This Article Abstract Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Permissions Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Sasamura, H. Articles by Saruta, T. Search for Related Content PubMed PubMed Citation Articles by Sasamura, H. Articles by Saruta, T.

(Hypertension. 1997;30:35-41.)
© 1997 American Heart Association, Inc.

Articles

Regulation of Vascular Type 1 Angiotensin Receptors by Cytokines

Hiroyuki Sasamura; Yuichi Nakazato; Tomoko Hayashida; Yudai Kitamura; Matsuhiko Hayashi; ; Takao Saruta

From the Department of Internal Medicine, School of Medicine, Keio University, Tokyo, Japan.

Correspondence to Hiroyuki Sasamura, MD, Department of Internal Medicine, School of Medicine, Keio University, 35 Shinanomachi, Shinjuku-ku, Tokyo 160, Japan.

	Abstract

Top Abstract Introduction Methods Results Discussion References

 
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-1 (220 U/mL [10 ng/mL]), tumor necrosis factor- (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-1 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- 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-1 with tumor necrosis factor- and interferon gamma. Although the upregulation of AT₁ by interleukin-1 was unaffected by pretreatment of cells with N-monomethyl-L-arginine or indomethacin, downregulation of AT₁ by interleukin-1 combined with tumor necrosis factor-/interferon gamma was inhibited by N-monomethyl-L-arginine (P<.01). Interleukin-1 treatment enhanced Ang II–induced [³H]uridine incorporation, whereas treatment with interleukin-1 combined with tumor necrosis factor-/interferon gamma attenuated Ang II–induced [³H]uridine and [³H]leucine incorporation. These results demonstrate that interleukin-1 upregulates AT₁ receptors and enhances Ang II–stimulated hypertrophic responses. However, a combination of interleukin-1 with tumor necrosis factor- and interferon gamma downregulates AT₁ receptors by a nitric oxide–dependent mechanism and reduces Ang II–stimulated trophic responses in vascular smooth muscle cells.

Key Words: angiotensin • receptors, angiotensin • cytokines • interleukin-1 • tumor necrosis factor • interferons

	Introduction

Top Abstract Introduction Methods Results Discussion References

 
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.

	Methods

Top Abstract Introduction Methods Results Discussion References

 
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 6x SSC (0.9 mol/L sodium chloride, 0.09 mol/L sodium citrate [pH 7.0]), 5x 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 AT_1a 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 AT_1a and AT_1b receptors,¹⁶ and the probe recognizes both AT_1a and AT_1b receptors. A 1.1-kb human GAPDH cDNA probe was purchased from Clontech. Probes were radiolabeled with [-³²P]dCTP by the random primer synthesis method (RadPrime DNA Labeling System, GIBCO BRL). After hybridization, the filter was washed in 0.2x 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.

Statistics
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.

	Results

Top Abstract Introduction Methods Results Discussion References

 
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).

View larger version (18K): [in this window] [in a new window]   Figure 1. Effects of IL-1 on the vascular AT1 receptor. a, Scatchard analysis of AT1 receptor binding in cells treated with vehicle or IL-1 (220 U/mL [10 ng/mL]) for 24 hours. b, Displacement of binding of 125I–[Sar1,Ile8]Ang II (sarile) (0.2 nmol/L) in IL-1–treated cells by cold sarile, losartan, and PD 123319. Results are mean±SEM of three to four determinations.

View larger version (17K): [in this window] [in a new window]   Figure 2. Dose dependency (a) and time course (b) of IL-1–induced upregulation of AT1 receptor. a, Cells were treated with the indicated doses of IL-1 for 24 hours, and AT1 receptor numbers were estimated as described in "Methods." b, Cells were treated with 220 U/mL (10 ng/mL) IL-1 for the indicated times, and AT1 receptor numbers were estimated as described in "Methods." Results are mean±SEM of three to four determinations. *P<.05, **P<.01 vs values in cells treated with vehicle alone.

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).

View larger version (25K): [in this window] [in a new window]   Figure 3. Effects of IL-1 on AT1 mRNA. Cells were treated with IL-1 (220 U/mL [10 ng/mL]) for the indicated times, and AT1 mRNA/GAPDH mRNA levels were estimated as described in "Methods." a, Representative Northern blot of AT1 and GAPDH mRNAs from cells treated with IL-1. Similar levels of 18S RNA were seen in all lanes, as shown in the middle panel. b, Mean±SEM of cumulative data from three independent experiments. *P<.05 vs values in vehicle-treated cells.

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).

View this table: [in this window] [in a new window]   Table 1. Effects of Cytokines on Angiotensin Type 1 Receptors

View larger version (15K): [in this window] [in a new window]   Figure 4. Scatchard analysis of AT1 receptor binding in cells treated with vehicle or a combination of IL-1, TNF, and IFN for 24 hours.

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).

View larger version (20K): [in this window] [in a new window]   Figure 5. Role of cytokines and prostaglandins in cytokine-induced regulation of AT1 receptor. Cells were pretreated with L-NMMA (3 mmol/L) or indomethacin (indo) (1 µmol/L) for 2 hours before treatment with IL-1 (220 U/mL [10 ng/mL]) or a combination of IL-1, TNF (T), and INF (I) for 24 hours. a, Effect of L-NMMA and indomethacin on IL-1–induced upregulation of the AT1 receptor. b, Effect of L-NMMA and indomethacin on IL-1/TNF/IFN–induced downregulation of the AT1 receptor. Results are mean±SEM of three to four determinations. **P<.01 vs values in cells treated with vehicle alone; ##P<.01 vs values in cells treated with IL-1/TNF/IFN alone.

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).

View larger version (24K): [in this window] [in a new window]   Figure 6. Effects of cytokines on Ang II–induced [3H]uridine and [3H]leucine incorporation. Cells were pretreated for 3 hours with either vehicle alone or IL-1 (a and b) or a combination of IL-1, TNF, and IFN (c and d). Cells then were stimulated with Ang II (1 µmol/L) for 19 hours as described in "Methods." [3H]Uridine (a and c) or [3H]leucine (b and d) was added during the last 4 hours of incubation, and incorporated radioactivity was measured. a, Effect of IL-1 on Ang II–induced [3H]uridine incorporation. b, Effect of IL-1 on Ang II–induced [3H]leucine incorporation. c, Effect of IL-1/TNF/IFN on Ang II–induced [3H]uridine incorporation. d, Effect of IL-1/TNF/IFN on Ang II–induced [3H]leucine incorporation. Results are mean±SEM of four determinations. *P<.05,**P<.01 vs values in cells treated with vehicle alone; ++P<.01 vs values in cells treated with IL-1 alone; and ##P<.01 vs values in cells treated with Ang II alone.

	Discussion

Top Abstract Introduction Methods Results Discussion References

 
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-B (NF-B).²⁵ Interestingly, Inagami et al²⁶ have reported the presence of NF-B 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-B 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 IFN = interferon gamma IL-1 = interleukin-1 L-NMMA = NG-monomethyl-L-arginine NO = nitric oxide SDS = sodium dodecyl sulfate TNF = tumor necrosis factor- VSMC = vascular smooth muscle cell

	Acknowledgments

 
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; first decision October 21, 1996; accepted December 19, 1996.

	References

Top Abstract Introduction Methods Results Discussion References

 
1. Geisterfer AAT, Peach MJ, Owens GK. Angiotensin II induces hypertrophy, not hyperplasia, of cultured rat aortic smooth muscle cells. Circ Res. 1988;62:745-756.

2. Griffin SA, Brown WCB, MacPherson F, McGrath JC, Wilson VG, Korsgaard N, Mulvany MJ, Lever AF. Angiotensin II causes vascular hypertrophy in part by a non-pressor mechanism. Hypertension. 1991;17:626-635.[Abstract/Free Full Text]

3. Kato H, Suzuki H, Tajima S, Ogata Y, Tominaga T, Sato A, Saruta T. Angiotensin II stimulates collagen synthesis in cultured vascular smooth muscle cells. J Hypertens. 1991;9:17-22.[Medline] [Order article via Infotrieve]

4. Ohishi M, Ueda M, Rakugi H, Higaki J, Kamide K, Naruko T, Becker AE, Ogihara T. Immunocytochemical localization of angiotensin II, ACE, and chymase in human coronary atherosclerotic lesions. J Hypertens. 1996;14(suppl 1):S136. Abstract.

5. Rakugi H, Kim D-K, Krieger JE, Wang DS, Dzau VJ, Pratt RE. Induction of angiotensin converting enzyme in the neointima after vascular injury. J Clin Invest. 1994;93:339-346.

6. Prescott MF, Webb RL, Reidy MA. Angiotensin-converting enzyme inhibitor versus angiotensin II AT1 receptor antagonist: effects on smooth muscle cell migration and proliferation after balloon catheter injury. Am J Pathol. 1991;139:1291-1296.[Abstract]

7. Morishita R, Gibbons GH, Ellison KE, Lee W, Zhang L, Yu H, Kaneda Y, Ogihara T, Dzau VJ. Evidence for direct local effect of angiotensin in vascular hypertrophy. J Clin Invest. 1994;94:978-984.

8. Dzau VJ, Sasamura H, Hein L. Heterogeneity of angiotensin synthetic pathways and receptor subtypes: physiological and pharmacologic implications. J Hypertens. 1993;11(suppl 3):S13-S18.

9. Sato A, Suzuki H, Murakami M, Nakazato Y, Iwaita Y, Saruta T. Glucocorticoid increases angiotensin II type 1 receptor and its gene expression. Hypertension. 1994;23:25-30.[Abstract/Free Full Text]

10. Sato A, Suzuki H, Iwaita Y, Nakazato Y, Kato H, Saruta T. Potentiation of inositol trisphosphate production by dexamethasone. Hypertension. 1992;19:109-115.[Abstract/Free Full Text]

11. Saruta T, Suzuki H, Handa M, Igarashi Y, Kondo K, Senba S. Multiple factors contribute to the pathogenesis of hypertension in Cushing's syndrome. J Clin Endocrinol Metab. 1986;62:275-279.[Abstract/Free Full Text]

12. Ross R. The pathogenesis of atherosclerosis: a perspective for the 1990s. Nature. 1993;362:801-809.[Medline] [Order article via Infotrieve]

13. Bourcier T, Dockter M, Hassid A. Synergistic interaction of interleukin-1beta and growth factors in primary cultures of rat aortic smooth muscle cells. J Cell Physiol. 1995;164:644-657.[Medline] [Order article via Infotrieve]

14. Markwell MA, Haas SM, Bieber LL, Tolbert NE. A modification of the Lowry procedure to simplify protein determination in membrane and lipoprotein samples. Anal Biochem. 1978;87:206-210.[Medline] [Order article via Infotrieve]

15. Chomczynski P, Sacchi N. Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Anal Biochem. 1987;162:156-159.[Medline] [Order article via Infotrieve]

16. Iwai N, Inagami T. Identification of two subtypes in the rat type 1 angiotensin II receptor. FEBS Lett. 1992;298:257-260.[Medline] [Order article via Infotrieve]

17. Itoh H, Pratt RE, Dzau VJ. Atrial natriuretic peptide inhibits hypertrophy of vascular smooth muscle cells. J Clin Invest. 1990;86:1690-1697.

18. Moyer CF, Sajuthi D, Tulli H, Williams JK. Synthesis of IL-1 alpha and IL-1 beta by arterial cells in atherosclerosis. Am J Pathol. 1991;138:951-960.[Abstract]

19. Barath P, Fishbein MC, Cao J, Berenson J, Helfant RH, Forrester JS. Detection and localization of tumor necrosis factor in human atheroma. Am J Cardiol. 1990;65:297-302.[Medline] [Order article via Infotrieve]

20. Hansson GK, Holm J, Jonasson L. Detection of activated T lymphocytes in the human atherosclerotic plaque. Am J Pathol. 1989;135:169-175.[Abstract]

21. Libby P, Warner SJC, Friedman GB. Interleukin 1: a mitogen for human vascular smooth muscle cells that induces the release of growth-inhibitory prostanoids. J Clin Invest. 1988;81:487-498.

22. Raines EW, Dower SK, Ross R. Interleukin-1 mitogenic activity for fibroblasts and smooth muscle cells is due to PDGF-AA. Science. 1989;243:393-396.[Abstract/Free Full Text]

23. Fujitani Y, Ninomiya H, Okada T, Urade Y, Masaki T. Suppression of endothelin 1-induced mitogenic responses of human aortic smooth muscle cells by interleukin-1beta. J Clin Invest. 1995;95:2474-2482.

24. Ullian ME, Linas SL. Angiotensin II surface receptor coupling to inositol trisphosphate formation in vascular smooth muscle cells. J Biol Chem. 1990;265:195-200.[Abstract/Free Full Text]

25. Bomsztyk K, Rooney JW, Iwasaki T, Rachie NA, Dower SK, Hopkins-Sibley C. Evidence that interleukin-1 and phorbol esters activate NF-B by different pathways: role of protein kinase C. Cell Regul. 1991;2:329-335.[Medline] [Order article via Infotrieve]

26. Inagami T, Guo D-F, Kitami Y. Molecular biology of angiotensin II receptors: an overview. J Hypertens. 1994;12(suppl 10):S83-S94.

27. Ichiki T, Kambayashi Y, Inagami T. Multiple growth factors modulate mRNA expression of angiotensin II type-2 receptor in R3T3 cells. Circ Res. 1995;77:1070-1076.[Abstract/Free Full Text]

28. Sedor JR, Nakazato Y, Konieczkowski M. Interleukin-1 and the mesangial cell. Kidney Int. 1992;41:595-599.[Medline] [Order article via Infotrieve]

29. Palmer JN, Hartogensis WE, Patten M, Fortuin FD, Long CS. Interleukin-1beta induces cardiac myocyte growth but inhibits cardiac fibroblast proliferation in culture. J Clin Invest. 1995;95:2555-2564.

30. Brock TA, Rittenhouse SE, Powers CW, Ekstein LS, Gimbrone MA, Alexander RW. Phorbol ester and 1-oleoyl-2-acetylglycerol inhibit angiotensin activation of phospholipase C in cultured vascular smooth muscle cells. J Biol Chem. 1985;260:14158-14162.[Abstract/Free Full Text]

31. Marumo T, Nakaki T, Nagata K, Miyata M, Adachi H, Esumi H, Suzuki H, Saruta T, Kato R. Dexamethasone inhibits nitric oxide synthase mRNA induction by interleukin-1alpha and tumor necrosis factor-alpha in vascular smooth muscle cells. Jpn J Pharmacol. 1993;63:361-367.[Medline] [Order article via Infotrieve]

32. Cahill PA, Redmond EM, Foster C, Sitzmann JV. Nitric oxide regulates angiotensin II receptors in vascular smooth muscle cells. Eur J Pharmacol. 1995;288:219-229.[Medline] [Order article via Infotrieve]

33. Kleinert H, Euchenhofer C, Ihrig-Biedert I, Foerstermann U. Glucocorticoids inhibit the induction of nitric oxide synthase II by down-regulating cytokine-induced activity of transcription factor nuclear factor-B. Mol Pharmacol. 1996;49:15-21.[Abstract]

This article has been cited by other articles:

				Y.-M. Kang, Y. Ma, J.-P. Zheng, C. Elks, S. Sriramula, Z.-M. Yang, and J. Francis Brain nuclear factor-kappa B activation contributes to neurohumoral excitation in angiotensin II-induced hypertension Cardiovasc Res, June 1, 2009; 82(3): 503 - 512. [Abstract] [Full Text] [PDF]

				S. Sriramula, M. Haque, D. S.A. Majid, and J. Francis Involvement of Tumor Necrosis Factor-{alpha} in Angiotensin II-Mediated Effects on Salt Appetite, Hypertension, and Cardiac Hypertrophy Hypertension, May 1, 2008; 51(5): 1345 - 1351. [Abstract] [Full Text] [PDF]

				I. A. Arenas, S. J. Armstrong, Y. Xu, and S. T. Davidge Tumor Necrosis Factor-{alpha} and Vascular Angiotensin II in Estrogen-Deficient Rats Hypertension, September 1, 2006; 48(3): 497 - 503. [Abstract] [Full Text] [PDF]

				K. Nithipatikom, B. B. Holmes, M. J. McCoy, C. J. Hillard, and W. B. Campbell Chronic administration of nitric oxide reduces angiotensin II receptor type 1 expression and aldosterone synthesis in zona glomerulosa cells Am J Physiol Endocrinol Metab, November 1, 2004; 287(5): E820 - E827. [Abstract] [Full Text] [PDF]

				C. Bregonzio, I. Armando, H. Ando, M. Jezova, G. Baiardi, and J. M. Saavedra Anti-inflammatory effects of angiotensin II AT1 receptor antagonism prevent stress-induced gastric injury Am J Physiol Gastrointest Liver Physiol, July 7, 2003; 285(2): G414 - G423. [Abstract] [Full Text] [PDF]

				R. T. Cowling, D. Gurantz, J. Peng, W. H. Dillmann, and B. H. Greenberg Transcription Factor NF-kappa B Is Necessary for Up-regulation of Type 1 Angiotensin II Receptor mRNA in Rat Cardiac Fibroblasts Treated with Tumor Necrosis Factor-alpha or Interleukin-1beta J. Biol. Chem., February 15, 2002; 277(8): 5719 - 5724. [Abstract] [Full Text] [PDF]

				G. Nickenig and D. G. Harrison The AT1-Type Angiotensin Receptor in Oxidative Stress and Atherogenesis: Part II: AT1 Receptor Regulation Circulation, January 29, 2002; 105(4): 530 - 536. [Full Text] [PDF]

				R. Shimizu-Hirota, H. Sasamura, M. Mifune, H. Nakaya, M. Kuroda, M. Hayashi, and T. Saruta Regulation of Vascular Proteoglycan Synthesis by Angiotensin II Type 1 and Type 2 Receptors J. Am. Soc. Nephrol., December 1, 2001; 12(12): 2609 - 2615. [Abstract] [Full Text] [PDF]

				M. Bucher, K.-P. Ittner, J. Hobbhahn, K. Taeger, and A. Kurtz Downregulation of Angiotensin II Type 1 Receptors During Sepsis Hypertension, August 1, 2001; 38(2): 177 - 182. [Abstract] [Full Text] [PDF]

				M. Mifune, H. Sasamura, R. Shimizu-Hirota, H. Miyazaki, and T. Saruta Angiotensin II Type 2 Receptors Stimulate Collagen Synthesis in Cultured Vascular Smooth Muscle Cells Hypertension, November 1, 2000; 36(5): 845 - 850. [Abstract] [Full Text] [PDF]

				G. Nickenig, K. Strehlow, S. Wassmann, A. T. Baumer, K. Albory, H. Sauer, and M. Bohm Differential Effects of Estrogen and Progesterone on AT1 Receptor Gene Expression in Vascular Smooth Muscle Cells Circulation, October 10, 2000; 102(15): 1828 - 1833. [Abstract] [Full Text] [PDF]

				R. Largo, D. Gomez-Garre, K. Soto, B. Marron, J. Blanco, R. M. Gazapo, J. J. Plaza, and J. Egido Angiotensin-Converting Enzyme Is Upregulated in the Proximal Tubules of Rats With Intense Proteinuria Hypertension, February 1, 1999; 33(2): 732 - 739. [Abstract] [Full Text] [PDF]

				M. Bucher, J. Hobbhahn, K. Taeger, and A. Kurtz Cytokine-mediated downregulation of vasopressin V1A receptors during acute endotoxemia in rats Am J Physiol Regulatory Integrative Comp Physiol, April 1, 2002; 282(4): R979 - R984. [Abstract] [Full Text] [PDF]

This Article Abstract Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Permissions Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Sasamura, H. Articles by Saruta, T. Search for Related Content PubMed PubMed Citation Articles by Sasamura, H. Articles by Saruta, T.