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(Circulation Research. 2005;96:965.)
© 2005 American Heart Association, Inc.

Molecular Medicine

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

Vanesa Esteban, Mónica Ruperez, Elsa Sánchez-López, Juan Rodríguez-Vita, Oscar Lorenzo, Heidi Demaegdt, Patrick Vanderheyden, Jesús Egido, Marta Ruiz-Ortega

From the Vascular and Renal Research Laboratory (V.E., M.R., E.S.-L., J.R.-V., O.L., J.E., M.R.-O.), Fundación Jiménez Diaz, Universidad Autónoma Madrid, Spain; and the Department of Molecular and Biochemical Pharmacology (H.D., P.V.), Institute for Molecular Biology and Biotechnology, Vrije Univerisiteit Brussel, Belgium.

Correspondence to Marta Ruiz-Ortega, PhD, Vascular and Renal Research Laboratory, Fundación Jiménez Díaz, Universidad Autónoma de Madrid Avda, Reyes Católicos, 2, 28040 Madrid, Spain. E-mail mruizo{at}fjd.es

	Abstract

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Inflammation is a key event in the development of atherosclerosis. Nuclear factor-B (NF-B) is important in the inflammatory response regulation. The effector peptide of the renin angiotensin system Angiotensin II (Ang II) activates NF-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-B and related genes) in cultured vascular smooth muscle cells (VSMCs). In these cells, Ang IV increased NF-B DNA binding activity, caused nuclear translocation of p50/p65 subunits, cytosolic IB degradation and induced NF-B–dependent gene transcription. Ang II activates NF-B via AT₁ and AT₂ receptors, but AT₁ or AT₂ antagonists did not inhibit NF-B activation caused by Ang IV. In VSMC from AT_1a receptor knockout mice, Ang IV also activated NF-B pathway. In those cells, the AT₄ antagonist divalinal diminished dose-dependently Ang IV–induced NF-B activation and prevented IB degradation, but had no effect on the Ang II response, indicating that Ang IV activates the NF-B pathway via AT₄ receptors. Ang IV also increased the expression of proinflammatory factors under NF-B control, such as MCP-1, IL-6, TNF-, ICAM-1, and PAI-1, which were blocked by the AT₄ antagonist. Our results reveal that Ang IV, via AT₄ receptors, activates NF-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.

Key Words: angiotensin IV • signal transduction • nuclear factor–B • vascular smooth muscle cell • insulin regulated aminopeptidase

	Introduction

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Activation of the renin angiotensin system (RAS) is involved in the pathogenesis of several cardiovascular diseases, including atherosclerosis and hypertension.¹ The classical view of atherosclerosis as a lesion composed by lipid deposits has now been changed to a chronic inflammatory disorder, regulated by the production of inflammatory mediators and immune cells, which are involved in the initiation, progression, and rupture of the plaque.² The inflammatory response includes endothelial dysfunction, increased adhesion molecule expression, and release of proinflammatory mediators (chemokines and cytokines). Inflammatory mechanisms have also recently been related to development of hypertension-induced vascular damage.^1,3

The nuclear factor-B (NF-B) is a pivotal transcription factor in inflammatory diseases and chronic immune responses.⁴ Many stimuli relevant to cardiovascular diseases, including cytokines (IL-6 and TNF-), growth factors, Angiotensin II (Ang II), signals elicited by ischemic stress (NO and oxygen free radicals), and mechanical forces, have been shown to activate NF-B. This transcription factor participates in vascular damage through the regulation of several genes, including adhesion molecules, cytokines, chemokines, angiotensinogen, and other products involved in proliferation and immune responses.⁴ In pathological conditions, elevated tissue NF-B activity correlated with inflammatory events has been observed.^1,3 NF-B inhibition suppresses the development of atherosclerotic lesions by preventing inflammation, vascular smooth muscle cell (VSMC) proliferation, and apoptosis.^4–7 Blockade of the RAS diminished tissue NF-B activation and upregulation of proinflammatory parameters. Ang II in vivo and in cultured cells activates NF-B and upregulates related genes.^1,3

Although Ang II has been considered the effector peptide of the RAS, other peptides, such as the N-terminal, Ang III, and Ang IV, and the C-terminal, Ang(1–7), degradation products also possess biological activities.⁸ However, their potential roles in pathophysiological processes are far from being completely understood. Ang III shares some Ang II functions, like blood pressure regulation, and participates in renal damage.^9,10 Ang(1–7) induces vasodilatation and antiproliferation.¹¹ Ang IV [the Val(3)-Phe(8) fragment of Ang II] has sparked of great interest because of its wide range of physiological effects. Ang IV mediates important physiological functions in the central nervous system, including blood flow regulation and processes attributed to learning and memory.^12,13 Ang IV participates in cell growth responses in cardiac fibroblasts, endothelial cells, and VSMCs.^14–16 Interestingly, an antiapoptotic role for Ang IV in neuronal cells was reported.^17,18

The existence of specific receptor subtypes for Ang peptides has been investigated.^19,20 Two main subtypes, AT₁ and AT₂ that bind mainly to Ang II and Ang III, have been cloned and extensively studied. A different and specific receptor for Ang(1–7), the mas receptor, has recently been described.¹¹ Ang IV binds to a specific receptor, the AT₄, that has recently been identified as insulin-regulated aminopeptidase (IRAP) by cross-linking to an Ang IV analogue, purifying the protein by chromatography and SDS-PAGE, and identifying tryptic peptides using mass spectrometry.¹² The presence of Ang IV–specific binding sites has been identified in various mammalian tissues and cells, including brain, blood vessels, heart, kidney, and cultured VSMCs.^16,19,20

In an endothelial model of balloon injury, Ang IV binding was increased in media, neointima, and reendothelialized cell layer, suggesting a role for Ang IV in vascular wall remodeling after damage.²¹ Ang IV could contribute to vascular damage through the stimulation of cell growth and plasminogen activator inhibitor (PAI-1) expression.²² Inflammation is a key event in the progression of atherosclerosis and hypertension. However, whether Ang IV possesses proinflammatory properties has not been investigated. Our aim was to investigate whether Ang IV could regulate proinflammatory mediators, evaluating the activation of NF-B pathway and proinflammatory NF-B–related genes in cultured VSMCs.

	Materials and Methods

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Materials
Ang peptides were obtained from BACHEM. Antibodies to NF-B subunits were from Santa Cruz; NF-B consensus (5'-AGT-TGAGGGGACTTTCCCAGGC-3') and mutant (5'-AGTTGAGG-CTCCTTTCCCAGGC-3') oligonucleotides were from Promega. -[³²P]dCTP (3000 Ci/mmol) and -[³²P]CTP (3000 Ci/mmol) were from Amersham. Cell culture reagents were from Life Technologies, Inc. The rest of the compounds were from Sigma-Aldrich.

Cell Cultures
Rat VSMCs were obtained from thoracic aorta of Wistar-Kyoto rats by collagenase method.²³ Murine VSMC were prepared from 2 different strains, AT₁ knockout mice (provided by Dr Sugaya, Tanabe Seiyaku Corp, Osaka, Japan) and their wild littermates.²⁴ For experiments, cells at 80% confluence from passages 2 to 5 were used.

Radioligand Binding and Enzyme Assay
In membranes, homogenates from rat VSMCs, prepared as described, binding experiments and determination of the cystinil aminopeptidase catalytic activity were done.²⁵

NF-B Activation
Nuclear and cytosolic extracts were prepared by homogenization and centrifugation.²³ NF-B DNA binding activity was determined in nuclear extracts by binding with labeled NF-B consensus and electrophoresis. To quantify cytosolic IB and phospho-IkB, Western blot analyses were done.²³ The quality of proteins and efficacy of protein transfer was evaluated by Red Ponceau staining (not shown). In all experiments, protein content was determined by the BCA method. For immunofluorescence, cells were fixed (3%PFA, 10 minutes and 0.1% Triton-X100, 1 minute) and incubated with antibodies against p50/p65 subunits and then with FITC-labeled secondary antibody. The absence of primary antibody was the negative control (not shown).

To study NF-B–dependent gene transcription, VSMCs were seeded in 6-well plates and 24 hours later cells were transient transfected with FUGENE (Roche Molecular Biochemicals) containing 1 µg NF-B/luc promoter and 1 µg TK-renilla as internal control (Clontech). After a 24-hour serum-starvation step, cells were stimulated for 24 hours, and luciferase/renilla activity was measured.

Gene and Protein Studies
Total RNA was isolated with Trizol and proinflammatory gene expression was analyzed by real-time PCR.²⁶ Assays on demand used were as follows: mice MCP-1, Mm00441242_m1; IL-6, Mm00446190_m1; TNF-, Mm00443258_m1; ICAM-1, Mm00516023_m1; PAI-1, Mm00435860_m1; and human IRAP, Hs00194378_m1. MCP-1 mRNA expression was also analyzed by Northern blot,²³ and MCP-1 release by ELISA (BD, Pharmingen). A cytokine/chemokine array kit (Ray Biotech, Inc) was used to detect a panel of 22 proteins in cell lysates from WT mice VSMCs.

Statistical Analysis
The autoradiographs were scanned using the GS-800 Calibrated Densitometer (Quantity One, Bio-Rad). Data of gene expression were normalized against those of the corresponding G3PDH. Results are expressed as n-fold increase over control as mean±SEM of the experiments made. Significance was established with GraphPAD Instat using Student t test (GraphPAD Software), Wilcoxon and Student-Newman-Keuls text. Differences were considered significant when P<0.05.

	Results

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Ang IV Activates NF-B in Cultured Rat VSMCs
Ang IV increased NF-B DNA-binding activity after 15 minutes, peaked at 1 hour, and diminished to control levels by 6 hours (Figure 1A). This response was dose-dependent, and maximal after 1 hour with 10^–9 mol/L Ang IV (Figure 1B). Ang IV response was higher than Ang II and with longer kinetics, remaining elevated after 2 hours (Figure 1A).

View larger version (34K): [in this window] [in a new window]   Figure 1. Ang IV activates the transcription factor NF-B in rat VSMCs. A, Time-course. VSMCs were incubated with 10–7 mol/L Ang IV and Ang II for different times. B, Dose response. Cells were treated for 1 hour with Ang IV (10–7 and 10–11 mol/L). After the incubation period, nuclear extracts were isolated, and NF-B activity was determined by binding assay with a labeled NF-B oligonucleotide and analyzed by EMSA. Competition assays with a 100-fold excess of unlabeled or mutant NF-B and unrelated (SP-1) oligonucleotides show specific NF-B complexes (marked by arrows). Position of free-oligonucleotide is indicated. Figure shows in the top panels a representative EMSA and in bottom panels values of mean±SEM from 4 to 5 different experiments. *P<0.05 vs control.

By supershift assays, we have observed that antibodies to p50 and p65, but not c-Rel, shifted the band to a higher molecular weight, showing that Ang IV–activated NF-B complex is a p50/p65 heterodimer (Figure 2A). In control cells, a diffuse cytoplasmic immunofluorescence was seen with p50 or p65 antibodies. When cells were treated for 1 hour with 10^–9mol/L Ang IV, an intense nuclear fluorescence was observed with both antibodies, showing nuclear translocation of NF-B (Figure 2B).

View larger version (25K): [in this window] [in a new window]   Figure 2. Identification of NF-B complexes induced by Ang IV in rat VSMCs. A, Nuclear extracts of Ang IV–treated VSMCs were preincubated for 1 hour with antibodies against the NF-B subunits p50, p65, and c-Rel (1 µg/mL), and protein complexes were resolved by electrophoresis. Supershifted bands are observed with anti-p50 and anti-p65 antibodies (marked by arrows). Figure shows a representative EMSA of 4 experiments. B, Localization of NF-B subunits in Ang IV–treated VSMCs. Cells were treated with 10–9 mol/L Ang IV or 10–7 mol/L Ang II for 1 hour and immunofluorescence using p50 and p65 antibodies was done.

NF-B activation involves dissociation of IB and subsequent degradation. In control cells, IB was found in cytosolic fraction as a protein of around 39 kDa (Western blot). After 1 hour of Ang IV stimulation, this band disappeared, indicating IB degradation, and caused phosphorylation of IB (Figure 3A). These effects were closely correlated with the time-course of Ang IV–induced NF-B activation and p50/p65 nuclear translocation. IB is degraded by 2 distinct pathways: I-kinase–dependent phosphorylation or calcium/calpain (calcium-activated neutral proteinase) process.²⁷ VSMCs were incubated with MG132 (proteasome inhibitor) or calpeptin (calpain inhibitor). Both inhibitors diminished IB proteolysis, suggesting a parallel contribution of both pathways. MG132 treatment, but not calpeptin, increased IB phosphorylation (Figure 3B). Calpain inhibitors can block proteolysis of IB and NF-B activation, while having no effect on the signal-induced phosphorylation of the inhibitor, as we have observed.²⁸

View larger version (23K): [in this window] [in a new window]   Figure 3. Effect of Ang IV on cytosolic IB and phospho-IB levels in VSMCs. A, Cells were treated with Ang IV for increasing periods of time. Cytosolic extracts were electrophoresed under reducing, denaturating conditions, stained with anti-IB, or anti–phospho-IB antibodies and visualized with enhanced chemiluminescence. B, Cells were pretreated with MG132 or Calpeptin (10–5mol/L) and then stimulated with 10–9 mol/L Ang IV for 1 hour. Figures show a representative Western blotting of IB and phospho-IB cytoplasmatic extract (top panel) and in bottom panel mean±SEM of 5 experiments. White and black bars correspond to IB and phospho-IB, respectively. *P<0.05 vs control, #P<0.05 vs Ang IV.

Ang IV Activates NF-B Pathway via AT₄ Receptor
We investigated the receptor subtype involved in NF-B pathway. Cells were preincubated with specific AT-R antagonists (Losartan for AT₁, PD123319 for AT₂, and divalinal for AT₄^19,20) and then stimulated with Ang II or Ang IV for 1 hour.

In rat VSMCs, both AT₁ or AT₂ antagonists partially blocked Ang II–induced NF-B DNA-binding activity, whereas when both antagonists were added together NF-B activation was completely abolished. In contrast, neither AT₁ nor AT₂ antagonists diminished Ang IV–induced NF-B activation (Figure 4A). Similar results were observed in VSMCs from WT mice (Figure 4B). In VSMCs from AT₁ knockout mice, Ang IV increased NF-B DNA-binding activity (Figure 4C), which was not blocked by the AT₂ antagonist, showing that Ang IV acts independently of AT₁ and AT₂ receptors.

View larger version (22K): [in this window] [in a new window]   Figure 4. Ang IV activates NF-B through AT4 receptors, whereas Ang II acts via AT1 and AT2 receptors in rat VSMCs. Cells were preincubated for 1 hour with the receptor antagonists: AT1 Losartan (10–6 mol/L), AT2 PD123319 (10–5 mol/L), or AT4 divalinal (10–8 mol/L; dose-response from 10–6 to 10–12 mol/L), and then were stimulated with 10–7 mol/L Ang II or 10–9 mol/L Ang IV for 1 hour. PMA (10–7 mol/L) and IL-1ß (10 U/mL) were used as positive controls Ang IV activates NF-B via AT4 receptors in VSMCs from WT (B) and AT1 knockout mice (C). Figures show data of mean±SEM of 6 experiments. *P<0.05 vs control; #P<0.05 vs Ang IV; ¥P<0.05 vs Ang II; P=NS vs control.

We have investigated the presence of AT₄/IRAP receptors in VSMCs. Ang IV–specific binding sites, antagonized by divalinal, have previously described in cultured bovine VSMCs.¹⁶ In cell membranes from rat VSMCs, we have found IRAP enzymatic activity and specific binding to [¹²⁵I]-Ang IV. IRAP mRNA expression was detected in human VSMCs (real-time PCR) (not shown). These data suggest that IRAP is present in VSMCs. The AT₄ antagonist divalinal dose-dependently diminished Ang IV–induced NF-B activation in VSMCs from rat, wild-type and AT₁ knockout mice, whereas it did not modify Ang II response. Divalinal alone did not activate NF-B, showing that this compound did not act as an agonist. These results clearly demonstrate that Ang IV acting via the AT₄ receptor, activates NF-B pathway in VSMCs.

AT₄ receptors are linked to tyrosine kinase phosphorylation pathways.^19,29 Ang II via AT₁ activates several kinases, including protein kinase C (PKC) and phosphotyrosine kinases (PTK).^19,20 We showed that AT₁/NF-B pathway is mediated by PTK, but not PKC, activation, whereas AT₂/NF-B is PTK and PKC independent.²³ We investigated whether these intracellular signals are involved in AT₄/NF-B pathway. Treatment with several PKC inhibitors did not modify NF-B activation induced by Ang IV (figure 5), suggesting that PKC is not involved in this process. The PTK inhibitors, genistein, erbstatin, and herbimycin A, caused a marked reduction in Ang IV–induced NF-B activation (Figure 5). These data suggest that AT₄/NF-B pathway is mediated by activation of tyrosine phosphorylation.

View larger version (14K): [in this window] [in a new window]   Figure 5. Molecular mechanisms of Ang IV–induced NF-B activation: role of tyrosine phosphorylation in AT4 signaling. Rat VSMCs were preincubated for 1 hour with the inhibitors and then stimulated with 10–9 mol/L Ang IV for 1 hour. PKC inhibitors: H-7 (10–5 mol/L) and BIP (10–7 mol/L). PTK inhibitors: genistein, erbstatin, and herbimycin A (10–6 mol/L). Figure shows data of mean±SEM of 3 experiments. *P<0.05 vs control; #P<0.05 vs Ang IV.

Effect of Ang IV on NF-B–Mediated Gene Transcription
We have investigated whether Ang IV regulates NF-B–mediated gene expression by transient transfection with a luciferase reporter plasmid (NF-B/luc). In mice VSMCs, Ang IV potently increased NF-B promoter activity (Figure 6), as observed with Ang II, IL-1ß, and TNF-. In AT₁ knockout VSMCs, Ang IV also increases NF-B–mediated gene expression. The AT₄ antagonist divalinal significantly diminished Ang IV–induced luciferase activity, showing that NF-B promoter activation was mediated by AT₄ receptors.

View larger version (14K): [in this window] [in a new window]   Figure 6. Ang IV activates NF-B dependent transcription in VSMCs from WT and AT1 knockout mice. VSMCs were transfected with NF-B/luc promoter and TK-renilla. After 24-hour serum starvation, VSMCs were incubated with 10–9 mol/L Ang IV, 10–7 mol/L Ang II, 10 U/mL IL-1ß, and 100 U/mL TNF- for 24 hours, and then luciferase/renilla activity was measured. Some cells were preincubated for 1 hour with 10–8 mol/L divalinal. Figures show data of mean±SEM of 2 replicates in 5 independent experiments. *P<0.05 vs control; #P<0.05 vs Ang IV.

We have investigated whether Ang IV regulates some proinflammatory factors under NF-B control. By real-time PCR, we have found that in AT₁ knockout VSMC Ang IV upregulated gene expression of the adhesion molecule ICAM-1, the cytokines IL-6 and TNF-, the chemokine MCP-1, and the prothrombotic factor PAI-1 (Figure 7A). The AT₄ antagonist significantly diminished Ang IV–mediated gene overexpression (P<0.05). Using a protein cytokine array system a correlation between increasing levels of certain cytokines and chemokines with the Ang IV–increased gene overexpression were found (Figure 7B).

View larger version (45K): [in this window] [in a new window]   Figure 7. A, Ang IV upregulates several genes under NF-B control. VSMCs from AT1 knockout mice were treated with 10–9 mol/L Ang IV for 3 hours. Some cells were preincubated for 1 hour with 10–8 mol/L divalinal. Gene expression of MCP-1, IL-6, TNF-, ICAM-1, and PAI-1 was analyzed by real-time PCR. Results are expressed as mean±SEM of 4 experiments.*P<0.05 vs control; #P<0.05 vs Ang IV. B, Protein cytokine/chemokine array analysis of cell lysates from Ang IV–treated mice VSMCs. Boxes show cytokines (TNF-, IL-4, IL-10, and IFN-) and chemokines (MCP-1 and RANTES) that are upregulated (+) compared with untreated cells.

We have further investigated whether NF-B regulates Ang IV–induced proinflammatory gene expression, studying MCP-1 gene, an important mediator of monocyte infiltration.³⁰ In rat VSMCs, Ang IV stimulation rapidly increased MCP-1 mRNA levels after 30 minutes, being maximal at 3 hours (Figure 8A). Similar results were found in VSMCs from WT mice (not shown). Ang IV increased MCP-1 protein production, as shown in conditioned media from Ang IV–treated mice VSMC (Figure 8B). Some works have demonstrated that, in VSMCs, Ang II increases MCP-1 via AT₁ receptors.^23,31 In VSMCs from AT₁ knockout mice, Ang IV increases MCP-1 gene (Figure 8C) and protein release (not shown). The AT₄ antagonist completely abolished Ang IV–induced MCP-1 gene upregulation, clearly indicating AT₄ receptors are involved in this process. We have blocked NF-B pathway using the following NF-B inhibitors: pyrrolidine dithiocarbamate (PDTC, an NF-B inhibitor with antioxidant properties as a thiol agent), MG132 (a proteasome inhibitor of IB degradation), gliotoxin (an immunosuppressor), and parthenolide (a specific inhibitor of the IKK/NF-B pathway, which inhibits IKK activity, enhances stability of IB, and blocks nuclear translocation of NF-B). Pretreatment of rat VSMCs with those NF-B inhibitors diminished MCP-1 mRNA induction caused by Ang IV (Figure 8D).

View larger version (51K): [in this window] [in a new window]   Figure 8. A, Ang IV increases MCP-1 gene expression. Growth-arrested rat VSMCs were incubated with 10–9 mol/L Ang IV, 10–7mol/L Ang II, and 100 U/mL TNF- for different times. Figure shows a representative Northern blot (top panel) and mean±SEM of 5 experiments (bottom panels). *P<0.05 vs control. B, Ang IV increases MCP-1 protein release. VSMCs from WT mice were stimulated with 10–9 mol/L Ang IV, 10–7 mol/L Ang II, 10 U/mL IL-1ß, and 100 U/mL TNF- for 24 hours, and MCP-1 production was determined in supernatants by ELISA. Results are expressed as mean±SEM of 5 experiments done in duplicate. *P<0.05 vs control. C, Ang IV increases MCP-1 gene via AT4 receptors. VSMCs from AT1 knockout mice were preincubated for 1 hour with 10–8 mol/L divalinal and then treated with 10–9 mol/L Ang IV for 3 and 6 hours. Figure shows a representative Northern blot (top panel), and mean±SEM of 4 experiments (bottom panels). *P<0.05 vs control; #P<0.05 vs Ang IV. D, Effect of a NF-B inhibitors on MCP-1 mRNA overexpression caused by Ang IV. Cells were pretreated with different NF-B inhibitors PDTC, MG132, gliotoxin, and parthenolide (10–5 mol/L) and then stimulated with Ang IV for 6 hours. Quantification of MCP-1 mRNA was determined densitometrically and expressed as ratio MCP-1/G3PDH as n-fold over control.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

 
We have demonstrated for the first time that Ang IV activates the transcription factor NF-B and upregulates related genes involved in cardiovascular damage, such as the adhesion molecule ICAM-1, the cytokines IL-6 and TNF-, the chemokine MCP-1, and the prothrombotic factor PAI-1. The Ang IV response is mediated by activation of AT₄ receptors, independently of AT₁ and AT₂ receptors. These results reveal novel concepts of RAS in the cardiovascular system, suggesting that Ang IV, via AT₄ receptors, could play an active role in the inflammatory process associated with vascular damage.

NF-B regulates many genes that play key roles in the inflammatory process.^3,4 We have observed that in cultured VSMCs, Ang IV increased NF-B DNA-binding activity, which is functional in its ability to transactivate B containing promoters, and caused nuclear translocation of p50/p65 heterodimer and degradation of cytosolic IB, mediated by I-kinase–dependent phosphorylation and calpain pathways. NF-B inhibitors ameliorate end-organ damage in models characterized by activated tissue RAS.^3,4,26 We have observed that NF-B inhibitors diminished Ang IV–induced gene overexpression (exemplified by MCP-1), showing a NF-B–mediated transcriptional mechanism. The AT₄ antagonist blocked Ang IV–induced NF-B activation and markedly inhibited NF-B–mediated gene transcription (transfection of NF-B/luc reporter) and mRNA overexpression of ICAM-1, IL-6, TNF-, MCP-1, and PAI-1, revealing that NF-B pathway activation and upregulation of NF-B–related genes are mediated by AT₄ receptors. In vascular diseases, ACE inhibition diminishes local NF-B activation and proinflammatory parameters.^30,31 The beneficial effects of ACE inhibition could be due not only to the blockade of Ang II generation but also to the formation of Ang degradation products, such as Ang IV,^32,33 and thereby inhibiting NF-B activation (and upregulation of proinflammatory genes) induced by this peptide.

A common feature of all stages of atherosclerosis is inflammation of the vessel wall. The involvement of RAS in the pathogenesis of human atherosclerosis has already been demonstrated. RAS blockade diminished the presence of inflammatory cells and proinflammatory parameters in the lesion, serum, and circulating monocytes.^3,30,31 In human atherosclerotic plaques, the monocytes/macrophages presented high ACE activity associated with elevated levels of proinflammatory cytokines, such as IL-6.^34,35 In the atheroma plaques, there is an elevated production of Ang II and activation of proteases (mainly by activated macrophages) than can generate Ang degradation products, including Ang IV. Other evidences suggest that Ang IV could contribute to vascular damage. In a model of balloon injury, Ang IV binding in the lesion was increased, mainly in VSMCs and endothelial cells.²¹ ACE is expressed at the shoulder region of atherosclerotic plaques, and ACE activity is enhanced in unstable plaques; therefore, Ang peptides may participate in the plaque instability. Thus, in VSMCs, Ang IV, via AT₄ activation, caused gene overexpression of ICAM-1, IL-6, TNF-, MCP-1, and PAI-1, and could contribute to the progression and rupture of the plaque by activation of inflammation and thrombosis.

The inflammatory process is also important in the development of vascular damage in hypertension. Activation of leukocytes and monocytes was found in patients with essential hypertension, presenting elevated adherence to endothelial cells and producing higher levels of cytokines, IL-1ß, TNF-, and TGF-ß on stimulation.^2,3,36 In experimental models of hypertension, such as SHR, Ang II infusion and double-transgenic rats for renin and angiotensinogen, monocyte/macrophage infiltration, and overexpression of proinflammatory parameters into the vessel wall, heart, kidney, and brain have been described. ACE inhibitors decrease both inflammatory factors and monocyte/macrophage infiltration.³¹ Many studies have clearly demonstrated that Ang II, by direct activation of immune cells or by production of inflammatory mediators, contributes to tissue damage in hypertension. Our data disclose that the Ang degradation product Ang IV also activates proinflammatory parameters, enlarging the contribution of RAS activation in the pathogenesis of hypertension.

AT₁ and AT₂ are 7 transmembrane–spanning G protein–coupled receptors and elicit different intracellular signaling, although both receptors share a common molecular pathway: the activation of NF-B.^1,3,23 AT₁ receptor, similar to classical cytokines, activates protein kinases, such as PKC, PTK, phosphatidylinositol 3 kinase (PI3K), Akt, focal adhesion kinases (FAK), and mitogen-activated protein kinases (MAPK), among other signals. AT₂ receptor activates protein phosphatases, kinin/NO/cGMP system, and production of ceramides.^1,19,20 Recently, AT₄ binding sites have been characterized as being insulin-regulated aminopeptidase and can therefore be denoted as AT₄/IRAP. IRAP is also known as oxytocinase (OTase) or placental leucine aminopeptidase (P-LAP).¹² These different denominations are related to its independent "discovery" by several research teams and related to its enzymatic properties. AT₄/IRAP is a type II integral membrane protein and a member of the M1 family of zinc-dependent metallopeptidases. Some data indicate that after homodimer formation, IRAP can act as a classical receptor, transferring extracellular information across the cell membrane. These observations therefore may lead to the classification of this protein as a mixed enzyme/receptor. In addition or alternatively, Ang IV can exert its effects by inhibiting the catalytic activity of IRAP,^12,13 possibly through an allosteric mechanism.³⁷ As a consequence, Ang IV could reduce the cleavage of some central peptides, an effect of paramount importance in brain physiology. By this mechanism, Ang IV may extend the half-life of endogenous neuropeptides that increase memory.³⁸ The AT₄ antagonist divalinal was designed by Harding’s laboratory,³⁹ and binds only to AT₄, but not AT₁ or AT₂ receptors, with high affinity to a similar number of binding sites as [¹²⁵I]-Ang IV. Divalinal is an antagonist of Ang IV–mediated actions in a variety of physiological systems,^39–41 as we have observed in Ang IV–induced NF-B activation in VSMCs, although some authors have found agonistic properties in other systems.⁴⁰ AT₄/IRAP binds with high affinity to the Ang IV analogue, [¹²⁵I]-Nle¹-Ang IV, and can be selectively cross-linked with an Ang IV–related photoaffinity label. The correspondence between AT₄ binding sites and IRAP was further evidenced by the similar regional distribution of the enzyme’s mRNA and immunoreactivity and the binding of [¹²⁵I]-Nle¹-Ang IV in thin sections of mouse brain.¹² Ang IV–specific binding sites, antagonized by divalinal, have been described in VSMCs.^16,19–21 In line with these observations, we found in the present study that in these cells there is gene expression of IRAP and, in cellular membranes, specific binding to [¹²⁵I]-Ang IV and IRAP enzymatic activity. These data confirm that AT₄/IRAP is also expressed in VSMCs used in this study.

Although, as outlined earlier, the intracellular signaling of AT4/IRAP is not completely established, Ang IV is described to mediate a number of effects via this enzyme/receptor. Early works demonstrated that Ang IV is a vasodilator in kidney and brain.^42,43 Ang IV activates several protein kinases.^44,45 In endothelial cells, Ang IV increases PI3K, PI-dependent kinase-1 (PDK-1), extracellular signal-related kinases (ERK1/2), protein kinase B-/Akt (PKB-), and p70 ribosomal S6 kinase (p70S6K) activities.⁴⁶ Endothelium-dependent vasodilation is mediated by NO and cGMP production,³⁰ intracellular calcium release, and activation phospholipase C and PI3K.²⁹ In rat aorta, PTK inhibition and membrane L-type Ca²⁺ channels blockade diminished Ang IV contractile effects.⁴⁴ In tubular cells, Ang IV increases tyrosine phosphorylation of the focal adhesion-associated proteins, p125-FAK and paxillin. However, Ang IV does not affect cAMP or cGMP production and does not increase cytosolic calcium concentrations.⁴⁵ These data suggest differences in Ang IV signaling depending on the cell type, suggesting that future studies are needed to clearly define Ang IV intracellular mechanisms. Whereas AT₁ and AT₂ receptors present some well-established features of relevance to health and disease,^19,20 the existence of separate receptors for Ang fragments offers exciting possibilities for new therapeutics to target the diverse actions of these peptides.

Our data afford a novel intracellular mechanism involved in Ang IV signaling in VSMCs: the activation of the NF-B pathway. We have also observed that PTK inhibitors diminished Ang IV–induced NF-B activation, showing that tyrosine phosphorylation mediates this process.

In summary, our results demonstrate that, in cultured VSMCs, Ang IV via AT₄ receptors activates the NF-B pathway and upregulates several genes as ICAM-1, IL-6, TNF-, MCP-1, and PAI-1. Although Ang II has long been considered to represent the end product of RAS, there is accumulating evidence showing additional effector peptides with diverse functions. Many works support the importance of Ang IV in the fields of cognition, cardiovascular, and renal metabolism and pathophysiological conditions such as diabetes, atherosclerosis, and hypertension. Our data suggest that Ang IV could contribute to inflammatory events in cardiovascular diseases via NF-B pathway and the regulation of proinflammatory genes.

	Acknowledgments

 
This work has been supported by grants from Fondo de Investigación Sanitaria (PI020513, 01/3130), Comunidad Autónoma de Madrid (08.4/0018/2001), Red Cardiovascular (RECAVA, MP04), and European Project (QLG1-CT-2002–01215). There are no conflicts of interest. J. R-V, E. S-L, M.R., and V.E are fellows of FIS. H.D. has a grant from the Institute for the Promotion of Innovation through Science and Technology in Flanders (I.W.T.-Vlaanderen) and P.V. is holder of a VUB research fellowship.

	Footnotes

 
Original received June 7, 2004; resubmission received February 18, 2005; revised resubmission received April 5, 2005; accepted April 6, 2005.

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