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(Hypertension. 2000;36:845.)
© 2000 American Heart Association, Inc.

Scientific Contributions

Angiotensin II Type 2 Receptors Stimulate Collagen Synthesis in Cultured Vascular Smooth Muscle Cells

Mizuo Mifune; Hiroyuki Sasamura; Ryoko Shimizu-Hirota; Hitoshi Miyazaki; Takao Saruta

From the Department of Internal Medicine (M.M., H.S., R.S.-H., T.S.), School of Medicine, Keio University, Tokyo, Japan; and Institute of Applied Biochemistry (H.M.), University of Tsukuba, Ibaraki, Japan.

Correspondence to Hiroyuki Sasamura, MD, PhD, Department of Internal Medicine, School of Medicine, Keio University, 35 Shinanomachi, Shinjuku-ku, Tokyo 160-8582, Japan. E-mail sasamura{at}mc.med.keio.ac.jp

	Abstract

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Abstract—Previously, we and others have shown that angiotensin II enhances vascular smooth muscle cell extracellular matrix synthesis via stimulation of the angiotensin II type 1 (AT₁) receptor. Recently, expression of the type 2 (AT₂) receptor has been confirmed in the adult vasculature, but its role has not yet been fully defined. The aim of the present study was to examine the effects of stimulation of AT₂ receptors on collagen synthesis in vascular smooth muscle cells. Retroviral gene transfer was used to supplement adult vascular smooth muscle cells with AT₂ receptors to mimic the vasculature in vivo. The treatment of these cells with the AT₂ receptor agonist CGP42212A (10^-7 mol/L) alone did not cause a significant change in p42/p44 MAP kinase activity but caused a modest (30% to 50%) decrease in protein tyrosine phosphatase activity. Treatment with CGP42112A also caused a dose- and time-dependent increase in both cell-associated and secretory collagen synthesis (148±17% of control at 48 hours, P<0.05), which was completely inhibited by the AT₂ receptor antagonist PD123319, unaffected by the AT₁ receptor antagonist losartan, and attenuated by treatment with pertussis toxin or G_i antisense oligonucleotides. Interestingly, studies in other cell lines demonstrated that CGP42112A caused similar results in transfected mesangial cells but had essentially opposite effects in fibroblasts (NIH-3T3-AT₂). These results suggest that AT₂ receptor stimulation can increase collagen synthesis in vascular smooth muscle cells via a G_i-mediated mechanism and provide evidence for heterogeneity in the effects of AT₂ receptor stimulation in different tissues.

Key Words: angiotensin II • receptor, angiotensin II • collagen • muscle, smooth, vascular • fibroblasts

	Introduction

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Angiotensin II (Ang II) plays a central role in the regulation of systemic blood pressure and fluid homeostasis through its multiple effects on the vasculature, adrenals, kidneys, and brain. These pleiotropic actions of Ang II are mediated by specific receptors located on target tissues. The presence of 2 subtypes of angiotensin receptors (AT₁ and AT₂) has been demonstrated through pharmacological and molecular biology studies,^{1 2} Most of the "classic" hypertensive actions of the Ang II, namely vasoconstriction, stimulation of aldosterone secretion, and increased renal tubular sodium reabsorption, have been shown to be mediated by the AT₁ receptor.² On the other hand, several lines of evidence have suggested that the AT₂ receptor has predominantly antagonistic functions to the AT₁ receptor.

Although the AT₂ receptor is well known to be widely expressed in fetal tissues, histological confirmation of the expression of these receptors in the vasculature of normal human adults as well as rats has been provided by several groups with immunohistochemistry, in situ hybridization, and receptor binding studies.^{3 4 5} A recent study with adult hypertensive rats demonstrated that the stimulation of these receptors can increase the hypotensive actions of AT₁ receptor antagonists, providing evidence for a role for these receptors in blood pressure control in adults.⁶

In addition to being a vasoconstrictor and pressor hormone, Ang II is thought to play a direct role in the development of vascular hypertrophy and fibrosis and, consequently, of the vascular thickening that is a hallmark of hypertensive and atherosclerotic vascular disease. In regard to this relationship between Ang II and vascular remodeling, previous studies from our and other laboratories have shown that the stimulation of AT₁ receptors causes hypertrophy and increased extracellular matrix synthesis in vascular smooth muscle cells (VSMCs).^{7 8} In contrast, the effects of AT₂ receptor stimulation on extracellular matrix synthesis in the vasculature have not been well defined. In vivo studies have produced only discrepant results, with some groups reporting that AT₂ receptor blockade increases vascular hypertrophy,⁹ whereas other groups have reported that AT₂ receptor blockade inhibits vascular hypertrophy and fibrosis in vivo.^{4 10} It is particularly important to characterize the effects of prolonged AT₂ receptor stimulation on the vasculature at this time in view of the recent increase in the use of AT₁ receptor antagonists for the treatment of hypertension. The use of these agents is associated with a feedback activation of the renin-angiotensin system, which results in increased stimulation of the "unprotected" vascular AT₂ receptors. Thus, an understanding of the effects of AT₂ receptor stimulation on different tissues is increasing in clinical relevance.

Because in vivo experiments have produced confusing results regarding the role of AT₂ receptors in vascular extracellular matrix synthesis, our aim in the present study was to use a direct approach to examine the effects of AT₂ receptor stimulation on collagen synthesis by VSMCs in vitro. We also examined the signal transduction mechanisms involved in the AT₂ receptor–mediated control of collagen synthesis and then compared the effects of AT₂ receptor stimulation on collagen synthesis in 4 different cell types.

	Methods

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VSMC Culture and Transfection of AT₂ Receptor
Rat VSMCs were prepared from the thoracic aortas of 6-week-old male Wistar rats and cultured in DMEM supplemented with 10% FCS.¹¹ For transfection of the AT₂ receptor, a retrovirus construct (pLXSN-AT₂) was constructed through ligation of the rat AT₂ receptor cDNA¹² into the EcoRI-XhoI site of the retroviral vector pLXSN (Stratagene). pLXSN-AT₂ was stably transfected into the packaging cell line GP+E-86 (kindly provided by Dr Arthur Bank, Columbia University, New York, NY) through calcium phosphate transfection followed by G418 selection to produce the AT₂ receptor retrovirus–producing cell line GP+E-86-AT₂. Supernatant from these cells was used to transfect the VSMCs in the presence of Polybrene (4 µg/mL); then, the transfectants were isolated through selection with G418 (400 µg/mL).

Analysis of AT₁ and AT₂ Receptor mRNA and Binding
Expressions of AT₁ and AT₂ receptor mRNA and binding in VSMC-AT₂ cells were assessed with RT-PCR and whole cell receptor binding studies as described in detail previously.^{11 13}

p42/p44 Mitogen-Activated Protein Kinase and Protein Tyrosine Phosphatase Assays
Mitogen-activated protein kinase (MAPK) activity in CGP42112A- or Ang II–treated cells was assessed with the p42/p44 MAPK enzyme assay system (Amersham) as described previously.¹⁴ Protein tyrosine phosphatase (PTP) activity was assessed through dephosphorylation of a PTP-specific synthetic phosphorylated peptide with a commercially available kit (Takara).

Determination of Cell Proliferation
VSMCs on 24-well plates were made quiescent through placement for 48 hours in serum-free DMEM. The cells were treated with CGP42112A (10^-7 mol/L) for 48 hours, and cell counts were determined with a cytometer. In parallel experiments, cells were treated in a similar manner, and then thymidine incorporation was determined on the basis of trichloroacetic acid precipitation.¹¹

Determination of Collagen Synthesis
Synthesis of cell-associated and secreted collagen was determined as reported previously.¹⁴ In brief, quiescent VSMCs on 24-well plates were stimulated with CGP42112A (10^-7 mol/L unless otherwise stated) in the presence of ascorbic acid (50 µg/mL) for the indicated times. [³H]Proline (4 µCi/mL) was added during the last 24 hours of stimulation. De novo collagen synthesis was determined on the basis of the collagenase-sensitive proline incorporation.

Antisense Oligonucleotide Experiments
G_i1 and G_i3 antisense experiments were performed with the primers and protocols described by Wang et al.¹⁵ The sequences of the oligonucleotides were G_i1 sense, 5'-GGCGACGCTCGGCCAC-CATG-3'; G_i1 antisense, 5'-CATGGTGGCCGAGCGTCGCC-3'; G_i3 sense, 5'-CCTCTCCGGCCGCCGTCATG-3'; and G_i3 antisense 5'-CATGACGGCGGCCGGAGAGG-3'. Oligonucleotides (30 µmol/L) were added to the medium 24 hours before stimulation with CGP42112A.

Studies on Mesangial, NIH-3T3, and NRK49F Cells
Mesangial cells from Sprague-Dawley rats were obtained through enzymatic digestion¹⁴ and cultured in RPMI 1640 supplemented with 10% FCS. The kidney interstitial fibroblast cell line NRK-49F was obtained from American Type Culture Collection and cultured in DMEM plus 10% FCS. NIH-3T3 cells stably transfected with the AT₂ receptor¹⁶ were cultured in DMEM plus 10% FCS. The transfection of mesangial and NRK-49F cells with the AT₂ receptor was performed as described for VSMCs.

Materials
CGP42112A was obtained from Research Biochemicals International. Cell culture reagents were obtained from GIBCO BRL. RT-PCR reagents were from Perkin–Elmer Cetus. Radiochemicals were from Amersham. All other chemicals were from Sigma Chemical Co, unless otherwise stated.

Statistical Analysis
Results are expressed as the mean±SEM. Statistical comparisons were made by ANOVA followed by Fisher’s PLSD test for comparison between groups. Values of P<0.05 were considered statistically significant.

	Results

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Establishment of AT₂ Receptor–Expressing VSMCs
As shown in Figure 1, neither AT₂ receptor mRNA nor binding was detectable in wild-type adult VSMCs. However, in VSMC-AT₂ cells that had been transfected with AT₂ receptor retrovirus, both AT₂ receptor mRNA and binding were clearly detectable. Relative levels of AT₁ and AT₂ receptor expression in these cells were 2:1 on binding assay (AT₁ 664±64, AT₂ 280±48 fmol/mg protein).

View larger version (37K): [in this window] [in a new window]   Figure 1. AT1 and AT2 receptor expression in wild-type and AT2 receptor–transfected VSMC. a, Analysis of AT1 and AT2 receptor mRNA with RT-PCR. b, Analysis of AT1 and AT2 receptor binding with receptor binding assay. Data represent mean±SEM (n=4). ND indicates no significant difference from nonspecific binding.

Effects of CGP42112A on MAPK and PTP Activity in VSMC-AT₂
The treatment of VSMC-AT₂ with CGP42112A (10^-7 mol/L) did not cause significant changes in MAPK activity (MAPK activity at 0 minutes 4565±442, 5 minutes 4773±276, 10 minutes 4854±250, 15 minutes 4875±106 cpm/µg protein; n=4). On the other hand, the treatment of VSMC-AT₂ with the AT₂ receptor antagonist PD123319 did cause a significant increase in Ang II–stimulated MAPK activity, as has been reported by other groups¹⁷ (Figure 2a). A small (30% to 50%), dose-dependent decrease in PTP activity was also detected after CGP42112A stimulation in these cells, which was inhibited by pretreatment with the AT₂ receptor antagonist PD123319 but not the AT₁ antagonist losartan (Figures 2b and 2c). Further studies showed that the CGP42112A-induced decrease in PTP activity was not seen when the cells were pretreated with pertussis toxin (PTX) (without PTX: control 42.9±2.1, CGP42112A treated 29.9±4.0*x10⁵ U/µg; with PTX: control 37.4±4.4, CGP42112A treated 38.2±4.3 x10⁵ U/µg; n=4; *P<0.05 versus control).

View larger version (24K): [in this window] [in a new window]   Figure 2. Effects of AT2 receptor stimulation on MAPK and PTP activity in VSMC-AT2. a, Effects of Ang II (10-7 mol/L) on MAPK activity. b, Effects of CGP42112A on PTP activity. c, Effects of AT receptor antagonists on CGP42112A-induced changes in PTP activity. PD indicates cells pretreated with PD123319 (10-6 mol/L); Losartan, cells pretreated with losartan (10-6 mol/L). Results shown are the mean±SEM (n=4 per assay point). *P<0.05 vs control cells.

Effects of CGP42112A on Cell Proliferation in VSMC-AT₂
Treatment of VSMC-AT₂ with CGP42112A caused only small, statistically insignificant changes in cell numbers (from 22 900±900 to 21 600±1400 cells per well, n=4) and thymidine incorporation (from 3294±210 to 3568±106 cpm/well, n=4), suggesting that CGP42112A did not have a major effect on proliferation in these cells.

Effects of CGP42112A on Collagen Synthesis in VSMC-AT₂
In control VSMCs transfected with the vector alone, CGP42112A treatment did not cause any change in collagen synthesis, as shown in Figure 3a. On the other hand, the stimulation of VSMC-AT₂ with CGP42112A caused a significant increase in collagen synthesis that was completely inhibited by the AT₂ receptor antagonist PD123319 and unaffected by the AT₁ receptor antagonist losartan, as shown in Figure 3b, confirming AT₂ receptor–mediated enhancement of collagen synthesis. For comparison, experiments were also performed with the agonist Ang II in the presence of AT₁ and AT₂ receptor antagonists (Figure 3c). These experiments confirmed that an increase in collagen synthesis (1.4-fold) was seen in cells treated with an alternate method of the AT₂ receptor stimulation, namely a combination of Ang II and losartan. Stimulation of the AT₁ receptors alone, with a combination of Ang II and PD123319, caused a 1.7-fold increase in collagen synthesis. Interestingly, the stimulation of both receptors with Ang II in the absence of antagonists caused a 2.6-fold increase in collagen synthesis, suggesting a synergistic effect of the 2 receptor subtypes on collagen synthesis in these cells.

View larger version (19K): [in this window] [in a new window]   Figure 3. Effects of CGP42112A and Ang II on collagen synthesis in VSMC-AT2. Vector-transfected VSMCs (a) or VSMC-AT2 (b) were treated with CGP42112A (10-7 mol/L) for 48 hours before collagen assay. c, VSMC-AT2 were treated with Ang II (10-7 mol/L) for 48 hours before collagen synthesis assay. Results shown are the mean±SEM (n=4 per assay point). *P<0.05, **P<0.01 vs respective groups.

Studies on the time course and dose dependency of the effect of CGP42112A on collagen synthesis showed a dose- and time-dependent effect on both cell-associated and secretory collagen synthesis (Figure 4). The AT₂ receptor–mediated effect on collagen synthesis was unaffected by pretreatment of cells with the tyrosine phosphatase inhibitor okadaic acid or the serine/threonine phosphatase inhibitor sodium orthovanadate. On the other hand, the CGP42112A-mediated enhancement of collagen synthesis was attenuated in cells pretreated with PTX (Figure 4c). To further clarify the role of G_i in the AT₂-mediated effect, antisense experiments were performed with the method of Wang et al.¹⁵ As shown in the Table, the CGP42112A-induced enhancement of collagen synthesis was seen in cells pretreated with G_i1 or G_i3 sense oligonucleotides but not in cells pretreated with the corresponding antisense oligonucleotides.

View larger version (33K): [in this window] [in a new window]   Figure 4. Characterization of CGP42112A-induced increase in collagen synthesis in VSMC-AT2. VSMC-AT2 were treated with (a) CGP42112A (10-7 mol/L) for various times or (b) various doses of CGP42112A for 48 hours. c, Cells were pretreated with the serine/threonine phosphatase inhibitor okadaic acid (10 nmol/L), the tyrosine phosphatase inhibitor sodium orthovanadate (20 µmol/L), or PTX (400 ng/mL) before stimulation with CGP42112A (10-7 mol/L) and collagen synthesis assay. Results shown are the mean±SEM (n=4 per assay point). *P<0.05, **P<0.01 vs control.

View this table: [in this window] [in a new window]   Table 1. Effects of Gi Sense and Antisense Oligonucleotides on CGP42112A-Induced Increase in Collagen Synthesis in VSMC-AT2

Effects of CGP42112A on Collagen Synthesis in Mesangial, NIH-3T3, and NRK-49F Cells
To further examine the effects of CGP42112A on collagen synthesis in other cell lines, AT₂ receptor–expressing mesangial cells and NRK-49F cells were produced. We also used a previously characterized AT₂ receptor–expressing embryonal fibroblast cell line, NIH-3T3-AT₂.¹⁶ Values of AT₂ receptor binding in these cells were 158±6 fmol/mg protein for AT₂ receptor–transfected mesangial cells, 182±5 fmol/mg protein for NRK-49F cells, and 247±10 fmol/mg protein for NIH-3T3-AT₂ cells. The effects of CGP42112A on AT₂ receptor–expressing mesangial cells were essentially identical to the effects on VSMC-AT₂ (Figure 5a). Thus, CGP42112A induced a significant increase in collagen synthesis by mesangial cells that was attenuated by PD123319 but not losartan. On the other hand, the effects on NIH-3T3-AT₂ fibroblasts were markedly different. As shown in Figure 5b, CGP42112A was found to cause a significant decrease in collagen synthesis in these cells, which was attenuated by PD123319. In the case of the fibroblast cell line NRK-49F transfected with the AT₂ receptor, the changes in collagen synthesis did not attain statistical significance (untreated 147±5 cpm/µg protein; CGP42112A treated 133±5 cpm/µg protein, n=4).

View larger version (15K): [in this window] [in a new window]   Figure 5. Effects of CGP42112A on collagen synthesis in mesangial and fibroblast cell lines. AT2 receptor–transfected mesangial cells (a) or fibroblasts (NIH-3T3) (b) were treated with CGP42112A (10-7 mol/L) for 48 hours before collagen synthesis assay. Results shown are the mean±SEM (n=4 per assay point). *P<0.05 vs respective groups.

	Discussion

Top Abstract Introduction Methods Results Discussion References

 
The vasoactive peptide Ang II is known to act on 2 types of receptor: AT₁ and AT₂. The AT₁ receptor is widely expressed in tissues that are known physiological targets for Ang II action, such as the heart, kidneys, adrenal, and brain, as well as the vasculature.

The AT₂ receptor has also been shown to be expressed in adult blood vessels, both in the rat and in humans. In particular, expression of the AT₂ receptor in the small to medium-sized arteries of the kidney and mesenteric vasculature was confirmed by several groups through the use of in situ hybridization, immunohistochemistry, and binding assays.^{3 4 5} Physiological studies have suggested that these vascular AT₂ receptors may be involved in vasodilation and control of regional hemodynamics,¹⁸ as well as blood pressure control.⁶

Although the AT₂ receptor is also a member of the superfamily of G protein–coupled heptahelical receptors, its signal transduction appears to be quite different from that of other receptors of this family. Interestingly, several studies have shown that the actions of the AT₂ receptor can be abolished with PTX pretreatment, suggesting that the receptor is coupled to G_i.¹ Further support for AT₂ receptor–G_i coupling is provided in studies that show the intracellular application of anti-G_i antibodies can attenuate AT₂ receptor–induced activation of potassium¹⁹ as well as T-type calcium currents.²⁰ Further downstream, AT₂ receptor stimulation has been shown to modulate PTP activity (for a review, see Inagami¹ ).

In terms of the effects of Ang II on extracellular matrix, previous studies from our and other laboratories have shown that Ang II causes hypertrophy as well as increased collagen synthesis in VSMC via an AT₁ receptor–mediated mechanism.^{7 8} It is puzzling that the AT₂ receptor has been suggested to inhibit vascular hypertrophy in vivo by some investigators⁹ and to enhance vascular hypertrophy and collagen accumulation by others.^{4 10} These findings were based on experiments conducted with the AT₂ receptor antagonist PD123319 in vivo.

In the present study, we designed an in vitro strategy to examine the direct effects of AT₂ stimulation on collagen synthesis in VSMCs. To circumvent problems caused by potential incomplete inhibition of receptors at low concentrations of losartan and PD123319 on the one hand and by potential cross-inhibition of AT receptor subtypes at high concentrations on the other, we stimulated our cells with the AT₂-specific ligand CGP42112A. The fact that simultaneous stimulation with Ang II and losartan produced the same effect as CGP42112A suggested that the latter acted as an agonist, as reported previously.¹⁶ This was underscored in the present study by the fact that the AT₂ receptor antagonist PD123319 inhibited the CGP42112A-induced effects in our cells.

Although AT₂ receptors have been reported to be expressed in the vasculature of adult rats,⁵ their expression was undetectable with RT-PCR in cultured VSMCs, presumably because AT₂ receptors are easily lost after subculturing.²¹ We therefore supplemented the VSMCs with AT₂ receptors through gene transfer to mimic the situation of the vasculature in vivo. Previous studies have shown that pretreatment with AT₂ receptor antagonist PD123319 can enhance AT₁ receptor–stimulated MAPK activation,¹⁷ and we noted the same phenomenon in the present study with these cells. AT₂ receptor stimulation has been reported to be able to cause both an increase and a decrease in PTP activity¹ ; in the present study, we detected a small decrease in PTP activity after CGP42112A stimulation.

In the VSMC-AT₂ cells, stimulation of the AT₂ receptor caused a dose- and time-dependent increase in collagen synthesis. The mechanism was different from AT₁ receptor–induced enhancement of collagen synthesis, which has been shown to involve p42/p44 MAPK, because no significant change in p42/p44 MAPK activity was seen after CGP42112A stimulation. We found that the AT₂ receptor–mediated enhancement of collagen synthesis was attenuated by PTX treatment, suggesting the involvement of G_i in the AT₂-induced effects. Moreover, pretreatment of cells with anti-G_i antisense oligonucleotides provided similar results in response to PTX pretreatment, further supporting a role for G_i in the AT₂ receptor–mediated effect on collagen synthesis. PTX treatment was also effective in attenuating the CGP42112A-induced decrease in PTP activity that we noted in these cells. Interestingly, the experiments that compared the effects of AT₁ and AT₂ receptor stimulation suggested a synergistic effect of the 2 receptor subtypes on collagen synthesis in these cells. These data point to the need for further studies to clarify the intracellular signaling events downstream of G_i, which may be involved in the observed effects of the AT₂ receptor on collagen synthesis.

Recently, the effect of the AT₂ receptor on extracellular matrix synthesis in a nonvascular tissue was studied by another group. Ohkubo et al²² examined cardiac fibrosis in myopathic hamsters and found clear evidence that AT₂ receptor stimulation is associated with decreased collagen synthesis in the cardiac fibroblasts of these rats. To reconcile the conflicting reports concerning the effects of AT₂ receptor stimulation on extracellular matrix synthesis in vivo, we went on to examine the effects of AT₂ receptor stimulation in different cell types. In the case of mesangial cells, we found similar results to those with VSMCs. However, in fibroblasts, our results were markedly different from the results in VSMCs and mesangial cells and revealed tissue specificity in the actions of the AT₂ receptor on extracellular matrix synthesis. One caveat in the interpretation of these results is that the data reflect the situation in vitro with an "artificial" overexpression system. However, the fact that our in vitro results are in good agreement with in vivo reports that PD123319 decreased fibrosis in the rat vasculature and increased fibrosis in the hearts of cardiomyopathic hamsters^{4 10 22} suggests that our in vitro results are relevant to the in vivo situation.

What are the implications of the findings of the present study? First, as noted in the introduction, an increasing number of patients are starting on AT₁ receptor antagonists for the treatment of hypertension. The feedback activation of the renin-angiotensin system results in enhanced AT₂ receptor stimulation. The results of the present study suggest that this could have multiple effects on remodeling in different tissues.

Second, the results of the study have suggested the existence of an AT₂ receptor–induced and G_i-mediated pathway for the control of vascular collagen synthesis. Increased extracellular matrix synthesis plays an important role in the progression not only of hypertensive and atherosclerotic vascular disease but also of heart disease. Specific examples include myocardial scarring after myocardial infarction, cardiomyopathy associated with connective tissue diseases such as systemic sclerosis, and other causes of restrictive cardiomyopathy. Therefore, further study of the mechanisms involved may help to design new strategies to control the pathological accumulation of extracellular matrix in disease states.

Third, AT₂ receptor stimulation was found to cause apparently opposite effects on collagen synthesis in different tissues. To our knowledge, this is the first description of bidirectional control of extracellular matrix synthesis by a G protein–coupled receptor. At the present, we do not know the reason for the striking tissue variability in the effect of the AT₂ receptor, but we speculate that AT₂ receptor–mediated signal transduction may be strongly modulated by interaction with other tissue-specific factors. In conclusion, our results provide strong evidence that further study of the AT₂ receptor will provide us with a unique opportunity to understand previously unrecognized facets of the control of collagen synthesis in different tissues.

	Acknowledgments

 
This work was supported by grants from the Ministry of Education, Science, and Culture of Japan and a Special Grant-in-Aid for Innovative Collaborative Research Projects, Keio University, Japan.

Received February 28, 2000; first decision March 16, 2000; accepted May 10, 2000.

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