Juan Rodríguez-Vita

Angiotensin II Activates the Smad Pathway in Vascular Smooth Muscle Cells by a Transforming Growth Factor-β–Independent Mechanism

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 AT1 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 AT1 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.

Renal and vascular hypertension-induced inflammation: role of angiotensin II.

Purpose of reviewWe will focus on the recent findings concerning the inflammatory response in vascular and renal tissues caused by hypertension. Recent findingsAngiotensin 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-κB are the best known. SummaryThe 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.

Endothelin-1, via ETA Receptor and Independently of Transforming Growth Factor-β, Increases the Connective Tissue Growth Factor in Vascular Smooth Muscle Cells

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 ETA, but not ETB, antagonist diminished ET-1–induced CTGF expression gene and production. Several intracellular signals elicited by ET-1, via ETA 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-&bgr;– and angiotensin (Ang) II–induced fibrosis. In VSMCs, ET-1 did not upregulate TGF-&bgr; gene or protein. The presence of neutralizing transforming growth factor (TGF)-&bgr; antibody did not modify ET-1–induced CTGF production, showing a TGF-&bgr;–independent regulation. We have also found an interrelationship between Ang II and ET-1 because the ETA antagonist diminished CTGF upregulation caused by Ang II. Collectively, our results show that, in cultured VSMCs, ET-1, independently of TGF-&bgr; and through the activation of several intracellular signals via ETA receptors, regulates CTGF. This novel finding suggests that CTGF could be a mediator of the profibrotic effects of ET-1 in vascular diseases.

Angiotensin II activates the Smad pathway during epithelial mesenchymal transdifferentiation

Epithelial to mesenchymal transdifferentiation is a novel mechanism that promotes renal fibrosis and here we investigated whether known causes of renal fibrosis (angiotensin II and transforming growth factor beta, TGFbeta) act through this pathway. We infused angiotensin II into rats for 1 day and found that it activated the Smad pathway which persisted for up to 2 weeks in chronically infused rats. Renal TGF-beta mRNA expression was increased at 3 days and its protein at 2 weeks suggesting Smad pathway activation occurred earlier than TGF-beta upregulation. In cultured human tubuloepithelial cells, angiotensin II caused a rapid activation of Smad signaling independent of TGF-beta however, Smad-dependent transcription after 1 day was TGF-beta mediated. Two weeks of angiotensin II infusion activated genes associated with epithelial mesenchymal transdifferentiation. Stimulation with angiotensin II for 3 days caused transdifferentiation of the cultured epithelial cells by TGF-beta-mediated processes; however, early changes were independent of endogenous TGF-beta. Smad7 overexpression, which blocks Smad2/3 activation, diminished angiotensin II-induced epithelial mesenchymal transdifferentiation. Our results show that angiotensin II activates the Smad signaling system by TGF-beta-independent processes, in vivo and in vitro, causing renal fibrosis.

HMG-CoA Reductase Inhibitors Decrease Angiotensin II–Induced Vascular Fibrosis: Role of RhoA/ROCK and MAPK Pathways

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.

Angiotensin IV Activates the Nuclear Transcription Factor-κB and Related Proinflammatory Genes in Vascular Smooth Muscle Cells

Inflammation is a key event in the development of atherosclerosis. Nuclear factor-&kgr;B (NF-&kgr;B) is important in the inflammatory response regulation. The effector peptide of the renin angiotensin system Angiotensin II (Ang II) activates NF-&kgr;B and upregulates some related proinflammatory genes. Our aim was to investigate whether other angiotensin-related peptides, as the N-terminal degradation peptide Ang IV, could regulate proinflammatory factors (activation of NF-&kgr;B and related genes) in cultured vascular smooth muscle cells (VSMCs). In these cells, Ang IV increased NF-&kgr;B DNA binding activity, caused nuclear translocation of p50/p65 subunits, cytosolic I&kgr;B degradation and induced NF-&kgr;B–dependent gene transcription. Ang II activates NF-&kgr;B via AT1 and AT2 receptors, but AT1 or AT2 antagonists did not inhibit NF-&kgr;B activation caused by Ang IV. In VSMC from AT1a receptor knockout mice, Ang IV also activated NF-&kgr;B pathway. In those cells, the AT4 antagonist divalinal diminished dose-dependently Ang IV–induced NF-&kgr;B activation and prevented I&kgr;B degradation, but had no effect on the Ang II response, indicating that Ang IV activates the NF-&kgr;B pathway via AT4 receptors. Ang IV also increased the expression of proinflammatory factors under NF-&kgr;B control, such as MCP-1, IL-6, TNF-&agr;, ICAM-1, and PAI-1, which were blocked by the AT4 antagonist. Our results reveal that Ang IV, via AT4 receptors, activates NF-&kgr;B pathway and increases proinflammatory genes. These data indicate that Ang IV possesses proinflammatory properties, suggesting that this Ang degradation peptide could participate in the pathogenesis of cardiovascular diseases.

CTGF Promotes Inflammatory Cell Infiltration of the Renal Interstitium by Activating NF-κB

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.

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.

Essential Role of TGF-β/Smad Pathway on Statin Dependent Vascular Smooth Muscle Cell Regulation

Background The 3-hydroxy-3-methylglutaryl CoA reductase inhibitors (also called statins) exert proven beneficial effects on cardiovascular diseases. Recent data suggest a protective role for Transforming Growth Factor-β (TGF-β) in atherosclerosis by regulating the balance between inflammation and extracellular matrix accumulation. However, there are no studies about the effect of statins on TGF-β/Smad pathway in atherosclerosis and vascular cells. Methodology In cultured vascular smooth muscle cells (VSMCs) statins enhanced Smad pathway activation caused by TGF-β. In addition, statins upregulated TGF-β receptor type II (TRII), and increased TGF-β synthesis and TGF-β/Smad-dependent actions. In this sense, statins, through Smad activation, render VSMCs more susceptible to TGF-β induced apoptosis and increased TGF-β-mediated ECM production. It is well documented that high doses of statins induce apoptosis in cultured VSMC in the presence of serum; however the precise mechanism of this effect remains to be elucidated. We have found that statins-induced apoptosis was mediated by TGF-β/Smad pathway. Finally, we have described that RhoA inhibition is a common intracellular mechanisms involved in statins effects. The in vivo relevance of these findings was assessed in an experimental model of atherosclerosis in apolipoprotein E deficient mice: Treatment with Atorvastatin increased Smad3 phosphorylation and TRII overexpression, associated to elevated ECM deposition in the VSMCs within atheroma plaques, while apoptosis was not detected. Conclusions Statins enhance TGF-β/Smad pathway, regulating ligand levels, receptor, main signaling pathway and cellular responses of VSMC, including apoptosis and ECM accumulation. Our findings show that TGF-β/Smad pathway is essential for statins-dependent actions in VSMCs.