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Published online before print May 3, 2007
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From the Department of Medical Biochemistry and Immunology,* Cardiff University, Cardiff, United Kingdom; and the Institut für Biochemie, Christian-Albrechts-Universität zu Kiel, Kiel, Germany
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Abstract |
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50% decreased in IL-6–/– mice. Ang II infusion activated signal transducer and activator of transcription-3 in heart of WT and decreased Ang II receptor 1 (ATR1) expression in aorta. Both responses were unaffected by sgp130Fc and absent in IL-6–/– mice. In summary, we show that IL-6 trans-signaling is required for Ang II-dependent hypertension, but that hypertrophy, down-regulation of AT1R, and cardiac signal transducer and activator of transcription-3 activation are mediated via cognate IL-6R. These data show that IL-6 responses in a single disease context are governed by both modes of IL-6 signaling, with each pathway eliciting different outcomes. Inhibition of IL-6 signaling is suggested as a potential therapy for hypertension and cardiac hypertrophy.
Ang II has potent and diverse actions throughout the cardiovascular system including vasoconstriction, induction of vascular smooth muscle proliferation, cardiac hypertrophy, reactive oxygen species, and aldosterone production. Ang II responses are mediated predominantly via AT1 receptor activation (AT1R), a G protein-coupled receptor expressed on endothelial cells, monocytes, and vascular smooth muscle cells. However, whether IL-6 can mediate the biological and vascular damaging effects of Ang II is unknown.
Independently of Ang II signaling, an involvement of the IL-6 signal-transducing receptor gp130 in pressure overload and salt-sensitive models of hypertrophy has been found, whereas in vitro leukemia inhibitory factor and cardiotrophin-1 can stimulate cardiomyocyte hypertrophy via signal transducer and activator of transcription-3 (STAT3) and gp130.10,13-15 However, no studies to date have shown that IL-6 itself can cause vascular or cardiac hypertrophy either in vitro or in vivo. In addition, whether Ang II acting via IL-6 can activate STAT3 and gp130 signaling is unknown. Finally, the relative roles of the two IL-6 receptor isoforms, soluble (sIL-6R) and cognate IL-6R, in controlling the various Ang II responses have not been delineated.
The mechanisms by which IL-6 potentially affects hypertension or hypertrophy are unknown. To establish the relationship between Ang II and IL-6 activity, it is therefore essential to consider how IL-6 responses are governed in vivo. In the classic sense, IL-6 binds a membrane-bound cognate receptor (IL-6R) that then couples to gp130 to transmit its signal. This response can, however, be mimicked through IL-6 binding to its soluble receptor. The resulting IL-6/sIL-6R is then able to directly activate gp130 through a process termed IL-6 trans-signaling. The in vivo significance of this alternative mode of IL-6 activation is only fully appreciated when you consider the cellular distribution of both the cognate IL-6R and gp130. Although IL-6R displays a restricted expression profile, gp130 is ubiquitously expressed. IL-6 trans-signaling therefore affords IL-6 with the capacity to activate responses in cells that would inherently remain unresponsive to IL-6 itself. At present, the significance of this dual mode of activation remains unclear.
Herein, we examined in vivo responses to low-dose Ang II, which causes hypertension and hypertrophy during a 7-day period and reveals that IL-6–/– mice are very well protected against both responses in vivo. Mechanistic studies using soluble gp130Fc (sgp130Fc), an inhibitor of trans-signaling generated by linking sgp130 to the Fc domain of IgG1, were conducted to establish the mechanisms involved. The results provide insight into mechanisms of Ang II signaling in vascular disease and propose IL-6 as a potential target for treatment of hypertension, cardiac hypertrophy, and heart failure.
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Materials and Methods |
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Rabbit anti-human AT1-R, normal rabbit or goat IgG, and rabbit serum were from Santa Cruz Biotechnology (Santa Cruz, CA), goat anti-mouse IL-6R was from R&D Systems (Oxon, UK). Anti-rabbit IgG-Alexa 568 and anti-goat IgG-Alexa 568 were from Molecular Probes (Eugene, OR). Recombinant sgp130Fc was purified from the supernatant of stably transfected Chinese hamster ovary cells as described.16 Unless otherwise stated, all compounds were from Sigma (Poole, UK).
Animal Studies
All animal experiments were performed in accordance with the United Kingdom Home Office Animals (Scientific Procedures) Act of 1986. Experiments were performed using male C57BL/6J IL-6-deficient (IL-6–/–) mice.17 Age-matched wild-type (WT) male C57BL/6 mice (25 to 30 g) were obtained from Charles River, Margate, Kent, UK. All mice were kept in constant-temperature cages (20 to 22°C) and given free access to water and standard chow.
Hypertension Studies
Male 10- to 12-week-old WT and IL-6–/– mice were anesthetized via inhalation of 2% isoflurane (98% oxygen). Osmotic minipumps (Alzet model 1002; Durect Corp., Cupertino, CA) containing Ang II (infusion rate, 1.1 mg/kg/day) or vehicle were implanted subcutaneously in the midscapular area. Systolic blood pressure (BP) was monitored daily for 3 days before implantation (training period) and up to 7 days after implantation via tail cuff plethysmography (World Precision Instruments, Stevenage, Hertfordshire, UK) in conscious mice. Soluble gp130 was administered via intraperitoneal injection of sgp130Fc [100 µg/mouse–1 (3.33 mg/kg) days –1 and +1, 50 µg/mouse–1 (1.67 mg/kg) days +3 and +5].
Assessment of Heart/Body Weight Ratio, Aortic Medial Area, and Immunohistochemistry
Seven days after implantation, mice were sacrificed by cervical dislocation and heart and body weights recorded. The descending thoracic aorta was removed, cleaned of adipose tissue, and fixed in paraffin wax. For measurement of medial area, 15-µm sections were taken using a cryostat, placed on glass slides, fixed using acetone, and stained with hematoxylin. Images were acquired using a x10 air lens, with an Axiovert S100TV microscope and Hamamatsu Orca Digital Camera (Hamamatsu Corporation, Bridgewater, NJ), and medial area was calculated using Scion Image (Scion Corp., Frederick, MD). For each aorta, three separate sections were analyzed and averaged. For immunohistochemistry, 10-µmol/L transverse sections were methanol-fixed on glass slides, permeabilized using 0.1% (w/v) Triton X-100/phosphate-buffered saline (PBS), and blocked using 1% (w/v) bovine serum albumin/PBS (AT1R) or 10% rabbit serum (IL-6R). Negative controls used equivalent concentrations of isotype control IgG. Imaging was performed on an Axiovert S100TV inverted microscope connected to a Bio-Rad MRC 1024ES laser-scanning system (Bio-Rad Microscience, Hemel Hempstead, UK) and using standard analysis software (Lasersharp 2000; Bio-Rad Microscience). Images were acquired using a x10 air lens, with excitation at 568 nm and emission 595/35 nm. Quantification of receptor expression was determined by averaging pixel intensity across the area of antigen expression using Lasersharp 2000. Each group consisted of four to five separate animals. For each animal, three separate aortic sections were quantified with three to four separate determinations per section. Postacquisition processing was performed using Adobe Photoshop (Mountain View, CA).
Determination of Plasma sIL-6R
Seven days after implantation, mice were sacrificed by cervical dislocation and blood collected by cardiac puncture and chilled on ice. Whole blood was centrifuged (4°C, 3000 rpm, 10 minutes) to recover plasma supernatant. Soluble IL-6R (sIL-6R) expression was quantified using enzyme-linked immunosorbent assay with goat anti-mouse IL-6R (R&D Systems).
Electrophoretic Mobility Shift Assays for STAT1 and STAT3 Activation
Mouse hearts were ground to a fine powder in liquid nitrogen using a pestle and mortar. Cytosolic and nuclear extracts were then prepared from the powder using a rapid technique for the extraction of nuclear proteins.18
In brief, the powder was resuspended in ice-cold buffer A (10 mmol/L HEPES, pH 8, 1.5 mmol/L MgCl2, and 10 mmol/L KCl) containing 0.5 mmol/L dithiothreitol, 0.2 mmol/L phenylmethyl sulfonyl fluoride, 1 mmol/L sodium orthovanadate, 50 mmol/L NaF, and proteinase inhibitors (diluted 1:1000; Sigma) and incubated on ice for 30 minutes. The nuclei were pelleted by centrifugation, and the cytosolic fraction was removed. The nuclear pellet was resuspended in ice-cold buffer C (20 mmol/L HEPES, pH 8, 420 mmol/L NaCl, 1.5 mmol/L MgCl2, 0.2 mmol/L ethylenediaminetetraacetic acid, pH 8, and 25% glycerol) containing 0.5 mmol/L dithiothreitol, 0.2 mmol/L phenylmethyl sulfonyl fluoride, 1 mmol/L sodium orthovanadate, 50 mmol/L NaF, and proteinase inhibitors (diluted 1:1000; Sigma) and incubated on ice for 30 minutes to allow for high-salt extraction. Cellular debris was removed by brief high-speed centrifugation, and the resulting supernatants (nuclear extract) were collected. Protein concentrations were determined using the Bradford method. Electrophoretic mobility shift assays were performed using 10 µg of nuclear extract as previously described.19
Oligonucleotides containing the SIE STAT-binding GAS elements were annealed and labeled with [
32P]dTTP (Amersham Pharmacia) using the Klenow fragment of DNA polymerase I. The composition of STAT-DNA binding complexes was determined by supershift assays using rabbit polyclonal antibodies specific to STAT1 (M22, Sc-592) and STAT3 (C20, Sc-483) (both from Santa Cruz Biotechnology). Densitometry analysis was performed using the ImageJ 1.34s software (http://rsb.info.nih.gov/ij/). Alterations in STAT DNA-binding activity were expressed relative to control levels (equal to 1).
Isometric Tension Functional Studies
Male WT or IL-6–/– mice (25 to 30 g) were sacrificed by cervical dislocation. The thoracic aorta was removed and placed in Krebs-Henseleit buffer (120 mmol/L NaCl, 4.7 mmol/L KCl, 1.2 mmol/L MgSO4·7H2O, 24 mmol/L NaHCO3, 1.1 mmol/L KH2PO4, 10 mmol/L glucose, and 2.5 mmol/L CaCl2·2H2O). The aorta was dissected free of adipose tissue, taking care to retain adventitia, cut into rings (2 to 3 mm), and suspended in an isometric tension myograph (model 610; DMT, Aarhuis, Denmark) containing Krebs buffer at 37°C and gassed with 5% CO2/95% O2. A resting tension of 3 mN was maintained, and changes in isometric tension recorded via Myodaq software (DMT). After a 60-minute equilibration period, vessels were primed with 48 mmol/L KCl and rested for a further 30 minutes before a concentration (1 µmol/L) of phenylephrine (PE) producing 75% contraction was added. Once the response stabilized, 1 µmol/L acetylcholine (ACh) was added to assess endothelial integrity. Any rings that did not maintain contraction or relaxed <50% of the PE-induced tone were discarded. Rings were washed for 30 minutes, after which a cumulative concentration-response curve to PE was constructed (1 nmol/L to 10 µmol/L) to assess vasoconstrictor activity. The tissues were then washed for 60 minutes to restore basal tone, after which the vessels were constricted to 80% using 1 µmol/L PE. Once a stable response to PE was observed, cumulative concentration-response curves were constructed to 1 nmol/L to 10 µmol/L ACh to assess endothelium-dependent and -independent relaxations, respectively. Experiments were repeated in the presence of 300 µmol/L L-nitroarginine-methyl ester (L-NAME), 10 µmol/L indomethacin, or 1 µmol/L Ang II, which were added for 15 minutes before the end of wash periods. Constriction and dilation curves were then constructed as described previously. In some experiments the constriction response to a bolus dose of Ang II was examined. Responses were expressed as a percentage of either baseline tension (vasoconstriction) or contracted tension (vasodilation). ED50 for ACh relaxation is defined as the concentration of agonist giving half-maximal relaxation and was determined from experimental dose-response curves. Responses from patent rings of each animal (three to four rings) were combined to produce an average for each sample (n).
Statistical Analysis
Data are expressed as mean ± SEM. Groups are compared with WT group using two-way analysis of variance with Bonferroni’s posttest to isolate differences between groups. P < 0.05 or less was considered statistically significant.
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Results |
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No significant difference was noted between basal BP of WT and IL-6–/– mice (101.7 ± 1.4 mm Hg, n = 14, versus 102.2 ± 0.6 mm Hg, n = 14, for WT and IL-6–/–, respectively, mean ± SEM; Figure 1A
, days –3 to –1), and this remained unaltered after subcutaneous infusion of vehicle (saline) (Figure 1A)
. Infusion of Ang II (1.1 mg/kg–1/day–1) significantly increased systolic BP in WT mice from day +2 to +7 compared with WT controls (Figure 1A
, mean maximal pressure 139.4 ± 9.1 mm Hg days +2 to +6 versus 97 ± 0.93 mm Hg days +2 to +6, P < 0.001, compare with day 0). In contrast, the BP increase observed in IL-6–/– mice was significantly reduced (P < 0.01 compare days +5 to +6) when compared with WT mice. Further, in the IL-6–/– group there was no BP increase until day +4. These data demonstrate Ang II-dependent hypertension is reduced both in severity and onset in vivo in IL-6–/– mice.
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To examine the mechanisms of IL-6 involvement in Ang II-dependent hypertension, BP was measured in WT mice after administration of sgp130Fc, a selective inhibitor of IL-6 trans-signaling (100 µg/mouse–1 (approx 3.3 mg/kg–1) days –1 and +1, 50 µg/mouse–1 (1.7 mg/kg–1) days +3 and +5). In WT mice, BP increases to Ang II were significantly suppressed by sgp130Fc (P < 0.0001; P < 0.05 compare days +2 to +6), when compared with WT Ang II-treated mice (Figure 1B)
. There was no effect of sgp130Fc administered alone to WT mice (Figure 1B)
. These data demonstrate that IL-6 trans-signaling is required for the pressor effects of low-dose Ang II. Because small changes in food intake may alter salt intake, food consumption and body weight gain was monitored during sgp130Fc administration in WT and IL-6–/– mice. The data showed that neither parameter was affected during a 7-day period either before or during 7 days of sgp130Fc administration (data not shown).
Effect of sgp130Fc on Ang II-Induced Hypertension in IL-6–/– Mice
From days +4 to +6, a small rise in BP was noted in IL-6–/– mice with Ang II that appeared less than when sgp130Fc was administered to WT with Ang II. This was unexpected because complete blockade of IL-6 signaling as in the knockout should be at least as effective as inhibition of trans-signaling using sgp130Fc. Therefore, to examine this further, sgp130 was administered to IL-6–/– mice and BP examined during Ang II infusion. The data shows that sgp130 does not potentiate the effect of IL-6 deficiency, because a small increase in BP toward the end of the time course, similar to the response of IL-6–/– alone is noted (Figure 1C)
. Therefore, sgp130Fc is more effective at preventing Ang II-dependent hypertension in WT mice than the combination of sgp130Fc/IL-6–/–. One potential explanation is that, in the absence of IL-6, signaling by alternative gp130-activating cytokines, such as leukemia inhibitory factor, Oncostatin-M, or cardiotrophin-1, is up-regulated to compensate and that this would not inhibited by sgp130Fc because of its significantly greater kd for IL-6/sIL-6R than that of other IL-6-related cytokine receptors.20,21
Alternatively, the idea that sgp130Fc administration is more effective than IL-6 deficiency is primarily based on one data point (day 6), and analysis of variance analysis of WT/Ang II/sgp130Fc compared with IL-6–/–/Ang II, or IL-6–/–/Ang II/sgp130Fc finds no significant differences between these data sets (not shown).
IL-6 Deficiency Prevents Ang II-Induced Cardiac and Aortic Hypertrophy
Subcutaneous infusion of Ang II significantly increased heart:body weight ratio and aortic medial area in WT mice; however, no increases were observed in IL-6–/– mice (Figure 1, C and D)
. Furthermore, infusion of sgp130Fc did not prevent Ang II-induced hypertrophy in WT mice (Figure 1, D and E)
. These data suggest that Ang II-induced cardiac and aortic hypertrophy require IL-6 but that trans-signaling is not involved.
Expression of IL-6R in Aortic Tissue and Plasma Levels of sIL-6R Are Decreased in IL-6–/– Mice
The inability of sgp130Fc to influence changes in Ang II-induced cardiac and aortic hypertrophy suggests that regulation of these events is governed by classic IL-6R signaling. Consequently, expression of the cognate IL-6R was examined in aortic sections using immunohistochemistry. As shown, staining was apparent in aortae from both WT and IL-6–/– mice; however, the level of expression was markedly reduced (50%) in IL-6–/– vessels (Figure 2, A–G)
. Similarly, plasma levels of sIL-6R were 50% decreased in IL-6–/– mice versus WT (Figure 2H)
. Ang II infusion in the WT group did not change plasma sIL-6R or elevate IL-6, but in IL-6–/– mice its levels were significantly increased (P < 0.05; Figure 2H
and data not shown). These data indicate that IL-6 deficiency results in significant losses in circulating and tissue IL-6R. Furthermore, expression of cognate IL-6R on aortic tissue provides a mechanism for IL-6 regulation of hypertrophy, which is independent of IL-6 trans-signaling, in which circulating or tissue IL-6 directly activates vascular IL-6R.
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To substantiate signaling via the classic model of IL-6 activation, studies examined the regulation of gp130-mediated STAT3 activation in cardiac tissue. After nuclear extraction from heart tissue, STAT activation levels were examined using electrophoretic mobility shift assay. The data show a twofold increase in STAT activation with Ang II infusion in WT that is not inhibited by sgp130Fc infusion and is absent in IL-6–/– mice (Figure 3, A and B)
. Although there was a greater STAT activation with sgp130Fc, this difference was not statistically significant from Ang II infusion alone. Supershift analysis subsequently showed that the composition of this DNA/protein complex consisted predominantly of STAT3 (Figure 3C)
. These data show that Ang II infusion in vivo leads to an IL-6-dependent activation of STAT3 in heart that is not mediated by IL-6 trans-signaling and is probably involved in the hypertrophic response.
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To examine whether IL-6 deficiency altered Ang II signaling via changes in Ang II receptor expression, AT1R was examined by immunohistochemistry in aortic sections. Comparisons of pixel intensity in several sections showed that AT1R expression in IL-6–/– mice was significantly greater than in WT mice (P < 0.03, Figure 4
). After Ang II infusion, AT1R expression in both WT and sgp130Fc-treated WT mice, but not IL-6–/– mice, was significantly decreased (P < 0.0005, Figure 4
). These data indicate that IL-6 suppresses expression of AT1R, both basally and after Ang II infusion, and that this occurs via cognate IL-6R signaling.
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To examine whether IL-6 deficiency alters the mechanisms of vessel tone regulation in mice, the constriction/relaxation responses of thoracic aortic rings to the -receptor agonist PE and endothelial-dependent relaxant ACh were characterized in the presence and absence of the nitric-oxide synthase inhibitor L-NAME or the cyclo-oxygenase inhibitor indomethacin. Phenylephrine causeda concentration-dependent constriction in rings from WT and IL-6–/– mice, with no significant differences observed between the groups, either in the time to develop force or the ability to maintain constriction (Figure 5A)
. Similarly, no differences were found in vasodilation responses to ACh between strains, with ED50 for relaxation 8.5 x 10–8 or 9 x 10–8 mol/L for WT and IL-6–/– mice, respectively (Figure 5B)
. Vasorelaxation can be mediated by either nitric oxide (NO) or prostacyclin (PGI). To examine for differences in bioactivity of these mediators in IL-6 deficiency, responses were re-examined in the presence of either L-NAME or indomethacin to inhibit generation of NO or PGI, respectively. As expected from our previous studies with WT aortae,22
inclusion of L-NAME potentiated constriction and inhibited relaxation because of inhibition of eNOS, whereas indomethacin had a small inhibitory effect on constriction (Figure 5)
. Identical effects were found in rings from IL-6–/– mice, indicating that a similar balance of NO and PGI is required to sustain tone (Figure 5)
. Finally, no differences in DETA NONOate-dependent dilation were found between the WT and IL-6–/– rings, indicating that smooth muscle guanylate cyclase and cGMP signaling is preserved after IL-6 deletion (not shown). Collectively, these data indicate that the bioactivity of NO and PGI and the ability of vessels to maintain tone in response to physiological stimuli is unchanged in IL-6 deficiency.
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Ang II has direct vasoconstrictive effects in aortic vessels that could participate in the hypertensive response in vivo. To examine whether vascular Ang II signaling is altered in IL-6–/–, in vitro signaling by Ang II in WT and IL-6–/– aortae was characterized. First, direct vasoconstriction to three boluses of Ang II was measured and found to be identical in both groups (Figure 6A)
. It was not possible to construct a dose-response curve to Ang II because low doses appeared to induce desensitization to higher doses of the drug. Next, rings were preincubated with 1 µmol/L Ang II for 15 minutes before PE constriction and ACh relaxation. Preincubation with Ang II caused no significant change in vasoconstriction responses in either group (not shown) but led to a significant right-shift in the relaxation dose-response curve to lower concentrations of ACh (Figure 6, B, and C)
. This response in WT aortae, characterized as induction of endothelial dysfunction in vitro,23
appears to be unaffected by IL-6 deficiency with ED50 for relaxation at 7.5 x 10–7 or 6.5 x 10–7 for WT and IL-6–/–, respectively. These data indicate that IL-6 is not required for the acute vasoconstrictive effects of Ang II in aortae in vitro and furthermore suggest that this in vitro response may not be related to in vivo hypertensive responses of Ang II, which we have shown herein to require IL-6 trans-signaling.
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Discussion |
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To understand the contribution of these two pathways of IL-6R signaling in Ang II responses, we used sgp130Fc. With this approach, treatment of WT mice with sgp130Fc during the Ang II infusion selectively blocked hypertension (Figure 1B)
. In contrast, cardiac and aortic hypertrophy was unaffected, suggesting a role for IL-6 signaling through the cognate IL-6R. Although sgp130 can also inhibit leukemia inhibitory factor or cardiotrophin-1, the kd for interaction of sgp130 with sIL-6R is 100- to 1000-fold lower than that of sgp130’s interaction with other IL-6-related cytokines.20,21
In addition, the anti-hypertensive effect of sgp130 was mimicked by IL-6 deficiency. Therefore, the most likely effect to account for the bioactivity of sgp130 is inhibition of IL-6/sIL-6R signaling. These data indicate that both forms of IL-6 signaling participate in Ang II vascular effects and, furthermore, show that IL-6 signaling for hypertrophy is independent of its pressor effects.
A role for IL-6 in hypertension in vivo has been suggested by a study comparing BP responses in WT or IL-6–/– mice given high-dose Ang II (5.2 mg/kg per day, approximately fivefold higher than in our study), in combination with a high-salt diet.26
In contrast to our findings, deletion of IL-6 did not influence the pressor response to high-dose Ang II on a normal salt diet.27
Here, considerably lower Ang II doses that are still sufficient to generate pressor responses were used, and it was found that deletion of IL-6 was fully sufficient to prevent BP elevations throughout the first 4 days (Figure 1A)
. These data indicate that the mechanisms of Ang II-hypertension vary with dose, with high-dose Ang II elevating BP by IL-6-independent mechanisms. We found that aortae from IL-6–/– mice constrict to Ang II and display endothelial dysfunction when preincubated with Ang II, similar to WT vessels (Figure 6)
. This indicates that the direct vascular constrictive effects of Ang II are preserved with IL-6 deletion and are therefore unlikely to participate in the pressor response to low-dose Ang II. Perhaps at higher doses of Ang II, direct vasoconstriction by Ang II contributes to the rise in BP, negating the impact of IL-6.
Here, we show that that IL-6 deletion prevents both aortic and cardiac hypertrophy in vivo, in response to low-dose Ang II (Figure 1, C and D)
. IL-6 signals are coordinated after its binding to the membrane-bound cognate IL-6 receptor. This protein is expressed on the surface of a number of cells, including neutrophils, monocytes, hepatocytes, endothelial cells, adrenal cells, and lymphocytes. Expression of IL-6R in the vascular wall was found, consistent with localization in smooth muscle (Figure 2)
. Activation of gp130 on the cells would then mediate intracellular activation of Src and JAK, which in turn phosphorylate STAT3 and mediate gene induction.28-31
Indeed, STAT3 activation in cardiac tissue after Ang II infusion was found, which was absent in IL-6–/– and insensitive to sgp130Fc (Figure 3)
. These data propose the cognate IL-6R as a potential therapeutic target for treatment of Ang II-dependent cardiac hypertrophy and heart failure.
A study of the downstream regulation of receptors for both pathways (AT1R, IL-6R) by both Ang II and IL-6 in vivo showed that Ang II elevated plasma sIL-6R in IL-6–/– but not WT mice (Figure 2G)
. In addition, IL-6 deficiency was associated with decreased expression of both cognate IL-6R and plasma sIL-6R, suggesting that IL-6 regulates expression of IL-6R at the gene level (Figure 2)
. These data show that Ang II regulates sIL-6R in a manner that is antagonized by IL-6. On the other hand, before Ang II infusion, expression of AT1R was higher in IL-6–/– aortae, indicating a compensatory up-regulation, as previously shown.32
Interestingly, even with a twofold elevation of AT1R, responses to Ang II in this strain were either lower (BP/hypertrophy/STAT3 activation) or unaffected (in vitro constriction) (Figures 1 and 6)
. The elevation in AT1R could represent an attempt to compensate for attenuated Ang II responses in IL-6 deficiency, eg, by up-regulating components of the Ang II-signaling pathway, but this is not clarified herein. However, Ang II significantly decreased AT1R expression in WT but not IL-6–/–, and furthermore, this was not blocked by sgp130Fc (Figure 4M)
. These data indicate that IL-6 acting via the cognate IL-6R mediates Ang II-dependent down-regulation of AT1R in vivo. A novel mechanism of Ang II and IL-6 interaction in vivo, via a complex interdependence in receptor regulation is demonstrated. Exactly how Ang II stimulates IL-6 signaling is unclear. We found no increases in either circulating sIL-6R or plasma IL-6 (undetectable) after Ang II infusion (Figure 2A
and data not shown). Therefore, we propose that Ang II may regulate IL-6 signaling intracellularly, downstream of receptor/gp130 triggering (Figure 7)
. A further question regards the site of action where sIL-6R signaling mediates hypertension. Possibilities that will need to be addressed in future experiments include resistance arteries, where most of the vasoconstrictive effect of Ang II takes place, and the kidney, where sodium retention is an important contributor to hypertension in this model.
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. A hitherto unidentified relationship between IL-6-mediated STAT3 activation and Ang II signaling is established. Inhibition of IL-6 signaling is therefore suggested as a potential therapy for hypertension or cardiac hypertrophy.
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Footnotes |
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Supported by the Wellcome Trust (to V.B.O., S.A.J., and B.C.), the Deutsche Forschungsgemeinschaft, Bonn, Germany (grants SFB415 and TP-B5 to S.R.J. and J.S.), and Kidney Research UK (KRUK) (to C.A.F.).
Accepted for publication March 26, 2007.
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, WT sham group (n = 6); 
, WT Ang II-infused group (n = 8); 
, IL-6–/– sham group (n = 6); 
, IL-6–/– Ang II-infused group (n = 8). 
P < 0.001 compared with WT control group; 
P < 0.0001 compared with IL-6–/– group; 
P < 0.01 compared with WT Ang II-infused group; using two-way analysis of variance with Bonferroni’s posttest (mean ± SEM). B: Administration (intraperitoneally) of sgp130Fc inhibits Ang II-dependent hypertension in WT mice. Ang II (1.1 mg/kg per day) was infused into male, 10 to 12 weeks old, WT mice by osmotic minipump with or without administration of sgp130Fc (intraperitoneal injection on days –1 and +1 at 100 µg/mouse and days +3 and +5 at 50 µg/mouse), and BP was monitored as in A. 
, WT Ang II-infused sgp130-treated (n = 6). 
