall/lumen; (3) decreased number of adventitial, smooth muscle, and endothelial cells; (4) increased stiffness; (5) increased collagen deposition; and (6) diminished fenestrae area and number in the internal elastic lamina. Atorvastatin did not alter blood pressure but reversed all of the structural and mechanical alterations of mesenteric arteries, including collagen and elastin alterations. In mesenteric resistance arteries, Ang II increased vascular O(2)(.-) production and diminished endothelial NO synthase and CuZn/superoxide dismutase but did not modify extracellular-superoxide dismutase expression. Atorvastatin improved plasmatic and vascular oxidative stress, normalized endothelial NO synthase and CuZn/superoxide dismutase expression, and increased extracellular-superoxide dismutase expression, showing antioxidant properties. Atorvastatin also diminished extracellular signal-regulated kinase 1/2 activation caused by Ang II in these vessels, indicating an interaction with Ang II-induced intracellular responses. In vascular smooth muscle cells, collagen type I release mediated by Ang II was reduced by different antioxidants and statins. Moreover, atorvastatin downregulated the Ang II-induced NADPH oxidase subunit, Nox1, expression. Our results suggest that statins might exert beneficial effects on hypertension-induced vascular remodeling by improving vascular structure, extracellular matrix alterations, and vascular stiffness. These effects might be mediated by their antioxidant properties.
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-kappaB) 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-kappaB activity. Administration of CTGF increased renal expression of chemokines (MCP-1 and RANTES) and cytokines (INF-gamma, IL-6, and IL-4) that recruit immune cells and promote inflammation. Treatment with a NF-kappaB 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-kappaB pathway and the cascade of mitogen-activated protein kinases, demonstrating crosstalk between these signaling pathways. CTGF, via mitogen-activated protein kinase and NF-kappaB 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-kappaB pathway.
Chronic kidney disease is characterized by accumulation of extracellular matrix in the tubulointerstitial area. Fibroblasts are the main matrix-producing cells. One source of activated fibroblasts is the epithelial mesenchymal transition (EMT). In cultured tubular epithelial cells, transforming growth factor-? (TGF-?1) induced Gremlin production associated with EMT phenotypic changes, and therefore Gremlin has been proposed as a downstream TGF-?1 mediator. Gremlin is a developmental gene upregulated in chronic kidney diseases associated with matrix accumulation, but its direct role in the modulation of renal fibrosis and its relation with TGF-? has not been investigated.
Recent studies have described that the Notch signaling pathway is activated in a wide range of renal diseases. Angiotensin II (AngII) plays a key role in the progression of kidney diseases. AngII contributes to renal fibrosis by upregulation of profibrotic factors, induction of epithelial mesenchymal transition and accumulation of extracellular matrix proteins. In cultured human tubular epithelial cells the Notch activation by transforming growth factor-?1 (TGF-?1) has been involved in epithelial mesenchymal transition. AngII mimics many profibrotic actions of TGF-?1. For these reasons, our aim was to investigate whether AngII could regulate the Notch/Jagged system in the kidney, and its potential role in AngII-induced responses. In cultured human tubular epithelial cells, TGF-?1, but not AngII, increased the Notch pathway-related gene expression, Jagged-1 synthesis, and caused nuclear translocation of the activated Notch. In podocytes and renal fibroblasts, AngII did not modulate the Notch pathway. In tubular epithelial cells, pharmacological Notch inhibition did not modify AngII-induced changes in epithelial mesenchymal markers, profibrotic factors and extracellular matrix proteins. Systemic infusion of AngII into rats for 2 weeks caused tubulointerstitial fibrosis, but did not upregulate renal expression of activated Notch-1 or Jagged-1, as observed in spontaneously hypertensive rats. Moreover, the Notch/Jagged system was not modulated by AngII type I receptor blockade in the model of unilateral ureteral obstruction in mice. These data clearly indicate that AngII does not regulate the Notch/Jagged signaling system in the kidney, in vivo and in vitro. Our findings showing that the Notch pathway is not involved in AngII-induced fibrosis could provide important information to understand the complex role of Notch system in the regulation of renal regeneration vs damage progression.
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