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Acute respiratory distress syndrome (ARDS), an inflammatory condition with high mortality rates, is common in severe COVID-19, whose risk is reduced by metformin rather than other anti-diabetic medications. Detecting of inflammasome assembly in post-mortem COVID-19 lungs, we asked whether and how metformin inhibits inflammasome activation while exerting its anti-inflammatory effect. We show that metformin inhibited NLRP3 inflammasome activation and interleukin (IL)-1β production in cultured and alveolar macrophages along with inflammasome-independent IL-6 secretion, thus attenuating lipopolysaccharide (LPS)- and SARS-CoV-2-induced ARDS. By targeting electron transport chain complex 1 and independently of AMP-activated protein kinase (AMPK) or NF-κB, metformin blocked LPS-induced and ATP-dependent mitochondrial (mt) DNA synthesis and generation of oxidized mtDNA, an NLRP3 ligand. Myeloid-specific ablation of LPS-induced cytidine monophosphate kinase 2 (CMPK2), which is rate limiting for mtDNA synthesis, reduced ARDS severity without a direct effect on IL-6. Thus, inhibition of ATP and mtDNA synthesis is sufficient for ARDS amelioration., Graphical abstract, The exact mechanism by which metformin exerts its anti-inflammatory effects is still not known. Xian et al. shows that metformin’s inhibition of ETCCI blocks ATP-dependent mtDNA synthesis, cytoplasmic ox-mtDNA generation and NLRP3 inflammasome activation in macrophages, independent of AMPK and NF-κB. By limiting IL-1β production, metformin blunts pulmonary inflammation.

Acute respiratory distress syndrome (ARDS) is a leading cause of COVID-19 death. Long-term metformin usage decreases COVID-19 mortality through an obscure mechanism unrelated to glycemic control. It is unclear whether and how short-term metformin-based intervention can block acute inflammation. We show that metformin inhibits IL-6 and IL-1β secretion from LPS or SARS-CoV-2 Spike protein primed macrophages undergoing NLRP3 inflammasome activation, a key to ARDS initiation. Reduced IL-6 production correlates with blunted NFAT and C/EBPβ/NFIL-6 activation, whereas inhibition of IL-1β secretion reflects interference with NLRP3 inflammasome activation. By targeting electron transfer chain complex 1, but independently of AMPK and mitophagy, metformin inhibits ATP-dependent mitochondrial DNA synthesis and blocks generation of oxidized mitochondrial DNA, a trigger of inflammasome assembly. Correspondingly, short-term metformin treatment abrogates LPS-induced ARDS, lung macrophage recruitment, damage and mortality. We suggest that metformin, a cheap and safe drug, can be used to prevent ARDS onset in COVID-19 patients. Funding: The UCSD Tissue Technology Shared Resource supported by a National Cancer Institute Cancer Center Support Grant (CCSG P30CA23100). M.A. was supported by NIH (R01AI072726). E.S-L. was supported by NIAMS (K01AR077111). Research was supported by NIH grant awards to M.K. and M.A. (U54CA260591), and M.K. (R01A1043477, P42ES010337), who is an American Cancer Research Society Professor and holds the Ben and Wanda Hildyard Chair for Mitochondrial and Metabolic Diseases. Conflict of Interest: M.K. is a founder of Elgia Pharmaceuticals and receives research support from Gossamer Bio and Jansen Pharmaceuticals. All other authors declare no competing interests. Ethical Approval: All mouse studies were conducted in accordance with UCSD and NIH guidelines and regulations for the housing and treatment of laboratory animals using protocols approved by the UCSD Institutional Animal Care and Use Committee.

The tumor microenvironment (TME) exerts critical pro-tumorigenic effects through cytokines and growth factors that support cancer cell proliferation, survival, motility and invasion. Insulin-like growth factor-1 (IGF-1) and signal transducer and activator of transcription 3 (STAT3) stimulate colorectal cancer development and progression via cell autonomous and microenvironmental effects. Using a unique inhibitor, NT157, which targets both IGF-1 receptor (IGF-1R) and STAT3, we show that these pathways regulate many TME functions associated with sporadic colonic tumorigenesis in CPC-APC mice, in which cancer development is driven by loss of the Apc tumor suppressor gene. NT157 causes a substantial reduction in tumor burden by affecting cancer cells, cancer-associated fibroblasts (CAF) and myeloid cells. Decreased cancer cell proliferation and increased apoptosis were accompanied by inhibition of CAF activation and decreased inflammation. Furthermore, NT157 inhibited expression of pro-tumorigenic cytokines, chemokines and growth factors, including IL-6, IL-11 and IL-23 as well as CCL2, CCL5, CXCL7, CXCL5, ICAM1 and TGFβ; decreased cancer cell migratory activity and reduced their proliferation in the liver. NT157 represents a new class of anti-cancer drugs that affect both the malignant cell and its supportive microenvironment.

Sanchez-Niño, Maria D. et al., Apoptosis is a driving force of diabetic end-organ damage, including diabetic nephropathy (DN). However, the mechanisms that modulate diabetes-induced cell death are not fully understood. Heat shock protein 27 (HSP27/HSPB1) is a cell stress protein that regulates apoptosis in extrarenal cells and is expressed by podocytes exposed to toxins causing nephrotic syndrome. We investigated the regulation of HSPB1 expression and its function in podocytes exposed to factors contributing to DN, such as high glucose and angiotensin (Ang) II. HSPB1 expression was assessed in renal biopsies from patients with DN, minimal change disease or focal segmental glomerulosclerosis (FSGS), in a rat model of diabetes induced by streptozotocin (STZ) and in Ang II-infused rats. The regulation of HSPB1 was studied in cultured human podocytes and the function of HSPB1 expressed in response to pathophysiologically relevant stimuli was explored by short interfering RNA knockdown. Total kidney HSPB1 mRNA and protein expression was increased in rats with STZ-induced diabetes and in rats infused with Ang II. Upregulation of HSPB1 protein was confirmed in isolated diabetic glomeruli. Immunohistochemistry showed increased glomerular expression of HSPB1 in both models and localized glomerular HSPB1 to podocytes. HSPB1 protein was increased in glomerular podocytes from patients with DN or FSGS. In cultured human podocytes HSPB1 mRNA and protein expression was upregulated by high glucose concentrations and Ang II. High glucose, but not Ang II, promoted podocyte apoptosis. HSPB1 short interfering RNA (siRNA) targeting increased apoptosis in a high-glucose milieu and sensitized to Ang II or TGFß1-induced apoptosis by promoting caspase activation. In conclusion, both high glucose and Ang II contribute to HSPB1 upregulation. HSPB1 upregulation allows podocytes to better withstand an adverse high-glucose or Ang II-rich environment, such as can be found in DN., This work was supported by the grants FIS 06/0046, FIS PS09/00447, PI081564, EUproject DIALOK: LSHBCT-2007-036644, ISCIII-RETIC REDinREN/RD06/0003, REDinREN/RD06/0004, Fondecyt (Chile) 1080083. Comunidad de Madrid/FRACMS-BIO0283/2006, SAF 2007/63648 y CAM S-GEN-0247/2006. The salary support was provided by the FIS to MDSN, ABS, Programa Intensificación Actividad Investigadora (ISCIII/Agencia Lain-Entralgo/CM) to AO.

Author(s): Chen, Yun; Choi, Michael Y; Chen, Liguang; Yu, Jian; Zhang, Ling; Ghia, Emanuela M; Sanchez-Lopez, Elsa; Widhopf II, George F; Messer, Karen; Rassenti, Laura Z; Jamieson, Catriona; Kipps, Thomas J | Abstract: Abstract ROR1 is an oncoembryonic receptor tyrosine kinase expressed on chronic lymphocytic leukemia (CLL) B cells, but not on most normal post-partum tissues. It functions as a receptor for Wnt5a, which is present at high levels in the plasma of patients with CLL relative to that of age-matched controls. Wnt5a/ROR1 is known to activate pro-survival signals in CLL cells, but detailed mechanisms are not fully understood. We found that monocyte-derived Nurse-Like Cells (NLC) could induce CLL cells to activate STAT3 (pY705) and that this effect could be blocked by neutralizing antibodies to Wnt5a, which we found expressed at high levels by NLC. We observed that this effect also could be blocked by cirmtuzumab, a humanized mAb that can inhibit ROR1-signaling, indicating this effect of NLC on CLL cells was Wnt5a and ROR1 dependent. We performed a time-course study examining for pSTAT3 in isolated serum-starved CLL cells treated with exogenous Wnt5a. This revealed that Wnt5a induced delayed activation of STAT3, appearing first at 3 hours. As such, we hypothesized that the noted activation of STAT3 in CLL cells was indirect, being caused by a factor(s) made by isolated CLL cells in response to Wnt5a. We examined harvested supernatants of CLL cells cultured with or without Wnt5a using a human cytokine array assay. Multiple proinflammatory factors including IL-6, IL-8, CCL2, CCL3, CCL4, and CXCL1 were detected in the medium of CLL cells cultured for 24 hours with Wnt5a that were either not detected or found at lower levels in the medium of CLL cells cultured without Wnt5a. Moreover, the medium harvested from CLL cells cultured with Wnt5a, but not the medium of CLL cells cultured without, could induce pSTAT3 within 30 minutes in serum-starved CLL cells; this effect could be inhibited by the anti-IL-6-receptor mAb, tocilizumab, but not by cirmtuzumab, even when used at concentrations that could block the capacity of Wnt5a to induce latent activation of STAT3 and production of IL-6. Since the genes encoding these proinflammatory factors are targets of nuclear factor kappa B (NF-κB), we hypothesized that they were induced through Wnt5a/ROR1-dependent activation of NF-κB. Consistent with this notion, we found that BAY 11-7082 or BMS-345541, which can each inhibit activation of NF-κB, also could block Wnt5a-induced CLL-cell activation of STAT3 and production of IL-6. Furthermore, Wnt5a could induce phosphorylation of NF-κB p65 in CLL cells within 30 minutes, and this effect could be blocked by cirmtuzumab. Using real-time PCR array to evaluate for expression of NF-κB target genes, we found Wnt5s could induce up-regulation of NF-κB target genes in CLL cells, and that this effect could be blocked by cirmtuzumab. Moreover, these studies revealed that Wnt5a/ROR1/NF-κB signaling could induce expression of genes encoding the noted proinflammatory factors in CLL cells. To examine the in vivo significance of these findings, we collected plasma and CLL cells from patients treated with cirmtuzumab in a phase I clinical trial (Choi, MY, et al, Cell Stem Cell 22:951, 2018). RNAseq and ELISA respectively were used to examine the transcriptomes of negatively-selected CLL cells and the concentrations of IL-6 in plasma collected before and after treatment. Consistent with our in vitro findings, treatment with cirmtuzumab downregulated CLL-cell expression of NF-κB target genes in vivo by gene set enrichment analysis (n = 3, NES = 2.10, FDR q = 0.01). The levels of IL-6 in plasma also were significantly decreased in patients after therapy (p = 0.02, n = 5, Paired Student t test). Collectively, these studies indicate that Wnt5a/ROR1-dependent signaling induced by NLC may play a major role in the noted activation of NF-κB in CLL, leading to the production of factors, such as IL-6, which are posted to contribute to pathogenesis. Moreover, these data suggest that some of the noted clinical effects of therapy with cirmtuzumab may be due to suppression of Wnt5a-induced, ROR1-dependent activation of NF-κB in patients with CLL. Disclosures Choi: Genentech: Speakers Bureau; Pharmacyclics: Consultancy, Research Funding, Speakers Bureau; Gilead: Speakers Bureau; AbbVie, Inc: Consultancy, Speakers Bureau; Rigel: Consultancy. Kipps:AbbVie: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Janssen: Honoraria, Membership on an entity's Board of Directors or advisory committees; Verastem: Membership on an entity's Board of Directors or advisory committees; Verastem: Membership on an entity's Board of Directors or advisory committees; Janssen: Honoraria, Membership on an entity's Board of Directors or advisory committees; Genentech Inc: Consultancy, Research Funding; Gilead: Consultancy, Honoraria, Research Funding; Pharmacyclics: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Celgene: Consultancy; F. Hoffmann-La Roche Ltd: Consultancy, Research Funding.

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.

Tubulointerstitial fibrosis is a final common pathway to end-stage chronic kidney diseases, which are characterized by elevated renal angiotensin II (AngII) production. This peptide participates in kidney damage inducing fibrosis and epithelial mesenchymal transition (EMT). Our aim was to describe potential therapeutic targets in AngII-induced EMT, investigating the blockade of different intracellular pathways. Studies were done in human tubular epithelial cells (HK2 cell line), evaluating changes in phenotype and EMT markers (Western blot and immunofluorescence). Treatment of HK2 cells with AngII for 3 days caused transdifferentiation into myofibroblast-like cells. The blockade of MAPKs cascade, using specific inhibitors of p38 (SB203580), extracellular signal-regulated kinase1/2 (ERK; PD98059) and Jun N-terminal kinase (JNK) (SP600125), diminished AngII-induced EMT. The blockade of RhoA/ROCK pathway, by transfection of a RhoA dominant-negative vector or by ROCK inhibition with Y-27632 or fasudil, inhibited EMT caused by AngII. Connective tissue growth factor (CTGF) is a downstream mediator of AngII-induced EMT. MAPKs and ROCK inhibitors blocked CTGF overexpression induced by AngII. HMG-CoA reductase inhibitors, although blocked AngII-mediated kinases activation, only partially diminished EMT and did not regulate CTGF. These data suggest a potential therapeutic use of kinase inhibitors in renal fibrosis.

3-hydroxy-3-methylglutaryl (HMG)-coenzyme A (CoA) reductase inhibitors (statins) present beneficial effects in cardiovascular diseases. Angiotensin II (Ang II) contributes to cardiovascular damage through the production of profibrotic factors, such as connective tissue growth factor (CTGF). Our aim was to investigate whether HMG-CoA reductase inhibitors could modulate Ang II responses, evaluating CTGF expression and the mechanisms underlying this process. In cultured vascular smooth muscle cells (VSMCs) atorvastatin and simvastatin inhibited Ang II–induced CTGF production. The inhibitory effect of statins on CTGF upregulation was reversed by mevalonate and geranylgeranylpyrophosphate, suggesting that RhoA inhibition could be involved in this process. In VSMCs, statins inhibited Ang II–induced Rho membrane localization and activation. In these cells Ang II regulated CTGF via RhoA/Rho kinase activation, as shown by inhibition of Rho with C3 exoenzyme, RhoA dominant-negative overexpression, and Rho kinase inhibition. Furthermore, activation of p38MAPK and JNK, and redox process were also involved in Ang II–mediated CTGF upregulation, and were downregulated by statins. In rats infused with Ang II (100 ng/kg per minute) for 2 weeks, treatment with atorvastatin (5 mg/kg per day) diminished aortic CTGF and Rho activation without blood pressure modification. Rho kinase inhibition decreased CTGF upregulation in rat aorta, mimicking statin effect. CTGF is a vascular fibrosis mediator. Statins diminished extracellular matrix (ECM) overexpression caused by Ang II in vivo and in vitro. In summary, HMG-CoA reductase inhibitors inhibit several intracellular signaling systems activated by Ang II (RhoA/Rho kinase and MAPK pathways and redox process) involved in the regulation of CTGF. Our results may explain, at least in part, some beneficial effects of statins in cardiovascular diseases.

Nuclear factor κB (NF-κB), a key activator of inflammation, primes the NLRP3-inflammasome for activation by inducing pro-IL-1β and NLRP3 expression. NF-κB, however, also prevents excessive inflammation and restrains NLRP3-inflammasome activation through a poorly defined mechanism. We now show that NF-κB exerts its anti-inflammatory activity by inducing delayed accumulation of the autophagy receptor p62/SQSTM1. External NLRP3-activating stimuli trigger a form of mitochondrial (mt) damage that is caspase-1- and NLRP3-independent and causes release of direct NLRP3-inflammasome activators, including mtDNA and mtROS. Damaged mitochondria undergo Parkin-dependent ubiquitin conjugation and are specifically recognized by p62, which induces their mitophagic clearance. Macrophage-specific p62 ablation causes pronounced accumulation of damaged mitochondria and excessive IL-1β-dependent inflammation, enhancing macrophage death. Therefore, the "NF-κB-p62-mitophagy" pathway is a macrophage-intrinsic regulatory loop through which NF-κB restrains its own inflammation-promoting activity and orchestrates a self-limiting host response that maintains homeostasis and favors tissue repair.

We will focus on the recent findings concerning the inflammatory response in vascular and renal tissues caused by hypertension.Angiotensin II is one of the main factors involved in hypertension-induced tissue damage. This peptide regulates the inflammatory process. Angiotensin II activates circulating cells, and participates in their adhesion to the activated endothelium and subsequent transmigration through the synthesis of adhesion molecules, chemokines and cytokines. Among the intracellular signals involved in angiotensin II-induced inflammation, the production of reactive oxygen species and the activation of nuclear factor-kappaB are the best known.The pharmacological blockade of angiotensin II actions, by angiotensin-converting enzyme inhibitors or angiotensin receptor antagonists, results in beneficial organ protective effects, in addition to the effects of these agents on blood pressure control, that can be explained by the blockade of the angiotensin II-induced pro-inflammatory response. These data provide a rationale for the use of blockers of the renin-angiotensin system to prevent vascular and renal inflammation in patients with hypertension.

The Rho-kinase pathway regulates angiotensin II-induced renal damage Background Angiotensin II (AngII) is a key factor in the pathogenesis of renal damage. AngII via AngII type 1 receptors activates several intracellular signaling systems, including the small guanosine triphosphatase Rho and its downstream effector Rho-dependent serine-threonine kinase (Rho-kinase). The Rho/Rho-kinase pathway contributes to inflammatory and proliferative changes observed in cardiovascular diseases. However, the data on renal diseases are scarce. The aim of this study was to investigate the effect of Rho-kinase inhibition in AngII-induced renal damage. Methods We used the model of systemic AngII infusion into normal rats (100 ng/kg per minute; subcutaneous osmotic minipumps), and some animals were treated with the Rho-kinase inhibitor Y-27632 (30 mg/kg per day). In the kidneys of these animals, we evaluated renal lesions, transcription factor activity (by electrophoretic mobility shift assay), and messenger RNA (by polymerase chain reaction) and protein expression levels (by Western blot and/or immunohistochemistry) of proinflammatory and profibrotic factors. Results Rats infused with AngII for three days present renal inflammatory cell infiltration and slight tubular damage, which were diminished by treatment with the Rho-kinase inhibitor Y-27632. AngII activates nuclear factor-κB and causes overexpression of proinflammatory factors, including cytokines (tumor necrosis factor α) and chemokines (monocyte chemotactic protein-1), and of profibrotic factors (connective tissue growth factor). Treatment of AngII-infused rats with Y-27632 decreases the upregulation of these proinflammatory and profibrotic mediators. Conclusion These data demonstrate that the Rho-kinase pathway is involved in renal damage caused by AngII through the regulation of proinflammatory and profibrotic mediators. These results suggest that inhibition of the Rho-kinase pathway represents a novel therapy for renal diseases associated with local AngII generation.

ObjectivesLittle is known about targeting the metabolome in non-cancer conditions. Choline kinase (ChoKα), an essential enzyme for phosphatidylcholine biosynthesis, is required for cell proliferation and has been implicated in cancer invasiveness. Aggressive behaviour of fibroblast-like synoviocytes (FLS) in rheumatoid arthritis (RA) led us to evaluate whether this metabolic pathway could play a role in RA FLS function and joint damage.MethodsCholine metabolic profile of FLS cells was determined by 1H magnetic resonance spectroscopy (1HMRS) under conditions of ChoKα inhibition. FLS function was evaluated using the ChoKα inhibitor MN58b (IC50=4.2 μM). For arthritis experiments, mice were injected with K/BxN sera. MN58b (3 mg/kg) was injected daily intraperitoneal beginning on day 0 or day 4 after serum administration.ResultsThe enzyme is expressed in synovial tissue and in cultured RA FLS. Tumour necrosis factor (TNF) and platelet-derived growth factor (PDGF) stimulation increased ChoKα expression and levels of phosphocholine in FLS measured by Western Blot (WB) and metabolomic studies of choline-containing compounds in cultured RA FLS extracts respectively, suggesting activation of this pathway in RA synovial environment. A ChoKα inhibitor also suppressed the behaviour of cultured FLS, including cell migration and resistance to apoptosis, which might contribute to cartilage destruction in RA. In a passive K/BxN arthritis model, pharmacologic ChoKα inhibition significantly decreased arthritis in pretreatment protocols as well as in established disease.ConclusionsThese data suggest that ChoKα inhibition could be an effective strategy in inflammatory arthritis. It also suggests that targeting the metabolome can be a new treatment strategy in non-cancer conditions.

Introduction: The nuclear factor-κB (NF-κB) is an important regulator of the inflammatory response. Angiotensin II (Ang II) activates the NF-κB pathway linked to renal inflammation. Although both AT1 and AT2 receptors are involved in Ang II-mediated NF-κB activation, the biological processes mediated by each receptor are not fully characterized. Interleukin-1β (IL-1β) is an important macrophage-derived cytokine that regulates immune and inflammatory processes, activating intracellular pathways shared with Ang II, including the NF-κB. Materials and methods: In vitro studies were done in primary cultured rat mesangial cells. NF-κB pathway was evaluated by phosphorylated levels of p65/IκB and DNA binding activity. The Ang II receptor subtype was determined by pretreatment with AT1 and AT2 antagonists. Results: In mesangial cells the simultaneous presence of Ang II and IL-1β caused a synergistic activation of the NF-κB pathway and a marked upregulation of proinflammatory factors under NF-κB control, including monocyte chemoattractant protein-1. The AT1, but not AT2, antagonist abolished the synergistic effect on NF-κB activation and proinflammatory genes caused by coincubation of Ang II and IL-1β. Conclusions: These data indicates that Ang II, via AT1/NF-κB pathway activation, cooperates with IL-β to increase the inflammatory response in mesangial cells, This work was supported by grants from the Instituto de Salud Carlos III (ISCIIIRETIC REDinREN RD06/0016, RD12/0021, PI11/01854), Comunidad de Madrid (Fibroteam S2010/BMD- 2321, S2010/BMD-2378), and Research Institute Queen Sophia (IRSIN). Programa Intensificación Actividad Investigadora (ISCIII/Agencia Laín-Entralgo/CM) to AO. MA and ESL are supported by a ‘Sara Borrell’ postdoctoral contract from Instituto de Salud Carlos III (CD10/00347 and CD09/00066, respectively)

Endothelin (ET)-1 is a potent vasoconstrictor that participates in cardiovascular diseases. Connective tissue growth factor (CTGF) is a novel fibrotic mediator that is overexpressed in human atherosclerotic lesions, myocardial infarction, and experimental models of hypertension. In vascular smooth muscle cells (VSMCs), CTGF regulates cell proliferation/apoptosis, migration, and extracellular matrix (ECM) accumulation. Our aim was to investigate whether ET-1 could regulate CTGF and to investigate the potential role of ET-1 in vascular fibrosis. In growth-arrested rat VSMCs, ET-1 upregulated CTGF mRNA expression, promoter activity, and protein production. The blockade of CTGF by a CTGF antisense oligonucleotide decreased FN and type I collagen expression in ET-1–treated cells, showing that CTGF participates in ET-1–induced ECM accumulation. The ET A , but not ET B , antagonist diminished ET-1–induced CTGF expression gene and production. Several intracellular signals elicited by ET-1, via ET A receptors, are involved in CTGF synthesis, including activation of RhoA/Rho-kinase and mitogen-activated protein kinase and production of reactive oxygen species. CTGF is a mediator of TGF-β– and angiotensin (Ang) II–induced fibrosis. In VSMCs, ET-1 did not upregulate TGF-β gene or protein. The presence of neutralizing transforming growth factor (TGF)-β antibody did not modify ET-1–induced CTGF production, showing a TGF-β–independent regulation. We have also found an interrelationship between Ang II and ET-1 because the ET A antagonist diminished CTGF upregulation caused by Ang II. Collectively, our results show that, in cultured VSMCs, ET-1, independently of TGF-β and through the activation of several intracellular signals via ET A receptors, regulates CTGF. This novel finding suggests that CTGF could be a mediator of the profibrotic effects of ET-1 in vascular diseases.

Background— Angiotensin II (Ang II) participates in vascular fibrosis. Transforming growth factor-β (TGF-β) is considered the most important fibrotic factor, and Smad proteins are essential components of the TGF-β signaling system. Our aim was to investigate whether Ang II activates the Smad pathway in vascular cells and its potential role in fibrosis, evaluating connective tissue growth factor (CTGF) and extracellular matrix (ECM) proteins. Methods and Results— Systemic infusion of Ang II into Wistar rats increased aortic Smad2, phosphorylated-Smad2, and Smad4 expression, associated with CTGF upregulation. In growth-arrested vascular smooth muscle cells, Ang II treatment for 20 minutes caused Smad2 phosphorylation, nuclear translocation of phosphorylated-Smad2 and Smad4, and increased Smad DNA-binding activity. Ang II also caused Smad overexpression and Smad-dependent gene transcription. The AT 1 antagonist losartan diminished Ang II-induced Smad activation. The blockade of endogenous TGF-β did not modify the activation of Smad caused by Ang II. The p38 mitogen-activated protein kinase (MAPK) inhibitor SB203580 diminished Ang II-induced Smad2 phosphorylation. These data show that Ang II activates the Smad pathway via AT 1 receptors and MAPK activation independently of TGF-β. Transient transfection with Smad7, which interferes with receptor-mediated activation of Smad2, diminished Ang II-induced CTGF promoter activation, gene and protein expression, and fibronectin and type-1 procollagen overexpression, showing that Smad activation is involved in Ang II-induced fibrosis. Conclusions— Our results show that Ang II activates the Smad signaling system in vascular cells in vivo and in vitro. Smad proteins are involved in Ang II-induced CTGF and ECM overexpression independently of TGF-β. This novel finding suggests that Smad activation could be involved in the profibrogenic effects of Ang II in vascular diseases.