Direct Angiotensin II Type 2 Receptor Stimulation Acts Anti-Inflammatory Through Epoxyeicosatrienoic Acid and Inhibition of Nuclear Factor κB
Angiotensin II type 2 (AT2) receptors can be regarded as an endogenous repair system, because the AT2 receptor is upregulated in tissue damage and mediates tissue protection. A potential therapeutic use of this system has only recently come within reach through synthesis of the first selective, orally active, nonpeptide AT2 receptor agonist, compound 21 (C21; dissociation constant for AT2 receptor: 0.4 nM; dissociation constant for angiotensin II type 1 receptor: >10 000 nM). This study tested AT2 receptor stimulation with C21 as a potential future therapeutic approach for the inhibition of proinflammatory cytokines and of nuclear factor κB. C21 dose-dependently (1 nM to 1 μmol/L) reduced tumor necrosis factor-α–induced interleukin 6 levels in primary human and murine dermal fibroblasts. AT2 receptor specificity was controlled for by inhibition with the AT2 receptor antagonist PD123319 and by the absence of effects in AT2 receptor–deficient cells. AT2 receptor–coupled signaling leading to reduced interleukin 6 levels involved inhibition of nuclear factor κB, activation of protein phosphatases, and synthesis of epoxyeicosatrienoic acid. Inhibition of interleukin 6 promoter activity by C21 was comparable in strength to inhibition by hydrocortisone. C21 also reduced monocyte chemoattractant protein 1 and tumor necrosis factor-α in vitro and in bleomycin-induced toxic cutaneous inflammation in vivo. This study is the first to show the anti-inflammatory effects of direct AT2 receptor stimulation in vitro and in vivo by the orally active, nonpeptide AT2 receptor agonist C21. These data suggest that pharmacological AT2 receptor stimulation may be an orally applicable future therapeutic approach in pathological settings requiring the reduction of interleukin 6 or inhibition of nuclear factor κB.
Pharmacological treatment usually intends to eliminate a hazard (bacteria and tumor cells) or to disrupt deleterious pathogenetic mechanisms causative for a respective disease (eg, angiotensin-converting enzyme inhibitors or angiotensin II type 1 (AT1) receptor blockers in hypertension and end organ damage). Despite the fact that the human body possesses an impressive capability of self-healing and self-repair, drug therapy only very rarely aims at supporting and reinforcing endogenous defense or healing mechanisms, such as in immunotherapy in cancer or in therapeutic approaches involving stem cells.1,2
There is now good evidence that the angiotensin II (Ang II) type 2 (AT2) receptor (AT2R) represents such a body’s own protective system. Although data about AT2R-coupled functions are still somewhat controversial, the majority support the concept that the AT2R acts antiproliferative, anti-inflammatory, antifibrotic, and antiapoptotic.3,4 These features of the AT2R are in contrast to what is usually associated with the renin-angiotensin system (RAS), namely, hypertension, inflammation, fibrosis, and end-organ damage. However, these unfavorable actions of Ang II are mediated via the AT1 receptor (AT1R). The AT2R in many ways “cross-talks” with the AT1R, thus interfering with the detrimental signaling cascades coupled to the AT1R.3–5 Tissue-protective properties of the AT2R, for example, in stroke, myocardial infarction, atherosclerosis, or neuronal damage, have been demonstrated in various in vivo studies in genetically altered animals or using the selective AT2R antagonists PD123319 or PD123177, with anti-inflammation being an important underlying molecular mechanism.6–9 Strikingly, in the adult organism, the AT2R is only sparsely expressed in healthy tissue; however, in case of tissue damage, receptor expression is strongly upregulated. This phenomenon again supports a role of the AT2R as an endogenous tissue protective system.3,4
Elucidation of AT2R-related effects has been very difficult in the past because of the lack of a specific and selective AT2R agonist. The natural ligand of the AT2R, Ang II, binds with equal affinity to both AT1R and AT2R, but, because of the prevailing expression of AT1Rs in most adult tissues and cultured cells, stimulation with Ang II usually elicits an AT1R-mediated response. Thus, in the majority of tissues, examination of AT2R-coupled effects requires specific AT2R stimulation.
The most commonly used AT2R-agonist so far is the peptide CGP42112A. However, not only is the suitability of peptides for in vivo use generally limited, CGP42112A also has partly antagonistic properties.10 Both features rendered CGP42112A a problematic tool for research and prevented its development for clinical use.
In 2004, synthesis of the first selective, orally active AT2R agonist, compound 21 (C21), has been published.11 Synthesis of this compound is not just a breakthrough in AT2R research enabling us for the first time to examine AT2R actions in vitro and in vivo by direct receptor stimulation, it also principally offers the possibility to use AT2R stimulation as a therapeutic tool thanks to the in vivo stability (4-hour plasma half-life) and oral bioavailability (20% to 30% in rats) of C21.11
This study aimed to confirm the AT2R agonistic character of C21, to examine the impact of AT2R stimulation by C21 on inflammatory responses in vitro and in vivo, and to elucidate signaling mechanisms mediating AT2R-coupled actions in inflammation.
For an extended version of the Methods, please see the online Data Supplement at http://hyper.ahajournals.org.
Human primary dermal fibroblasts, murine primary dermal fibroblasts, and human primary umbilical vein endothelial cells were isolated and cultured according to standard protocols.
In Vitro Experimental Protocols
Interleukin (IL) 6, monocyte chemoattractant protein (MCP) 1, and tumor necrosis factor (TNF)-α mRNA expressions in human and murine dermal fibroblasts were quantified by real-time RT-PCR after 12 hours and in human umbilical vein endothelial cells after 24 hours of stimulation. For a detailed description of the protocols please see the online Data Supplement.
Nuclear Factor κB Nuclear Translocation
For details, please see the online Data Supplement.
Transient Transfection and Luciferase Assay
For details, please see the online Data Supplement.
Quantitative Real-Time PCR and ELISA
Quantitative real-time PCR and ELISA were performed according to standard protocols/manufacturer’s instructions.
Toxic cutaneous inflammation was induced in mice (female C3H/He; 5 weeks old; Jackson Laboratories, Bar Harbor, ME) by daily SC injections of bleomycin (BLM) over 8 days (100 mg/mL; Hexal).12 Animals were divided into 3 treatment groups (n=8): vehicle (PBS), BLM+vehicle, and BLM+C21 (1 mg/kg).
Data are representative of ≥3 different experiments and are expressed as mean±SD (in vitro experiments) or as mean±SEM (in vivo experiments). Results are compared by t test for 2 comparisons, with P<0.05 considered significant. Representative images of cells were selected from n≥3 experiments.
Agonistic Properties and AT2R Specificity of C21
Direct AT2R Stimulation Inhibits TNF-α–Induced Cytokine Expression
To test whether C21 indeed has agonistic properties, the effect of AT2R stimulation on interleukin (IL) 6 mRNA generation was studied in human primary dermal fibroblasts in vitro by conventional AT2R stimulation, which is stimulation of cells with Ang II under concomitant AT1R blockade or by direct AT2R stimulation with the novel nonpeptide AT2R agonist C21 (Figure 1A). It was assumed that if the two approaches would give the same result, then C21 could be considered an AT2R agonist (not an antagonist). To facilitate detection of a potentially inhibitory effect of C21 on IL6 expression, cells were incubated with TNF-α (10 ng/mL) to increase IL6 levels (Figure 1A). Cells were coincubated with Ang II (plus irbesartan [Irb]) or C21 for stimulating the AT2R. AT2R stimulation by Ang II (100 nM) under concomitant AT1R blockade with Irb (10 μmol/L) significantly decreased TNF-α–induced IL6 upregulation (Figure 1A). The same result was obtained by direct AT2R stimulation with C21 (1 μmol/L), thus supporting its agonistic rather than antagonistic action on the AT2R. The inhibitory effect of C21 on IL6 expression was confirmed by ELISA on the protein level (Figure S1A, available in the online Data Supplement). Inhibition of IL6 expression by C21 was also seen when IL6 mRNA generation was promoted by Ang II (data not shown). C21 further suppressed TNF-α–induced MCP-1 and TNF-α mRNA levels (Figure S1B and S1C). These effects were also seen in cells more closely associated with the cardiovascular system, namely in human umbilical vein endothelial cells (Figure S2A through S2C).
Interestingly, AT1R antagonists (ARBs) with peroxisome proliferator-activated receptor-γ agonistic properties (like Irb and telmisartan) seem also able to inhibit TNF-α–induced cytokine levels, whereas ARBs that do not possess this additional mechanism of action do not (Figure S2D). Selective stimulation of the AT1R (Ang II plus PD123319) had no additional effect on TNF-α–induced IL6 expression (Figure 1A).
Incubation with the selective AT2R antagonist PD123319 (10 μmol/L), before stimulation with C21, abolished the inhibitory effect of C21 on TNF-α–induced IL6 expression, thus indicating that C21 indeed acts via the AT2R (Figure 1B). C21 (1 nM to 1 μmol/L) repressed TNF-α–induced IL6 expression in a dose-dependent manner (Figure S3A).
Expression levels of IL6 mRNA in response to TNF-α sometimes differed between experiments. Such differences in the general responsiveness between cells are unavoidable when using primary cells received from various donors.
As a kind of internal test of the assay, we corroborated the known stimulatory effect of Ang II, acting via the AT1R, on IL6 expression (Figure S3B). Incubation of cells with Ang II (100 nM) led to a significant increase in IL6 mRNA expression, which could be abolished by the AT1R antagonist Irb (10 μmol/L), whereas the AT2R antagonist PD123319 (10 μmol/L) had no effect.
AT2R Agonist C21 Is Ineffective in Dermal Fibroblasts Isolated From AT2R-Deficient Mice
Because C21 is a novel compound with only limited data available in the literature, we intended to confirm its selectivity for the AT2R by 2 approaches: by inhibiting its effect on IL6 expression with the established, selective AT2R antagonist PD123319 (see section above and Figure 1B) and by determining whether the effect of C21 on IL6 expression is absent in AT2R-deficient cells. For the latter purpose, primary dermal fibroblasts isolated from either wild-type or AT2R-knockout mice were stimulated with TNF-α (10 ng/mL) with or without concomitant incubation with C21 (1 μmol/L). Murine dermal fibroblasts expressing the AT2R showed a significant depression of TNF-α–induced IL6 expression by C21 (Figure 1C), as was observed in human fibroblasts (Figure 1A), whereas in fibroblasts obtained from AT2R-deficient animals, no such effect of C21 could be observed (Figure 1D).
Signaling Cascades Evoked by Direct AT2R Stimulation With C21
Direct AT2R Stimulation Involves Stimulation of Protein Phosphatases
Activation of phosphatases has been described as a signaling mechanism coupled to the AT2R.13 In fact, the inhibitory effect of C21 on TNF-α–induced IL6 expression in primary human dermal fibroblasts was absent under inhibition of serine/threonine or tyrosine phosphatases by okadaic acid (OA;10 nM) or sodium orthovanadate (Na3Vo4; 10 nM), respectively (Figure 2). Incubation with OA or Na3Vo4 had been performed 30 minutes before stimulation with TNF-α±C21 for an additional 12 hours. Treatment with either OA or Na3Vo4 alone did not affect IL6 expression (data not shown).
Arachidonic Acid Metabolite Epoxyeicosatrienoic Acid Is a Second Messenger in AT2R-Mediated Anti-Inflammation
In 1999, the arachidonic acid metabolite 11,12-epoxyeicosatrienoic acid (EET) has been reported in a landmark article to act as an anti-inflammatory mediator in vascular inflammation.14 Although the RAS represents another main player in vascular inflammation, the question has never been addressed whether AT2R stimulation in the context of inflammation is directly coupled to EETs acting as second messengers. Because EETs and 20-hydroxyeicosatetraenoic acid (20-HETE) were shown by other authors to exert opposite effects in vascular inflammation and vascular reactivity,14–17 we further tested whether 20-HETE is involved in AT1R-coupled proinflammatory actions.
To verify our hypotheses, we analyzed the effect of compounds that selectively block the synthesis of 20-HETE or EETs on AT1R- or AT2R-dependent signaling and the potential of exogenously added 20-HETE and EET to mimic the effects of AT1R or AT2R stimulation, respectively. Regarding AT2R-mediated signaling, the effect of C21 on TNF-α–mediated IL6-synthesis was abrogated by preincubation with PPOH (6-(2-propargyloxyphenyl)hexanoic acid; 10 μmol/L; 30 minutes), indicating that 11,12-EET acts as a second messenger for the AT2R in this setting (Figure 3A). Coincubation with EET (100 nM) and TNF-α tended to inhibit TNF-α–induced (10 ng/mL) IL6 expression, but this effect did not reach statistical significance. The failure of EET to significantly reduce IL6 expression may be attributed to the very limited stability of EET in culture medium. However, further experiments on nuclear factor κB (NF-κB) activation and translocation clearly substantiated the role of EETs as mediators in the anti-inflammatory pathways elicited on AT2R stimulation (see below). A role of 20-HETE in the AT2R coupled anti-inflammatory response was excluded showing that 20-HETE did not inhibit TNF-α–induced IL6-levels and that the effect of C21 was not diminished when 20-HETE (100 nM) synthesis was blocked by HET0016 (10 μmol/L; 30 minutes; Figure S4A).
Regarding AT1R-mediated signaling, the Ang II–induced increase in IL6 expression was inhibited by preincubation with the inhibitor of CYP4A/4F-dependent 20-HETE synthesis, HET0016 (10 μmol/L; 30 minutes; Figure 3B). In line with this observation, stimulation of human dermal fibroblasts with 20-HETE (100 nM) caused a significant increase in IL6 levels. A role for EET in AT1R-coupled signaling was excluded, showing that EET (100 nM) had no Ang II–like effects on basal IL6 expression and that the inhibitor of CYP2C/2J-dependent EET synthesis, PPOH (10 μmol/L; 30 minutes), did not inhibit Ang II–induced IL6 mRNA generation (Figure S4B). The increase in IL6 expression by PPOH can be attributed to inhibition of basal, endogenous EET synthesis (Figure S4B).
Direct AT2R Stimulation Inhibits NF-κB Activity
IL6 transcription is controlled by the transcription factor NF-κB. To investigate whether the respective alterations in IL6 expression mediated by the AT1R or the AT2R are in fact related to changes in NF-κB activity, the latter was examined by 2 approaches: qualitatively by monitoring nuclear translocation of the NF-κB p50 subunit by immunofluorescence staining and quantitatively by measuring NF-κB–dependent IL6 promoter transcriptional activity using a luciferase reporter assay. Furthermore, these experiments served to decide whether alterations in NF-κB activity are part of the AT1R- or AT2R-coupled signaling cascades involving 20-HETE or EET, respectively.
NF-κB p50 Subunit Nuclear Translocation
TNF-α (10 ng/mL) led to an intense increase in nuclear translocation of p50, which was inhibited by coincubation with C21 (1 μmol/L) or EET (100 nM; Figure S5). In fibroblasts further pretreated with either the AT2R antagonist, PD123319 (10 μmol/L), or the inhibitor of EET synthesis, PPOH (10 μmol/L), the effect of C21 was blocked, resulting in restoration of TNF-α–induced nuclear translocation of NF-κB (Figure S5).
Unstimulated control cells showed an even distribution of p50 in cytoplasm and nuclei. By contrast, Ang II (100 nM)–treated cells revealed a marked nuclear translocation of the p50 subunit that could be prohibited by AT1R blockade with Irb (10 μmol/L) or by disruption of AT1R-coupled signaling with HET0016 (10 μmol/L), the inhibitor of the 20-HETE synthesis. 20-HETE (100 nM) stimulation resulted in a nuclear translocation that was comparable in strength to the Ang II–induced translocation.
Luciferase Reporter Assay
To quantify NF-κB–related IL6 promoter activity, human dermal fibroblasts were transfected with a luciferase expression vector containing the human IL6 promoter with either the wild-type (IL6-pGL3) or a mutated (mutIL6-pGL3) NF-κB binding site. Unstimulated control cells showed a basal luciferase signal (Figure 4A and 4B). TNF-α caused (10 ng/mL) a strong augmentation of the relative luciferase activity through NF-κB activation, which was inhibited by costimulation with C21 (1 μmol/L; Figure 4A). The effect of C21 could be blocked by previous treatment with the AT2R antagonist PD123319 (10 μmol/L).
Ang II (100 nM) significantly stimulated relative luciferase activity, which was attenuated by pretreatment with the AT1R antagonist Irb (10 μmol/L; Figure S6). Inhibition of IL6 promoter activity by C21 was comparable in strength to inhibition by hydrocortisone applied in the same concentration (1 μmol/L each; Figure 4B).
Regarding the involvement of EET and 20-HETE, the TNF-α–induced increase in luciferase activity was attenuated by cotreatment with EET (100 nM) in a comparable way as by C21 (1 μmol/L; Figure 4C). Blockade of EET synthesis with PPOH (10 μmol/L) abrogated the inhibitory effect of C21 on NF-κB–dependent IL6 promoter activity.
20-HETE (100 nM) stimulated relative luciferase activity to a similar extent as Ang II (Figure 4D). The Ang II–induced activation of NF-κB could be inhibited by blockade of 20-HETE synthesis with HET0016 (10 μmol/L), indicating again that 20-HETE is a second messenger of the AT1R-mediated stimulation of IL6 transcription. Mutation of the NF-κB binding site completely inhibited the stimulatory effects of Ang II, 20-HETE, and TNF-α on IL6 promoter activity, confirming the dependence of IL6 transcription on NF-κB (Figure 4).
Direct AT2R Stimulation Inhibits IL6 Expression In Vivo
The effect of C21 on IL6 expression in vivo was studied using C3H/He mice presenting with a toxic cutaneous inflammatory reaction elicited by subcutaneous injections of BLM every second day over a period of 1 week. BLM injections resulted in significantly elevated IL6, MCP-1, and TNFα mRNA levels in murine skin when compared with controls (Figure 5). BLM-injected mice treated with C21 (1 mg/kg per day SC) for 1 week showed a significant reduction in IL6, MCP-1, and TNF-α expression.
All of the AT2R-related results are summarized in Table S1.
Ang II is commonly known as a cardiovascular hormone involved in the control of blood pressure and electrolyte homeostasis, as well as in the pathogenesis of hypertension and related end-organ damage. Although these “classic” Ang II actions are all mediated by the AT1R, the spectrum of AT2R-mediated actions contrasts those of the AT1R.3,4
Anti-inflammation is one feature that has been ascribed to the AT2R.13,18,19 However, there has always been some controversy about AT2R-mediated effects.4,20 Because of the lack of a selective, nonpeptide AT2R agonist, experimental induction of “true” AT2R effects had been difficult (in particular in vivo), and experimental approaches were often complex (stimulation with Ang II under concomitant AT1R blockade) or indirect (experiments using AT2R antagonists or studies in AT2R-deficient mice).6–8,20–22 Moreover, because no adequate compound for in vivo AT2R stimulation was available, AT2R stimulation has never seriously been considered as a therapeutic target.
In the present study, we describe anti-inflammatory effects of direct AT2R stimulation using the first specific and selective nonpeptide AT2R agonist, C21.11 We show that direct angiotensin AT2R stimulation by C21 acts anti-inflammatory by inhibiting cytokine levels in vitro and in vivo. AT2R stimulation by C21 or the natural ligand Ang II (applied under concomitant AT1R blockade to exclude AT1R-related effects) in vitro inhibited TNF-α–induced IL6 in a comparable way, thus supporting agonistic properties of C21 at the AT2R. AT2R specificity of C21-induced effects was demonstrated by their inhibition with the established AT2R antagonist, PD123319, or by their absence in AT2R-deficient cells.
Regarding AT2R-coupled signaling in the context of anti-inflammatory actions, we further found that AT2R-coupled signaling involves activation of protein phosphatases, CYP-dependent epoxidation of arachidonic acid to EETs, and inhibition of NF-κB. By contrast, the proinflammatory effect mediated by the AT1R involves CYP-dependent hydroxylation of arachidonic acid to 20-HETE and activation of NF-κB (for a schematic overview, see Figure S7).
It has to be pointed out that, in our experimental settings, AT2R stimulation did not counteract Ang II–induced, AT1R-mediated actions thus being restricted to “intra-RAS” effects, but it directly antagonized the effects of a cytokine, TNF-α. AT2R-mediated antagonism of TNF-α13 or of other non-RAS stimuli (eg, growth factors) has already been shown earlier by us and others.10,23,24 Thus, AT2R stimulation seems to be able to interfere with signaling cascades coupled to detrimental stimuli, which do not necessarily have to be considered part of the RAS. “Conventional” ARBs seem not to be able to repress cytokine-induced IL6 expression, as shown in this study and very recently by Tian et al.25 Apparently, additional peroxisome proliferator-activated receptor-γ–agonistic properties are necessary for a direct anti-inflammatory action of ARBs by a peroxisome proliferator-activated receptor-γ/NF-κB cross-talk.25
The molecular mechanism of interference of AT2R-coupled signaling with cytokine-coupled signaling is probably based on interruption of kinase-dependent phosphorylation cascades by dephosphorylation through activated phosphatases. Such an interference has already been shown for extracellular regulated kinase and signal transducer and activator of transcription signaling and for the deactivation of NF-κB.13,18,23 Reduced NF-κB activity resulting in decelerated IL6 transcription has also been observed in our study. The reduction of NF-κB dependent IL6 promoter activity by direct AT2R stimulation was comparable in strength to inhibition by the classic NF-κB inhibitor, hydrocortisone.
Increased NF-κB activity and/or IL6 levels are key characteristics of a broad variety of pathological conditions ranging from chronic inflammatory/rheumatic diseases or autoimmune disorders to insulin resistance, atherosclerosis, or even cancer.26–29 Because currently used drugs capable of IL6 and NF-κB inhibition, such as glucocorticoids and certain biologicals, have a high rate of severe adverse reactions or are orally ineffective, there is an extensive search for novel, orally active drug classes for inhibition of IL6 and NF-κB with a more favorable adverse effect profile.
Because C21 has been shown in our study to reduce IL6 levels and NF-κB activity and is an orally active small molecule with ≈30% bioavailability,11 it may have the potential to become a candidate drug in the context of chronic inflammatory diseases. This assumption of course has to be supported by more data from animal studies, but apart from our extensive in vitro data, we have already demonstrated an effective reduction in cytokine levels in dermal toxic inflammation (this study) and in myocardial inflammation postmyocardial infarction.24 Furthermore, Gelosa et al30 very recently reported an anti-inflammatory effect of C21 in hypertensive nephropathy in SHR-SP rats.
Apart from the C21-induced reduction of NF-κB activity, our data also indicated that EETs serve as second messengers of the AT2R in anti-inflammation. This novel finding is in agreement with previous reports showing the capacity of EETs to protect against cytokine-induced inflammation in human endothelial cells.14 Moreover, EETs have also become a drug target in current developments of anti-inflammatory medications. For example, an orally administered soluble epoxide hydrolase inhibitor, AR9281, which is currently tested in phase II clinical trials, acts by preventing EET degradation, thus enhancing EET tissue levels.15
Regarding a putative future therapeutic use, the low expression of AT2Rs in most healthy tissues but strong upregulation in damaged tissue offers the advantage of the strongest putative therapeutic effects of an AT2R agonist at the site of tissue damage and minimization of adverse effects.3,4 A low rate of adverse effects by C21 can also be assumed, because indirect AT2R stimulation by increased levels of Ang II is regarded to contribute to the therapeutic effect of ARBs, a drug class known for the placebo-like incidence of adverse reactions.31 Finally, pharmacological stimulation of AT2R by C21 can be regarded as reinforcement of an endogenous tissue-protective system, because, in case of tissue damage, increased levels of locally synthesized Ang II stimulate upregulated AT2Rs, a process instrumental in repair and regeneration.3,4
Taken together, our study features the novel nonpeptide AT2R agonist C21 as a long-needed, valuable tool for AT2R research. We used this novel tool to demonstrate that direct AT2R stimulation reduces TNF-α–induced IL6 expression by activation of protein phosphatases, increasing EET synthesis and inhibition of NF-κB activity. Moreover, this article describes a novel anti-inflammatory mechanism with a large therapeutic potential, distinct from but with potentially similar effectiveness as the one engaged by glucocorticoids and most likely fraught with considerably less adverse effects than the latter. Because the pharmacokinetic properties of C21 are in favor of a future clinical use of this compound, pathological settings involving chronic inflammation may be future indications for therapeutic AT2R stimulation.
B.D. has significant and T.U. has modest ownership interest in Vicore Pharma and received speaker fees. U.M.S. and M.Al. received speaker fees from Vicore Pharma.
T.U. and U.M.S. contributed equally to this work.
- Received November 16, 2009.
- Revision received December 4, 2009.
- Accepted January 19, 2010.
de Gasparo M, Catt KJ, Inagami T, Wright JW, Unger T. International Union of Pharmacology: XXIII–the angiotensin II receptors. Pharmacol Rev. 2000; 52: 415–472.
Iwai M, Liu HW, Chen R, Ide A, Okamoto S, Hata R, Sakanaka M, Shiuchi T, Horiushi M. Possible inhibition of focal cerebral ischemia by angiotensin II type 2 receptor stimulation. Circulation. 2004; 110: 843–848.
Iwai M, Chen R, Li Z, Shiuchi T, Suzuki J, Ide A, Tsuda M, Okumura M, Min L-J, Mogi M, Horiushi M. Deletion of angiotensin II type 2 receptor exaggerated atherosclerosis in apolipoprotein E-null mice. Circulation. 2005; 112: 1636–1643.
Lucius R, Gallinat S, Rosenstiel P, Herdegen T, Sievers J, Unger T. The angiotensin II type 2 (AT2) receptor promotes axonal regeneration in the optic nerve of adult rats. J Exp Med. 1998; 188: 661–670.
Wan Y, Wallinder C, Plouffe B, Beaudry H, Mahalingam AK, Wu X, Johansson B, Holm M, Botoros M, Karlen A, Pettersson A, Nyberg F, Fändriks L, Gallo-Payet N, Hallberg A, Alterman M. Design, synthesis, and biological evaluation of the first selective nonpeptide AT2 receptor agonist. J Med Chem. 2004; 47: 5995–6008.
Wu L, Iwai M, Li Z, Shiuchi T, Min L-J, Cui T-X, Li J-M, Okumura M, Nahmias C, Horiuchi M. Regulation of inhibitory protein-κB and monocyte chemoattractant protein-1 by angiotensin II type 2 receptor activated Src homology protein tyrosine phosphatase-1 in fetal vascular smooth muscle cells. Mol Endocrinol. 2004; 18: 666–678.
Node K, Huo Y, Ruan X, Yang B, Spiecker M, Ley K, Zeldin DC, Liao JM. Anti-inflammatory properties of cytochrome P450 epoxygenase-derived eicosanoids. Science. 1999; 285: 1276–1279.
Roman RJ. P-450 metabolites of arachidonic acid in the control of cardiovascular function. Physiol Rev. 2002; 82: 31–85.
Horiuchi M, Hayashida W, Akishita M, Tamura K, Daviet L, Lehtonen JY, Dzau VJ. Stimulation of different subtypes of angiotensin II receptors, AT1 and AT2 receptors, regulates STAT activation by negative crosstalk. Circ Res. 1999; 84: 876–882.
Ichihara S, Senbonmatsu T, Price Jr E, Ichiki T, Gaffney FA, Inagami T. Targeted deletion of angiotensin II type 2 receptor caused cardiac rupture after acute myocardial infarction. Circulation. 2002; 106: 2244–2249.
Tsuzuki S, Matoba T, Eguchi S, Inagami T. Angiotensin II type 2 receptor inhibits cell proliferation and activates tyrosine phosphatase. Hypertension. 1996; 28: 916–918.
Kaschina E, Grzesiak A, Li J, Foryst-Ludwig A, Timm M, Rompe F, Sommerfeld M, Kemnitz UR, Curato C, Namsolleck P, Tschöpe C, Hallberg A, Alterman M, Hucko T, Paetsch I, Dietrich T, Schnackenburg B, Graf K, Dahlöf B, Kintscher U, Unger T, Steckelings UM. Angiotensin II type 2 receptor stimulation: a novel option of therapeutic interference with the renin-angiotensin-system in myocardial infarction? Circulation. 2008; 118: 2523–2532.
Tian Q, Miyazaki R, Ichiki T, Imayama I, Inanaga K, Ohtsubo H, Yano K, Takeda K, Sunagawa K. Inhibition of tumor necrosis factor-α -induced interleukin-6 expression by telmisartan through cross-talk of peroxisome proliferator-activated receptor-γ with nuclear factor kappaB and CCAAT/enhancer-binding protein-β. Hypertension. 2009; 53: 798–804.
Sethi G, Sung B, Aggarwal BB. Nuclear factor-κB activation: from bench to bedsite. Exp Bio Med. 2008; 233: 21–31.