Angiotensin IV Activates the Nuclear Transcription Factor-B and Related Proinflammatory Genes in Vascular Smooth Muscle Cells
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循环研究杂志 2005年第5期
The Vascular and Renal Research Laboratory (V.E., M.R., E.S.-L., J.R.-V., O.L., J.E., M.R.-O.), Fundacie Jimeenez Diaz, Universidad Auteoma Madrid, Spain
the Department of Molecular and Biochemical Pharmacology (H.D., P.V.), Institute for Molecular Biology and Biotechnology, Vrije Univerisiteit Brussel, Belgium.
Abstract
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-BeCdependent 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 AT1a receptor knockout mice, Ang IV also activated NF-B pathway. In those cells, the AT4 antagonist divalinal diminished dose-dependently Ang IVeCinduced 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 factoreCB vascular smooth muscle cell insulin regulated aminopeptidase
Introduction
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.4eC7 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(1eC7), 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(1eC7) 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.14eC16 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(1eC7), 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 IVeCspecific 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-BeCrelated genes in cultured VSMCs.
Materials and Methods
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-BeCdependent gene transcription, VSMCs were seeded in 6-well plates and 24 hours later cells were transient transfected with FUGENE (Roche Molecular Biochemicals) containing 1 e NF-B/luc promoter and 1 e 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
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 10eC9 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).
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 IVeCactivated 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 10eC9mol/L Ang IV, an intense nuclear fluorescence was observed with both antibodies, showing nuclear translocation of NF-B (Figure 2B).
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 IVeCinduced NF-B activation and p50/p65 nuclear translocation. IB is degraded by 2 distinct pathways: I-kinaseeCdependent 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
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 AT419,20) and then stimulated with Ang II or Ang IV for 1 hour.
In rat VSMCs, both AT1 or AT2 antagonists partially blocked Ang IIeCinduced 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 IVeCinduced 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.
We have investigated the presence of AT4/IRAP receptors in VSMCs. Ang IVeCspecific 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 IVeCinduced 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 IVeCinduced NF-B activation (Figure 5). These data suggest that AT4/NF-B pathway is mediated by activation of tyrosine phosphorylation.
Effect of Ang IV on NF-BeCMediated Gene Transcription
We have investigated whether Ang IV regulates NF-BeCmediated 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-1, and TNF-. In AT1 knockout VSMCs, Ang IV also increases NF-BeCmediated gene expression. The AT4 antagonist divalinal significantly diminished Ang IVeCinduced luciferase activity, showing that NF-B promoter activation was mediated by AT4 receptors.
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 IVeCmediated gene overexpression (P<0.05). Using a protein cytokine array system a correlation between increasing levels of certain cytokines and chemokines with the Ang IVeCincreased gene overexpression were found (Figure 7B).
We have further investigated whether NF-B regulates Ang IVeCinduced 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 IVeCtreated 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 IVeCinduced 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).
Discussion
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-kinaseeCdependent 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 IVeCinduced gene overexpression (exemplified by MCP-1), showing a NF-BeCmediated transcriptional mechanism. The AT4 antagonist blocked Ang IVeCinduced NF-B activation and markedly inhibited NF-BeCmediated 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-BeCrelated 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-1, TNF-, and TGF- 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 transmembraneeCspanning G proteineCcoupled 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 IVeCmediated actions in a variety of physiological systems,39eC41 as we have observed in Ang IVeCinduced 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 IVeCrelated 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 IVeCspecific binding sites, antagonized by divalinal, have been described in VSMCs.16,19eC21 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 IVeCinduced 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 Investigacie Sanitaria (PI020513, 01/3130), Comunidad Auteoma de Madrid (08.4/0018/2001), Red Cardiovascular (RECAVA, MP04), and European Project (QLG1-CT-2002eC01215). 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.
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Krebs LT, Krame EA, Hanesworth JM, Sardinia MF, Ball AE, Wright JW, Harding JW. Characterization of the binding properties and physiological action of divalinal-angiotensin IV, a putative AT4 receptor antagonist. Regul Pept. 1996; 67: 123eC130.
Handa RK, Krebs LT, Harding JW, Handa SE. Angiotensin IV AT4-receptor system in the rat kidney. Am J Physiol. 1998; 274: F290eCF299.
Coleman JK, Krebs LT, Hamilton TA, Ong B, Lawrence KA, Sardinia MF, Harding JW, Wright JW. Autoradiographic identification of kidney angiotensin IV binding sites and angiotensin IVeCinduced renal cortical blood flow changes in rats. Peptides. 1998; 19: 269eC277.
Kramar EA, Krishnan R, Harding JW, Wright JW. Role of nitric oxide in angiotensin IVeCinduced increases in cerebral blood flow. Regul Pept. 1998; 74: 185eC192.
Loufrani L, Henrion D, Chansel D, Ardaillou R, Levy BI. Functional evidence for an angiotensin IV receptor in rat resistance artery. J Pharmacol Exp Ther. 1999; 291: 583eC588.
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Chen JK, Zimpelmann J, Harris RC, Burns KD. Angiotensin IV induces tyrosine phosphorylation of focal adhesion kinase and paxillin in proximal tubule cells. Am J Physiol Renal Physiol. 2001; 280: F980eCF988.
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the Department of Molecular and Biochemical Pharmacology (H.D., P.V.), Institute for Molecular Biology and Biotechnology, Vrije Univerisiteit Brussel, Belgium.
Abstract
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-BeCdependent 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 AT1a receptor knockout mice, Ang IV also activated NF-B pathway. In those cells, the AT4 antagonist divalinal diminished dose-dependently Ang IVeCinduced 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 factoreCB vascular smooth muscle cell insulin regulated aminopeptidase
Introduction
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.4eC7 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(1eC7), 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(1eC7) 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.14eC16 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(1eC7), 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 IVeCspecific 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-BeCrelated genes in cultured VSMCs.
Materials and Methods
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-BeCdependent gene transcription, VSMCs were seeded in 6-well plates and 24 hours later cells were transient transfected with FUGENE (Roche Molecular Biochemicals) containing 1 e NF-B/luc promoter and 1 e 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
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 10eC9 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).
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 IVeCactivated 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 10eC9mol/L Ang IV, an intense nuclear fluorescence was observed with both antibodies, showing nuclear translocation of NF-B (Figure 2B).
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 IVeCinduced NF-B activation and p50/p65 nuclear translocation. IB is degraded by 2 distinct pathways: I-kinaseeCdependent 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
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 AT419,20) and then stimulated with Ang II or Ang IV for 1 hour.
In rat VSMCs, both AT1 or AT2 antagonists partially blocked Ang IIeCinduced 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 IVeCinduced 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.
We have investigated the presence of AT4/IRAP receptors in VSMCs. Ang IVeCspecific 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 IVeCinduced 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 IVeCinduced NF-B activation (Figure 5). These data suggest that AT4/NF-B pathway is mediated by activation of tyrosine phosphorylation.
Effect of Ang IV on NF-BeCMediated Gene Transcription
We have investigated whether Ang IV regulates NF-BeCmediated 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-1, and TNF-. In AT1 knockout VSMCs, Ang IV also increases NF-BeCmediated gene expression. The AT4 antagonist divalinal significantly diminished Ang IVeCinduced luciferase activity, showing that NF-B promoter activation was mediated by AT4 receptors.
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 IVeCmediated gene overexpression (P<0.05). Using a protein cytokine array system a correlation between increasing levels of certain cytokines and chemokines with the Ang IVeCincreased gene overexpression were found (Figure 7B).
We have further investigated whether NF-B regulates Ang IVeCinduced 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 IVeCtreated 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 IVeCinduced 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).
Discussion
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-kinaseeCdependent 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 IVeCinduced gene overexpression (exemplified by MCP-1), showing a NF-BeCmediated transcriptional mechanism. The AT4 antagonist blocked Ang IVeCinduced NF-B activation and markedly inhibited NF-BeCmediated 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-BeCrelated 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-1, TNF-, and TGF- 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 transmembraneeCspanning G proteineCcoupled 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 IVeCmediated actions in a variety of physiological systems,39eC41 as we have observed in Ang IVeCinduced 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 IVeCrelated 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 IVeCspecific binding sites, antagonized by divalinal, have been described in VSMCs.16,19eC21 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 IVeCinduced 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 Investigacie Sanitaria (PI020513, 01/3130), Comunidad Auteoma de Madrid (08.4/0018/2001), Red Cardiovascular (RECAVA, MP04), and European Project (QLG1-CT-2002eC01215). 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.
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