Regulation of Vascular Type 1 Angiotensin Receptors by Cytokines
Abstract Although various cytokines are known to be expressed in atherosclerotic lesions, it is not known how these cytokines affect receptors for the peptide hormone angiotensin II (Ang II). We therefore examined the effects of interleukin-1α (220 U/mL [10 ng/mL]), tumor necrosis factor-α (280 U/mL [100 ng/mL]), and interferon gamma (100 U/mL) on Ang II type 1 (AT1) receptors expressed in rat vascular smooth muscle cells. Treatment with interleukin-1α caused a 1.4- to 1.7-fold increase in AT1 binding after 24 hours (P<.01) and a 2.3-fold increase in AT1 mRNA (P<.05). Tumor necrosis factor-α and interferon gamma did not cause a significant change in AT1 binding when administered alone but caused a 30% reduction in binding when administered together (P<.05). The maximal decrease in AT1 binding (60%, P<.01) was seen with the combination of interleukin-1α with tumor necrosis factor-α and interferon gamma. Although the upregulation of AT1 by interleukin-1α was unaffected by pretreatment of cells with N-monomethyl-l-arginine or indomethacin, downregulation of AT1 by interleukin-1α combined with tumor necrosis factor-α/interferon gamma was inhibited by N-monomethyl-l-arginine (P<.01). Interleukin-1α treatment enhanced Ang II–induced [3H]uridine incorporation, whereas treatment with interleukin-1α combined with tumor necrosis factor-α/interferon gamma attenuated Ang II–induced [3H]uridine and [3H]leucine incorporation. These results demonstrate that interleukin-1α upregulates AT1 receptors and enhances Ang II–stimulated hypertrophic responses. However, a combination of interleukin-1α with tumor necrosis factor-α and interferon gamma downregulates AT1 receptors by a nitric oxide–dependent mechanism and reduces Ang II–stimulated trophic responses in vascular smooth muscle cells.
The vasoconstrictor peptide Ang II plays an important role in the control of systemic blood pressure. Evidence suggests that Ang II is also directly involved in the process of vascular remodeling, which is a hallmark of both hypertensive vascular disease and atherosclerotic disease. Ang II has been shown to have trophic actions on the vasculature in vitro1 and in vivo2 and to cause increased extracellular matrix production.3 Increases in the expression of angiotensin-converting enzyme, which generates Ang II, have been demonstrated in human atherosclerotic lesions4 as well as the balloon injury model of vascular proliferation.5 Finally, animal experiments have shown that interventions to block the actions of Ang II decrease vascular proliferation in response to injury,6 whereas stimulation of the renin-angiotensin system increases vascular trophic responses.7
The effects of Ang II on VSMCs are mediated by two receptors: the AT1 and AT2 receptors.8 The AT2 receptor is expressed in fetal vessels but is not ordinarily expressed in adult VSMCs. In contrast, the AT1 receptor is expressed abundantly in adult VSMCs, and this receptor subtype has been shown to mediate the hypertrophic actions of Ang II on the vasculature.8
Previous studies from our laboratory have shown that hormonal changes, such as increases in glucocorticoid hormones, can cause increases in the vascular AT1 receptor,9 resulting in increases in responses to Ang II.10 This upregulation of AT1 receptor function probably plays an important role in the pathogenesis of the hypertension in Cushing’s syndrome11 —is a systemic disease caused by overproduction of glucocorticoid hormone by the adrenal gland.
The pathogenesis of the vascular remodeling seen with hypertensive vascular hypertrophy and atherosclerosis is more complex, in that the changes are not thought to occur because of a single hormonal imbalance. Instead, the changes seen in atherosclerotic lesions are thought to be long-term changes caused in part by an imbalance of local mediators, such as growth factors and cytokines.12 These factors are synthesized and/or secreted by several cell types, such as macrophages, endothelial cells, and VSMCs themselves, resulting in autocrine/paracrine changes in the properties of vessel wall cell components. One of the changes may be to enhance the sensitivity of VSMCs to growth factors such as fibroblast growth factor.13 With respect to the vasotrophic peptide hormone Ang II, it is unclear what effect these mediators have on the angiotensin receptor or on Ang II–induced trophic responses in the vasculature. Since studies using inhibitors of the renin-angiotensin system have highlighted the pivotal role of angiotensin in vascular remodeling processes, it is important to understand the mechanisms by which the actions of angiotensin may be modulated in the vessel wall. This would further our understanding of the processes by which cytokines exert their effects on the vasculature and might also suggest new therapeutic strategies for modulating the biology of vascular injury.
The aim of this study was therefore to examine whether the cytokines found to be expressed in the vascular wall in diseased vessels can cause changes in the number of receptors for the peptide hormone Ang II and whether they can modulate growth responses to Ang II. We also examined the mechanisms involved in the cytokine-induced regulation of vascular AT1 receptors. Our results suggest a potential mechanism by which cytokines could affect vascular growth processes through modulation of the AT1 receptor.
Culture of Rat VSMCs
Rat VSMCs were prepared from the thoracic aortas of 6-week-old male Wistar rats as previously described3 and cultured in DMEM supplemented with 10% fetal calf serum, 100 U/mL penicillin, and 68 mmol/L (100 mg/mL) streptomycin. Experiments were performed on cells from passages 5 to 12.
Receptor Binding Assays
Cells in 12-well plates were treated in DMEM with the cytokines IL-1 (220 U/mL [10 ng/mL]; generously provided by Dainippon Pharmaceutical Co), TNF (280 U/mL [100 ng/mL]), and IFN (100 U/mL) for 24 hours. The cells were then rinsed three times in phosphate-buffered saline (PBS). Binding assays were performed by incubating cells in 50 mmol/L Tris (pH 7.4), 100 mmol/L NaCl, 5 mmol/L MgCl2, 0.25% bovine serum albumin, 33 U/mL bacitracin, and 125I–[Sar1,Ile8]Ang II (sarile, 0.2 nmol/L) at 4°C for 2 hours. Losartan (10−6 mol/L) was added to some wells for measurement of nonspecific binding. Cells were then washed three times with ice-cold PBS and lysed in 0.5% SDS and 0.1 mol/L NaOH, and radioactivity was measured with a gamma counter. Values for nonspecific binding were subtracted from values for total binding. The lysate protein content was assayed by the modified Lowry method,14 and the binding data were normalized to protein content. In the experiments for Scatchard analyses, duplicate plates were prepared for the measurement of cell numbers using a cell counter, and data were represented as binding per million cells.
Isolation of Total RNA and Northern Blot Analysis
Total RNA was purified from VSMCs by the acid guanidinium/phenol/chloroform method15 and quantified by measurement of absorbance of 260 nm in a spectrophotometer. Total RNA (20 μg) from treated and untreated cells was denatured with formamide and formaldehyde at 65°C for 10 minutes and fractionated by electrophoresis through a 1.0% formaldehyde-agarose gel. RNA was stained with ethidium bromide to verify integrity and equal loading and then transferred to a nylon filter (Pall BioSupport) and cross-linked with a UV irradiator (Stratagene). Prehybridization was conducted at 42°C for 2 hours in a buffer containing 6× SSC (0.9 mol/L sodium chloride, 0.09 mol/L sodium citrate [pH 7.0]), 5× Denhardt’s solution (0.1% [wt/vol] polyvinylpyrrolidone, 0.1% [wt/vol] Ficoll type 400, and 0.1% [wt/vol] bovine serum albumin), 50% formamide, and 0.1% SDS and sheared, denatured salmon sperm DNA (100 μg/mL). Cloned rat AT1a cDNA was kindly provided by Dr T. Inagami (Nashville, Tenn). A 607-bp coding region fragment corresponding to bases 133 through 739 was produced from this cDNA by polymerase chain reaction amplification as previously described,16 characterized by restriction mapping, and used as probe. This region of the AT1 receptor corresponds to the region of high nucleotide homology (93%) between the AT1a and AT1b receptors,16 and the probe recognizes both AT1a and AT1b receptors. A 1.1-kb human GAPDH cDNA probe was purchased from Clontech. Probes were radiolabeled with [α-32P]dCTP by the random primer synthesis method (RadPrime DNA Labeling System, GIBCO BRL). After hybridization, the filter was washed in 0.2× SSC and 0.1% SDS at 42°C. Bands were visualized, and incorporated radioactivity was quantified by scanning with a laser image analyzer (model BAS 2000, Fuji Film Co). Values of AT1 mRNA were normalized to corresponding values of GAPDH mRNA.
Determination of Ang II–Induced [3H]Uridine and [3H]Leucine Incorporation
Ang II–induced [3H]uridine and [3H]leucine incorporation into trichloroacetic acid (TCA)–precipitable material was determined on the basis of a previously described method.17 Confluent VSMCs in 24-well plates were made quiescent by being placed for 48 hours in DMEM containing insulin (10 μg/mL), selenium (6.7 ng/mL), and transferrin (5.5 μg/mL). After stimulation with Ang II, cells were pulsed for 4 hours (15 to 19 hours after stimulation) with tritiated uridine (2 μCi/mL) or tritiated leucine (5 μCi/mL). After removal of the medium, cells were washed twice with cold PBS, twice with 10% (wt/vol) cold TCA, and incubated with 10% TCA at 4°C for 30 minutes. The cellular precipitate adhering to the wells was then rinsed twice with ethanol and solubilized in 0.1 mol/L NaOH and 0.5% SDS. Incorporated radioactivity was determined by liquid scintillation spectrometry.
Results are expressed as mean±SEM. Statistical comparisons were made by ANOVA followed by Scheffé’s F test for comparisons between groups. Values of P<.05 were considered statistically significant.
Effects of IL-1 on AT1 Receptors in VSMCs
We examined the effects of IL-1 on angiotensin receptor binding in cultured VSMCs. Cells were treated with IL-1 in DMEM for 24 hours, and receptor binding studies were performed using 125I-sarile with or without losartan to detect nonspecific binding. Scatchard analysis of the binding data revealed that treatment with IL-1 caused an increase in angiotensin receptor numbers (from 128 to 178 fmol/106 cells) without a change in the affinity constant (Fig 1a⇓). Competitive binding inhibition with the AT1- and AT2-specific antagonists losartan and PD 123319 demonstrated that the angiotensin receptors detected in IL-1–treated cells were exclusively AT1 receptors (Fig 1b⇓). Next, we performed dose dependency and time course studies and found that treatment with IL-1 at a dose of 220 U/mL (10 ng/mL) caused an approximately 1.7-fold increase in AT1 receptor number (P<.01). The increase in AT1 receptor number became apparent after 8 hours of treatment with IL-1 (Fig 2⇓).
Effects of IL-1 on AT1 Receptor mRNA in VSMCs
We performed studies to examine whether the IL-1–induced upregulation of the AT1 receptor was due to increases in AT1 receptor mRNA. Treatment of VSMCs with IL-1 caused an increase in AT1 receptor mRNA as determined by Northern blot analysis. Quantification of incorporated radioactivity using a laser image analyzer revealed a 2.3-fold increase in AT1 receptor mRNA after treatment with IL-1 for 24 hours (P<.05) (Fig 3⇓).
Effects of Cytokine Combinations on AT1 Receptors in VSMCs
We examined the effects of the cytokines IL-1, TNF, and IFN either alone or in combination on AT1 receptor binding in VSMCs. As shown in the Table⇓, when cells were treated with each cytokine alone, only IL-1 caused a significant increase in AT1 receptor binding. Although neither TNF nor IFN caused a significant change in basal AT1 receptor binding, both cytokines significantly attenuated the IL-1–induced AT1 receptor upregulation. Treatment with the combination of TNF and IFN caused a reduction in AT1 binding to 72% of control levels (P<.05). However, maximal downregulation of the AT1 receptor to 42% of control levels was seen with a combination of IL-1 with TNF and IFN (P<.01). This was comparable to the downregulation of AT1 receptors seen with the NO donors isosorbide dinitrate and sodium nitroprusside. Scatchard analysis of the binding data in VSMCs treated with the triple combination of IL-1 with TNF and IFN confirmed that receptor numbers were decreased without a change in the receptor affinity constant (Fig 4⇓). The downregulating effect of the three cytokines appeared to be synergistic and not simply an additive effect caused by using high total concentrations of cytokines, since further experiments using any one cytokine at triple the original concentrations did not cause significant downregulation of the AT1 receptors (AT1 binding in control cells: 245±16 cpm/μg protein; cells treated with IL-1 [660 U/mL (30 ng/mL)]: 281±24; cells treated with TNF [840 U/mL (300 ng/mL)]: 219±11; cells treated with IFN [300 U/mL]: 258±21; n=4).
Role of NO in Cytokine-Induced Downregulation of AT1 Receptors
We examined the roles of NO and prostaglandins in the effects of IL-1 on AT1 receptors in VSMCs. Cells were pretreated with the NO synthase inhibitor L-NMMA or the cyclooxygenase inhibitor indomethacin for 2 hours before addition of IL-1. Neither of these agents caused significant changes in AT1 receptor binding when administered alone (AT1 receptor binding in control cells: 163±6 cpm/μg protein; indomethacin-treated cells: 159±10; L-NMMA–treated cells: 171±12; n=4). Treatment with L-NMMA or indomethacin did not affect IL-1–induced upregulation of AT1 receptor binding. However, L-NMMA significantly attenuated the downregulation of AT1 receptor binding by IL-1 combined with TNF and IFN (P<.01) (Fig 5⇓).
Effects of Cytokines on Ang II–Induced [3H]Uridine and [3H]Leucine Incorporation
We examined the effects of IL-1 on Ang II–induced [3H]uridine and [3H]leucine incorporation. Quiescent cells were pretreated with IL-1 for 3 hours before the addition of Ang II (10−6 mol/L) for a further 19 hours and then were pulsed with [3H]uridine or [3H]leucine. As shown in Fig 6⇓, IL-1 significantly enhanced Ang II–induced [3H]uridine incorporation compared with cells not treated with IL-1 (P<.01). Similarly, Ang II–stimulated [3H]leucine incorporation appeared to be enhanced in IL-1–treated cells compared with cells that had not been treated with IL-1; however, the effect did not reach statistical significance. In contrast, cells pretreated with IL-1 combined with TNF and IFN showed significant attenuation of [3H]uridine or [3H]leucine incorporation in response to Ang II compared with cells not treated with cytokines (P<.01).
Hypertension is a risk factor for vascular pathology such as vessel wall hypertrophy and atherosclerotic disease. One of the hallmarks of lesions in diseased vessels is the presence of smooth muscle cell growth and hypertrophy, which contributes to the formation of stenoses in the blood vessels and plays a significant role in the pathophysiology of disorders such as coronary artery disease and cerebrovascular disease.
Atherosclerotic lesions are associated with overexpression of a variety of growth factors and cytokines.12 These terms designate the wide variety of locally synthesized autocrine/paracrine factors that influence the progression of atherosclerotic disease. The inflammatory cytokines IL-1, TNF, and IFN were originally described through their actions on the cells of the immune system but are now also thought to play an important role in the pathogenesis of atherosclerosis, which is in part an immune as well as a fibroproliferative response to injury.18 19 20 Both IL-1 and TNF have been shown to be associated with all the major cell components of atherosclerotic lesions, namely, macrophages, endothelial cells, and VSMCs.18 19 On the other hand, IFN is seen chiefly in and around infiltrating lymphocytes.20
Although the presence of these cytokines in atherosclerotic lesions strongly suggests that they play a role in modulating vascular growth responses, the exact mechanisms still remain to be defined. IL-1 may cause VSMC proliferation in the long term but not the short term because of the release of growth-inhibitory prostanoids21 and may exert its actions by modulating other growth factors, such as platelet-derived growth factor.22 On the other hand, a recent report has shown that IL-1 suppresses endothelin-induced VSMC proliferation.23
Presently, interactions between these cytokines and the trophic hormone Ang II have not been clarified, despite the fact that any effects on Ang II–induced responses may have important implications for our understanding of the mechanisms involved in vascular remodeling. In particular, it is important to examine the effects of cytokine combinations on AT1 receptor numbers in VSMCs because changes in AT1 receptor numbers cause marked changes in the responses to Ang II.10 24
We examined the effects of IL-1, TNF, and IFN on AT1 receptor binding in VSMCs. As shown in the Table⇑, of these cytokines, IL-1 was unique in causing upregulation of the AT1 receptor. Time course studies revealed that IL-1–induced upregulation of the AT1 receptor occurred over several hours, suggesting that the upregulation could be due to increased AT1 gene expression. Northern blot analyses revealed increased levels of AT1 mRNA in IL-1–treated cells, consistent with increased gene expression as the mechanism of IL-1–induced AT1 receptor upregulation. The effects of IL-1 on the regulation of gene expression can occur through the transcription factor nuclear factor-κB (NF-κB).25 Interestingly, Inagami et al26 have reported the presence of NF-κB recognition sites in the 5′-flanking region of the rat AT1 receptor. This suggests a possible mechanism for the upregulation of AT1 gene expression by IL-1. Further studies are required to confirm whether the IL-1–induced increases in AT1 gene expression seen in the present study were mediated through these NF-κB recognition sites.
Recently, Ichiki et al27 have found that the promoter region for the other major angiotensin receptor subtype, the AT2 receptor, contains a CCAAT enhancer binding protein site (C/EBP) motif, suggesting that the AT2 receptor might be regulated by IL-1. Indeed, AT2 receptor gene expression was upregulated by IL-1 treatment in the fibroblast-derived R3T3 cell line, which expresses AT2 receptors.27 However, in our studies we found increased expression of AT1 but not AT2 receptors in VSMCs after IL-1 treatment. Therefore, IL-1–induced upregulation of AT2 receptors may be a tissue-specific effect seen in cells that constitutively express AT2 receptors.
A number of signaling mechanisms have been described for IL-1–mediated effects, and the relative importance of these mechanisms in different cells is unclear. These include increases in 1,2-diacylglycerol formation, activation of protein kinase C, increases in prostaglandin synthesis, and NO-mediated effects, as well as others.28 29 It has been shown that the protein kinase C activator phorbol 12-myristate 13-acetate does not cause upregulation of VSMC AT1 receptors.30 We therefore examined the effects of the cyclooxygenase inhibitor indomethacin and the NO synthase inhibitor L-NMMA on IL-1–induced AT1 receptor upregulation. Neither of these inhibitors affected IL-1–induced AT1 receptor upregulation, suggesting that neither NO nor prostaglandins were involved in mediating this effect. The fact that NO was not involved in the process is consistent with previous studies from this laboratory which demonstrated that IL-1 administered alone did not cause significant increases in inducible NO synthase expression or nitrite synthesis in our VSMCs, whereas marked effects were seen when IL-1 was combined with other cytokines.31
Since in the in vivo situation the vascular wall may actually contain a mixture of several cytokines, we examined the effects of combinations of cytokines on AT1 receptor binding. Although neither TNF nor IFN caused a significant change in AT1 receptor binding, both of these cytokines attenuated the IL-1–mediated upregulation. Moreover, addition of TNF and IFN together caused a small but statistically significant decrease in AT1 receptor binding. Maximal reduction in AT1 receptor numbers was seen in cells treated with the combination of IL-1, TNF, and IFN. This downregulation of AT1 receptors was reversed by pretreatment of cells with L-NMMA, suggesting that downregulation of AT1 receptors was mediated by increased production of NO, presumably because of induction of inducible NO synthase (NO synthase II) by the cytokines. Our results using NO donors confirm the work of Cahill et al,32 who showed that these NO donors can downregulate vascular AT1 receptors. The fact that the effects of the different cytokines appeared synergistic rather than additive is consistent with previous studies from our laboratory and others showing a synergistic effect of the cytokines on the induction of NO synthase II. The intracellular mechanism of this synergistic interaction still requires further study.31 33
To examine the possibility that these findings may have relevance to vascular trophic responses to Ang II, we examined the effects of IL-1 on Ang II–mediated hypertrophic responses using increases in [3H]uridine incorporation and [3H]leucine incorporation as indexes of increased RNA synthesis (and/or decreased RNA degradation) and protein synthesis (and/or decreased protein degradation), respectively. IL-1 treatment alone caused a significant enhancement of Ang II–mediated increases in [3H]uridine incorporation, whereas the combination of IL-1 with TNF and IFN resulted in a decrease in Ang II–mediated [3H]-uridine and [3H]leucine incorporation. These results suggest that the cytokine-mediated changes in AT1 receptor regulation may be involved in modulating Ang II–mediated vascular growth effects.
The enhancement of Ang II–mediated growth effects contrasts with the inhibitory effect of IL-1 on endothelin-1–mediated VSMC proliferation, which has been shown to occur because of the increased prostanoid metabolism.23 On the other hand, IL-1 has been shown to enhance the proliferative effect of fibroblast growth factor.13 A unique feature of our study is the emphasis on the effects of combinations of cytokines, which may have effects that cannot be predicted from studies of each isolated cytokine. In particular, IL-1 may have opposing effects depending on the presence or absence of other cytokines. This is important because it is probable that several cytokines may be present together in a diseased lesion.
In summary, the following conclusions can be derived from the present study. First, cytokines can regulate AT1 receptors and Ang II–induced hypertrophic responses in VSMCs. The regulation can be bidirectional, ie, an enhancement or an attenuation, depending on the cytokine combination. The IL-1–induced upregulation of AT1 receptors occurs by an NO-independent pathway, whereas the cytokine-induced downregulation occurs by an NO-dependent pathway. The net effect of cytokines on AT1 receptors in a particular vascular lesion can therefore be altered by the mixture of cytokines present in that lesion.
Currently, the exact composition of the cytokines in atherosclerotic lesions and the temporal changes in the composition with the progression of the lesion remain unknown. Since the regulation of AT1 receptors is bidirectional, it is possible that the cytokines may enhance or attenuate Ang II–mediated effects differently, depending on the nature of the cytokine-producing cells present in that lesion. A potential mechanism by which cytokines may either enhance or attenuate vascular hypertrophy is by affecting peptide hormone receptors on the VSMC. These findings may have important implications for our understanding of how atherosclerotic lesions develop.
Selected Abbreviations and Acronyms
|Ang II||=||angiotensin II|
|AT1, AT2||=||angiotensin type 1, type 2 (receptor)|
|DMEM||=||Dulbecco’s modified Eagle’s medium|
|SDS||=||sodium dodecyl sulfate|
|TNF||=||tumor necrosis factor-α|
|VSMC||=||vascular smooth muscle cell|
This work was supported in part by grants from the Keio University Medical Science Fund, the Kanae Foundation, and the Ministry of Education, Science, and Culture, Japan.
- Received September 18, 1996.
- Revision received October 21, 1996.
- Accepted December 19, 1996.
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