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(Arteriosclerosis, Thrombosis, and Vascular Biology. 1999;19:1210-1217.)
© 1999 American Heart Association, Inc.

Vascular Biology

Production of Angiotensin II by Homogeneous Cultures of Vascular Smooth Muscle Cells From Spontaneously Hypertensive Rats

Noboru Fukuda; Chikara Satoh; Wen-Yang Hu; Masayoshi Soma; Atsushi Kubo; Hirobumi Kishioka; Yoshiyasu Watanabe; Yoichi Izumi; Katsuo Kanmatsuse

From the Second Department of Internal Medicine, Nihon University School of Medicine, Tokyo 173, Japan.

Correspondence and reprint requests to Noboru Fukuda, MD, PhD, Second Department of Internal Medicine, Nihon University School of Medicine, Ooyaguchi-kami 30-1, Itabashi-ku, Tokyo 173-8610, Japan.

	Abstract

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Abstract—Production of angiotensin II (Ang II) in spontaneously hypertensive rats (SHR)-derived vascular smooth muscle cells (VSMC) has now been investigated. A nonpeptide antagonist (CV-11974) of Ang II type 1 receptors inhibited basal DNA synthesis in VSMC from SHR, but it had no effect on cells from Wistar-Kyoto (WKY) rats. Ang II-like immunoreactivity, determined by radioimmunoassay after HPLC, was readily detected in conditioned medium and extracts of SHR-derived VSMC, whereas it was virtually undetectable in VSMC from WKY rats. Isoproterenol increased the amount of Ang II-like immunoreactivity in conditioned medium and extracts of SHR-derived VSMC, whereas the angiotensin-converting enzyme inhibitor delapril significantly reduced the amount of Ang II-like immunoreactivity in conditioned medium and extracts of these cells. Reverse transcription-polymerase chain reaction analysis revealed that the abundance of mRNAs encoding angiotensinogen, cathepsin D, and angiotensin-converting enzyme was greater in VSMC from SHR than in cells from WKY rats. The abundance of cathepsin D protein by Western blotting was greater in VSMC from SHR than in cells from WKY rats. Ang I-generating and acid protease activities were detected in VSMC from SHR, but not in cells from WKY rats. These results suggest that SHR-derived VSMC generate Ang II with increases in angiotensinogen, cathepsin D, and angiotensin-converting enzyme, which contribute to the basal growth. Production of Ang II by homogeneous cultures of VSMC is considered as a new mechanism of hypertensive vascular disease.

Key Words: angiotensin II • vascular smooth muscle • spontaneously hypertensive rats • cathepsin D • reverse transcription-polymerase chain reaction

	Introduction

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Increased growth of components of the cardiovascular system is apparent in individuals with essential hypertension¹ as well as in spontaneously hypertensive rats (SHR).² Cultured vascular smooth muscle cells (VSMC) from normotensive Wistar-Kyoto (WKY) rats and SHR exhibit distinct growth phenotypes, with SHR-derived cells showing a higher specific growth rate, abnormal contact inhibition, and an accelerated entry into the S phase of the cell cycle.³ SHR-derived VSMC also show a nonspecific hyperproliferation response to various growth factors, including calf serum, epidermal growth factor, and platelet-derived growth factor (PDGF).⁴ These observations indicate the presence of intrinsic abnormalities in VSMC from SHR. Transforming growth factor-ß1 (TGF-ß1)⁵ and PDGF A-chain⁶ mRNAs accumulate to a greater extent in VSMC from SHR compared with cells from WKY rats. We have previously demonstrated roles of TGF-ß1⁷ and PDGF A-chain⁸ in the exaggerated growth of SHR-derived VSMC by showing that antisense DNA complementary to the corresponding mRNAs inhibits the growth of these cells. Both TGF-ß and PDGF A-chain mRNAs are induced by angiotensin II (Ang II).^{9 10}

The renin-angiotensin (RA) system has been implicated in essential hypertension because of the antihypertensive effects of angiotensin-converting enzyme (ACE) inhibitors. Several lines of evidence have demonstrated the presence of a local tissue RA system that is independent of the circulating RA system.^{11 12} The tissue RA system has been detected in brain, heart, kidney,¹¹ and the vascular wall.¹² The RA system in the vascular wall acts not only to regulate vessel tone and blood flow, but also to induce vascular growth.¹³ The vascular wall contains renin, ACE, angiotensinogen, Ang I, and Ang II as components of the tissue RA system.^{11 12} Vessel walls are composed of endothelium, vascular smooth muscle, and extracellular matrix, and the smooth muscle contains renin, angiotensinogen, the Ang II receptors, and a small amount of ACE.¹⁴ Both Ang II type 1 (AT1) and type 2 receptors have been cloned,^{15 16 17} but VSMC express only the AT1 receptor,¹⁷ through which the growth-promoting effect of Ang II in these cells is mediated.¹⁸

The amounts of both renin¹⁹ and Ang II²⁰ in the vascular wall are increased in SHR compared with those in WKY rats. In addition, blood pressure in SHR is correlated with the amount of renin in the arterial wall but not with the plasma renin activity.²¹ Thus increased activity of the RA system in the vascular wall might be associated with hypertension in SHR. We have recently shown that the nonpeptide AT1 receptor antagonist CV-11974 as well as the ACE inhibitor delapril reduced basal DNA synthesis (in the absence of serum) in VSMC from SHR, but not in VSMC from WKY rats.²² These observations suggest that VSMC from SHR may generate Ang II, which promotes their basal growth in an autocrine-paracrine manner by acting at the AT1 receptor.

We have now investigated whether SHR-derived VSMC possess an Ang II-generating system and actually produce Ang II in a homogeneous cell culture system.

	Methods

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Cell Culture
VSMC were obtained by an explant method²³ from aortas of 10-week-old male SHR/Izm and WKY/Izm rats (SHR Corporation, Funabashi, Chiba, Japan), as described previously.³ They were seeded and grown in Dulbecco's modified Eagle's medium (DMEM) with 10% calf serum (Gibco Life Technologies), penicillin (100 U/mL), and streptomycin (100 mg/mL). The cells achieved confluence after 7 to 10 days, at which time we confirmed the hill-and-valley pattern characteristic of smooth muscle cells in culture, and immunofluorescence of -smooth muscle–specific actin using a monoclonal antibody specific for the VSMC -actin isoform (DAKO A/S) as previously described.²⁴ The cultures were uniformly composed of cells that stain positive for the presence of smooth muscle–specific -actin in VSMC from WKY rats (Figure 1A) and SHR (Figure 1B). They were passaged by trypsinization with 0.05% trypsin (Gibco) in Ca²⁺- and Mg²⁺-free Dulbecco's PBS and incubated in 80-cm² tissue culture flasks at a density of 10⁵ cells/mL. Experiments were performed after 3 to 5 passages.

View larger version (78K): [in this window] [in a new window]   Figure 1. Immunofluorescence demonstration of the expression of smooth muscle–specific -actin in culture of VSMC from WKY rats (A) and SHR (B). Third passaged VSMC were permeabilized and fixed in absolute methanol. Cells were then stained by indirect immunofluorescence using monoclonal antibody against the smooth muscle–specific isoform of -actin. Bar indicates 50 µm.

Quiescence was established by transferring cells to 10- or 2-cm² wells culture plates, and incubating first for 24 hours in DMEM containing 10% calf serum and then for 48 to 72 hours in DMEM with 0.2% calf serum.

Determination of DNA Synthesis
[³H]Thymidine incorporation into newly synthesized DNA was assayed as described previously.²⁵ Briefly, quiescent VSMC in 2-cm² wells were incubated for 24 hours in DMEM containing various concentrations of CV-11974 in the absence or presence of 10% calf serum. The medium was then changed to DMEM containing 0.5 mCi/mL of [methyl-³H]thymidine (specific activity, 20 Ci/mmol) (NEN), and cells were incubated for an additional 2 hours. Each well was then washed with 1 mL of 150 mmol/L NaCl, and the cells were fixed with 1 mL of ethanol and acetic acid (3:1, vol/vol) for 10 minutes. After washing the cells with 1 mL of H₂O, acid-insoluble material was precipitated with 1 mL of ice-cold perchloric acid, and DNA was then extracted into 1.5 mL of perchloric acid by heating at 90°C for 20 minutes. The solubilized DNA was transferred to a scintillation vial, and the associated radioactivity was measured with a liquid scintillation spectrometer.

Preparation of Conditioned Medium and Cell Extracts
VSMC (10⁶) from SHR and WKY rats were inoculated in 10-cm² wells with DMEM containing 10% calf serum and incubated for 24 hours. The cells were then washed twice with PBS and incubated first for 24 hours with DMEM without serum and then for 48 hours with 10 mL of fresh serum-free DMEM in the absence or presence of various concentrations of isoproterenol or delapril. The culture medium was then collected and centrifuged at 600g for 10 minutes, and the resulting supernatant (conditioned medium) was collected. The cells were washed twice with PBS, mechanically scraped into 10 mL of PBS, and disrupted by sonication (Handy Sonic, Tomy Seiko). The cell homogenates were centrifuged at 10 000g for 15 minutes, and the resulting supernatants (cell extracts) were collected. The conditioned medium and cell extracts were treated with 1 mmol/L each of aprotinin, leupeptin, and pepstatin A as well as 0.1 mmol/L phenylmethylsulfonyl fluoride (PMSF), and stored at -80°C until analysis for Ang II by radioimmunoassay (RIA).

Measurement of Angiotensin Peptides
Determination of angiotensin peptides in collected conditioned medium and cell extracts was performed as described previously.²⁶ Samples were applied to a Sep-Pak C18 cartridge (Waters Associates), and peptides were eluted with 3 mL of methanol-water-trifluoroacetic acid (80:19.9:0.1, vol/vol). The eluate was dried in a vacuum centrifuge, and angiotensin peptides were then separated by reversed-phase HPLC (RP-HPLC). Samples were loaded on a Shodex column (OPD-50; Showa Denko), and peptides were eluted with an exponential gradient of acetonitrile from 20% to 50% (vol/vol) in 0.05% trifluoroacetic acid at a flow rate 1 mL/min. Fractions (0.5 mL) were collected and dried in a vacuum centrifuge, and the residues were redissolved in 0.1 mol/L Tris-HCl (pH 7.4), and subjected to RIA for Ang I and Ang II.

The antiserum to Ang II (Amersham) showed <1% cross-reactivity with Ang I but 100% cross-reactivity with Ang III (heptapeptide), Ang II(3–8) (hexapeptide), and Ang II(4–8) (pentapeptide). The sensitivity of detection of Ang I and Ang II was 1 fmol per tube. The recoveries of Ang I and Ang II (70% and 80%, respectively) were monitored by the addition of [³H]Ang I or [³H]Ang II (Amersham) to conditioned medium and cell extracts.

Reverse Transcription-Polymerase Chain Reaction Analysis of mRNAs Encoding Angiotensinogen, Renin, Tonin, Kallikrein, Cathepsin D, Cathepsin E, or ACE
Quiescent VSMC at a density of 10⁵ cells/cm² in 2-cm² wells were washed with PBS and lysed in 800 mL of RNAzol B (Biotecx). Cell lysates were mixed with 80 mL of chloroform, incubated at 4°C for 15 minutes, and centrifuged at 12 000g for 15 minutes to extract total RNA. A portion (300 mL) of each aqueous phase was mixed with an equal volume of isopropanol, incubated at -20°C for 45 minutes, and centrifuged at 12 000g for 15 minutes at 4°C to precipitate the RNA. The RNA pellet was washed twice with 500 mL of 75% (vol/vol) ethanol by vortex mixing and centrifugation at 7500g for 8 minutes at 4°C, and then dried and dissolved in 10 mL of a solution containing 10 mmol/L Tris-HCl (pH 8.0) and 1 mmol/L EDTA for incubation for 15 minutes at 65°C. Each sample was then treated with 0.5 U of DNase (Gibco) in 0.5 mL of DNase buffer (20 mmol/L Tris-HCl [pH 8.3], 50 mmol/L KCl, and 2.5 mmol/L MgCl₂) at room temperature for 45 minutes, after which the DNase was inactivated by adding 0.5 mL of 20 mmol/L EDTA and heating at 98°C for 10 minutes.

Reverse transcription-polymerase chain reaction (RT-PCR) was performed as described previously.²⁷ Briefly, aliquots of RNA (1 mg/20 mL) were reverse-transcribed into single-stranded cDNA by incubation for 10 minutes at 30°C, 30 minutes at 42°C, and 5 minutes at 99°C in a final volume of 20 mL containing 5 U of avian myeloblastoma virus reverse transcriptase (Life Sciences), 10 mmol/L Tris-HCl (pH 8.3), 5 mmol/L MgCl₂, 50 mmol/L KCl, 1 mmol/L of each deoxynucleotide triphosphate, and 2.5 mmol/L random hexamer. The diluted cDNA products (5 mL) were then subjected to PCR in a final volume of 25 mL containing 10 mmol/L Tris-HCl (pH 8.3), 50 mmol/L KCl, 4 mmol/L MgCl₂, 0.625 U of Taq DNA polymerase (Takara), and 0.2 mmol/L each of upstream sense and downstream antisense primers. The primers used for PCR for angiotensinogen,²⁸ renin,²⁹ tonin,³⁰ kallikrein,³⁰ cathepsin D,³¹ cathepsin E,³² and ACE³³ were as previously described and are shown in Table 1. Rat ribosomal protein L19 mRNA was used as an internal control.³⁴ Because exons 3 and 4 of rat preprorenin cDNA have common sequences with cathepsin D and E cDNAs, the specific primers for amplification of renin cDNA were targeted to exons 7 and 9. Similarly, because exons 3 to 5 of rat tonin cDNA have common sequences with kallikrein cDNA, the specific primers for amplification of tonin cDNA were targeted to exons 2 and 3. PCR was performed in a DNA Thermal Cycler (Perkin-Elmer). Because the amount of PCR product corresponding to each of the target mRNAs increased in a linear manner from 20 to 35 cycles, PCR was performed for 30 cycles according to the profiles shown in Table 2. PCR products were separated by electrophoresis through 1.5% agarose gels and stained with ethidium bromide.

View this table: [in this window] [in a new window]   Table 1. PCR Primers and Sizes of Specific PCR Products

View this table: [in this window] [in a new window]   Table 2. Thermal Cycle Profiles for PCR

Western Blot Analysis for Cathepsin D Protein in VSMC
Quiescent VSMC at a density of 10⁵ cells/cm² in 10-cm² wells were washed with PBS and lysed in lysis buffer (50 mmol/L Tris-HCl [pH 8.0], 150 mmol/L NaCl, 0.02% sodium azide, 100 mg/mL PMSF, 1 mg/mL aprotinin, 1% Triton X). After centrifugation the protein concentration in the supernatant was determined by the method of Lowry et al.³⁵ Western blot analysis was performed on 5 mg of protein with 20 mL of sample buffer. The samples were boiled and subjected to 10% polyacrylamide gel electrophoresis. The proteins were transferred to a nitrocellulose membrane and incubated with the mouse monoclonal antibody specific for cathepsin D (Oncogene Research Products) diluted 100 vol in 5% nonfat milk in 10 mmol/L Tris-HCl (pH 8.0) with 150 mmol/L NaCl and 0.05% Tween 20 (TBST) or the mouse monoclonal antibody specific for -tubulin as a control (Sigma BioScience) diluted 500 vol in 5% nonfat milk in TBST overnight at 4°C. The membrane was incubated with the goat anti-mouse IgG (Bio-Rad Laboratories) diluted 3000 vol in 10 mmol/L NaH₂PO₄ in saline for 1 hour at room temperature, then washed with TBST 3x5 minutes. Immune complexes on the membrane were detected with protein A-gold (Bio-Rad Laboratories) as described previously.³⁶ The membrane was incubated with protein A-gold in 4 volumes of protein gold buffer (20 mmol/L Tris-HCl, pH 8.1, 150 mmol/L NaCl, 0.1% BSA, 0.1% Tween 20, and 0.02% sodium azide) for 1 hour at 20°C. Subsequently the membrane was washed 1x10 minutes with protein A-gold buffer, 1% Triton X-100, 2x5 minutes with protein gold buffer, 2x1 minute with double-distilled H₂O to remove chloride ions, and once with citrate buffer (0.2 mol/L citric acid, 0.2 mol/L sodium citrate, pH 3.7). The membrane then was colored by silver lactate dissolved in 20 mL of hydroquinone solution (170 mg of hydroquinone in 20 mL of citrate buffer).

Measurement of Ang I-Generating Activity
Conditioned medium (1 mL) from 2x10⁵ VSMC from WKY rats and SHR in 10-cm² wells that had been exposed to only serum-free DMEM was incubated without or with 1 mmol/L renin inhibitor or 1 mmol/L pepstatin A in the absence or presence of angiotensinogen (50 ng/mL) at 37°C for 2 hours. Ang I in each sample was separated with a Sep-Pak C18 cartridge and RP-HPLC, and measured by RIA with Ang I antiserum as described above. Ang I-generating activity was calculated as picograms of Ang I with angiotensinogen minus picograms of Ang I without angiotensinogen per hour.

Measurement of Acid Protease Activity
Quiescent VSMC (10⁶) from WKY rats and SHR in 10-cm² wells were washed several times with PBS, and then scraped and collected into tubes containing PBS. The cells were centrifuged for 10 minutes at 12 000g and resuspended in 1 mL of 50 mmol/L Tris-HCl (pH 7.4) containing 10 mmol/L mercaptoethanol. After sonication for four 15-s bursts, the protein concentration of the cell lysate was measured by the method of Lowry et al.³⁵ The lysate was centrifuged for 10 minutes at 12 000g, and the resulting supernatant was assayed for acid protease activity as described by Afting et al.³⁷ Briefly, 60 mL of the cell extracts in the absence or presence of 1 mmol/L pepstatin A (an inhibitor of aspartyl proteases) was incubated at 37°C for 30 minutes, with 80 mL of borate buffer (50 mmol/L Na₂B₄O₇ [pH 8.6] and 1 mol/L NaCl) containing hemoglobin (10 mg/mL) as substrate. The reaction was stopped by the addition of 100 mL of 10% (wt/vol) trichloroacetic acid. After 15 minutes on ice, each sample was centrifuged at 1200g for 3 minutes, and 100 mL of the resulting supernatant was neutralized with 50 mL of 1 mol/L NaOH and assayed for protein according to Lowry et al.³⁵ Absorbance at 595 nm was plotted as a function of time to confirm the linearity of the assay for each sample. One unit of acid protease activity was defined as the amount of enzyme that caused a rate of change in 0.1 absorbance unit per 60 minutes under the incubation conditions described.

Compounds
CV-11974 and delapril were purchased from Takeda Pharmaceutical; aprotinin, pepstatin A, renin-specific inhibitor (Na-Cbz-Arg-Pro-Phe-His-Sta-Ile-His-Ne-Boc-Lys methyl ester), PMSF, and hemoglobin were from Sigma; and leupeptin, angiotensinogen, Ang I, Ang II, and Ang III were purchased from Peptide Institute.

Statistical Analysis
Values are given as means±SEM. The level of significance for difference between means was evaluated by Student's t test for unpaired data, or by 2-way ANOVA followed by Duncan's multiple range test.

	Results

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Effect of CV-11974 on VSMC Growth
Because our previous demonstration of the inhibitory effect of CV-11974 on the growth of VSMC from WKY rats and SHR was for animals obtained from Charles River Japan,²² we examined the effect of increasing concentrations of CV-11974 on basal and stimulated DNA synthesis in VSMC from WKY/Izm rats and SHR/Izm. In the absence of serum, 10^-5 mol/L CV-11974 significantly (P<0.05) inhibited DNA synthesis in VSMC from SHR by 27%, but the drug had no effect on DNA synthesis in VSMC from WKY rats (Figure 2A). Incubation of VSMC from both SHR and WKY rats with 10% calf serum resulted in a marked increase in DNA synthesis, the effect being greater for SHR cells. However, CV-11974 did not significantly inhibit serum-induced DNA synthesis in cells from either rat strain (Figure 2B).

View larger version (14K): [in this window] [in a new window]   Figure 2. Effect of CV-11974 on basal (A) and serum-stimulated (B) [3H]thymidine incorporation into DNA of VSMC from SHR () and WKY rats (). Quiescent cells were incubated for 24 hours in the absence (A) or presence (B) of 10% calf serum with the indicated concentrations of CV-11974 before determination of [3H]thymidine incorporation. Data are means±SEM (n=4). * indicates P<0.05 vs basal values by 2-way ANOVA followed by Duncan's multiple range test.

Detection of Ang II-Like Immunoreactivity in VSMC
Figure 3A shows separation of a mixture of synthetic Ang I, Ang II, and Ang III by RP-HPLC as detected by absorbance at 214 nm. Ang II-like immunoreactivity (Ang II-LI) was detected by RIA in the conditioned medium and extracts of VSMC from SHR at a position in the HPLC profile corresponding to that of synthetic Ang II (Figure 3B). The serial dilution curves obtained with Ang II-LI in the conditioned medium and cell extracts of VSMC from SHR were parallel to the standard curve obtained with synthetic Ang II (Figure 3C).

View larger version (12K): [in this window] [in a new window]   Figure 3. A, Separation of a mixture of synthetic Ang I, Ang II, and Ang III by RP-HPLC. B, Representative HPLC profile of Ang II-LI determined by RIA in conditioned medium () and extracts () of VSMC from SHR. C, Typical RIA standard curve of Ang II () and dilution curves for conditioned medium () and cell extracts () of VSMC from SHR. B/BO indicates % radioactivity bound to antibody.

The amounts of Ang II-LI in conditioned medium and extracts of VSMC were significantly (P<0.01) greater for SHR than for WKY rats (Table 3). Exposure to 10^-5 mol/L isoproterenol significantly increased the amounts of Ang II-LI in the conditioned medium (P<0.05) (about twofold) and cell extracts (P<0.01) (about fivefold) of VSMC from SHR, but it had no such effect on VSMC from WKY rats. Addition of 10^-5 mol/L delapril significantly (P<0.05) reduced the amount of Ang II-LI in the conditioned medium and cell extracts of VSMC from SHR. Delapril had no effect on the amount of Ang II-LI in the conditioned medium or extracts of VSMC from WKY rats (Table 3).

View this table: [in this window] [in a new window]   Table 3. Effect of Isoproterenol and Delapril on Amounts of Ang II-LI in Conditioned Medium and Extracts of VSMC From WKY Rats and SHR

Detection of Angiotensinogen mRNA in VSMC
VSMC from WKY rats and SHR were incubated for 20 hours in the absence or presence of various concentrations (10^-9 to 10^-5 mol/L) of isoproterenol and then assayed for angiotensinogen mRNA by RT-PCR (Figure 4). Angiotensinogen mRNA was detected in VSMC from WKY rats only after incubation with 10^-5 mol/L isoproterenol. In contrast, although angiotensinogen mRNA was detected in SHR-derived VSMC incubated in the absence of isoproterenol, its abundance increased after exposure to this agent, with the maximal effect apparent at 10^-7 mol/L. These data were representative of 3 separate experiments.

View larger version (21K): [in this window] [in a new window]   Figure 4. RT-PCR analysis of angiotensinogen mRNA in VSMC from WKY rats and SHR. Quiescent VSMC were incubated for 20 hours in the presence of the indicated concentrations of isoproterenol, after which angiotensinogen and ribosomal protein L19 (internal control) mRNAs were analyzed by RT-PCR.

RT-PCR Analysis of mRNAs Encoding Renin, Tonin, Kallikrein, Cathepsin D, Cathepsin E, or ACE in VSMC
Transcripts encoding renin, tonin, kallikrein, or cathepsin E were not detected by RT-PCR in VSMC from either SHR or WKY rats (data not shown). In contrast, VSMC from both rat strains contained cathepsin D mRNA, the abundance of which was significantly (P<0.05) greater in cells from SHR than in those from WKY rats (Figure 5). The abundance of mRNA encoding ACE (Figure 6) was also significantly (P<0.01) greater in VSMC from SHR than in cells from WKY rats.

View larger version (27K): [in this window] [in a new window]   Figure 5. Abundance of cathepsin D mRNA in VSMC from WKY rats and SHR. A, Cathepsin D and ribosomal protein L19 mRNAs were analyzed by RT-PCR. The position of molecular size standards (in base pairs) are indicated (lane M). B, The ratio of the abundance of cathepsin D mRNA to that of L19 mRNA was evaluated by densitometric analysis. Data are means+SEM from 4 experiments. * indicates P<0.05 vs WKY rats.

View larger version (34K): [in this window] [in a new window]   Figure 6. Abundance of ACE mRNA in VSMC from WKY rats and SHR. A, ACE and ribosomal protein L19 mRNAs were analyzed by RT-PCR. The position of molecular size standards (in base pairs) are indicated (lane M). B, The ratio of the abundance of ACE mRNA to that of L19 mRNA was evaluated by densitometric analysis. Data are means+SEM of values from 4 experiments. * indicates P<0.01 vs WKY rats.

Expression of Cathepsin D Protein in VSMC
Western blot analysis detected apparent cathepsin D protein with a molecular weight of 48 kDa in VSMC from WKY rats and SHR. The abundance of cathepsin D protein was significantly (P<0.01) greater in VSMC from SHR than in cells from WKY rats (Figure 7).

View larger version (26K): [in this window] [in a new window]   Figure 7. Abundance of cathepsin D protein in VSMC from WKY rats and SHR. A, Cathepsin D protein or -tubulin protein in quiescence VSMC from WKY rats and SHR were analyzed by Western blotting. The positions of molecular weight markers are indicated. The arrow indicates the 48-kDa cathepsin D protein or 55-kDa -tubulin protein as an internal control. B, The ratio of the abundance of cathepsin D protein to that of -tubulin protein was evaluated by densitometric analysis. Data are means+SEM from 6 experiments. * indicates P<0.01 vs WKY rats.

Ang I-Generating and Acid Protease Activities in VSMC
We also measured Ang I-generating activity and acid protease (including cathepsin D) activity in VSMC from WKY rats and SHR. Ang I-generating activity was significantly (P<0.01) greater in the conditioned medium of VSMC from SHR than in the conditioned medium of VSMC from WKY rats. Renin inhibitor did not affect Ang I-generating activity in VSMC from SHR. An aspartyl protease inhibitor, pepstatin A, significantly (P<0.01) inhibited Ang I-generating activity in VSMC from SHR (Table 4). Acid protease activity in VSMC from SHR was markedly (P<0.01) greater than that in cells from WKY rats, and was significantly (P<0.01) inhibited by pepstatin A (Figure 8).

View this table: [in this window] [in a new window]   Table 4. Ang I-Generating Activity in VSMC From WKY Rats and SHR

View larger version (12K): [in this window] [in a new window]   Figure 8. Acid protease activity in VSMC from WKY rats and SHR. VSMC extracts were incubated with hemoglobin without (open columns) or with (closed columns) of 1 mmol/L pepstatin A for 30 minutes. Data are means+SEM of values from 4 experiments. * indicates P<0.01 for comparisons between paired columns; ND, not detectable.

	Discussion

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Previous studies have indicated that 3T3 fibroblasts,³⁸ leukocytes,³⁹ neuroblastoma cells,⁴⁰ Leydig cells,⁴¹ and cardiac valvular interstitial cells³¹ synthesize Ang II. Morishita et al⁴² showed that transfection of VSMC with ACE or renin cDNA resulted in an increase in DNA and RNA synthesis that was inhibited by an AT1 receptor antagonist, suggesting that synthesis of Ang II by these cells regulated their growth. However, this report is not concerned with Ang II synthesis by homogeneous cultures of VSMC. We have now detected Ang II in conditioned medium and extracts of SHR-derived VSMC, which were grown under serum-free conditions to prevent the uptake of RA system components, and which were in their third to fifth passage to prevent contamination with other cell types. Thus we have shown that SHR-derived VSMC synthesize Ang II de novo. As the Ang II-generating system, the abundance of transcripts encoding angiotensinogen, cathepsin D, or ACE was significantly greater in VSMC from SHR than in those from WKY rats, and the corresponding proteins are thus likely responsible for the increased Ang II synthesis by VSMC from SHR. Katwa et al³¹ recently showed that generation of Ang II by cardiac valvular interstitial cells is associated with the presence of angiotensinogen and cathepsin D mRNAs and proteins, whereas renin mRNA was not detected.

Expression of angiotensinogen in hepatocytes is induced by ß-adrenergic agonists, glucocorticoids, or interleukin-6.^{43 44} Isoproterenol-induced accumulation of angiotensinogen mRNA in mouse hepatoma cells is mediated by cAMP response element in the promoter of the angiotensinogen gene.⁴⁵ Rat VSMC have previously been shown to produce angiotensinogen.⁴⁶ In the present study, the abundance of angiotensinogen mRNA was increased by isoproterenol in VSMC from both SHR and WKY rats. However, the mechanism responsible for the increased amount of this mRNA species in VSMC from SHR compared with that in cells from WKY rats cells is not clear.

Angiotensinogen is converted to Ang I by proteases, such as renin, cathepsin D, cathepsin E, and pepsin,⁴⁷ and directly to Ang II by tonin, kallikrein, and cathepsin G.⁴⁸ Gohlke et al⁴⁹ concluded that renin present in the blood vessel wall is derived from circulating renin produced in the kidney. With the use of Northern blot analysis, Holycross et al³² detected cathepsin D and cathepsin E mRNAs, but not renin mRNA, in the medial layer of the thoracic aorta from Sprague-Dawley rats. These researchers also showed that renin mRNA was amplified less consistently from the aortic cDNA by RT-PCR, and, when present, was markedly less abundant than cathepsin D or cathepsin E mRNAs, suggesting that cathepsin D and cathepsin E are the predominant aspartyl proteases in vascular tissue. In the present study, we did not detect mRNAs encoding renin, tonin, kallikrein, or cathepsin E by RT-PCR in VSMC from SHR or WKY rats. In contrast, cathepsin D mRNA was apparent in VSMC from both rat strains and was present in significantly greater amounts in cells from SHR than in those from WKY rats. Western blotting also revealed an apparent expression of cathepsin D protein in VSMC from SHR, but it was faint in cells from WKY rats. Cathepsin D is a lysosomal protease that is constitutively produced in a wide variety of tissues and blood cells.⁵⁰ Although we did not detect renin mRNA in SHR-derived VSMC, Ang I-generating activity was apparent in the conditioned medium of these cells, which was inhibited by pepstatin A, an aspartyl protease inhibitor, but not by renin inhibitor. In addition, acid protease activity (including the activity of cathepsin D) was markedly higher in VSMC from SHR than in cells from WKY rats, and it was inhibited by pepstatin A. These results indicate that cathepsin D mediates the conversion of angiotensinogen to Ang I in VSMC from SHR.

The amount of ACE mRNA was greater in VSMC from SHR than in cells from WKY rats in the present study. The vascular ACE activity has also been reported to be greater in SHR than in WKY rats in vivo.⁵¹ ACE is an ectoenzyme that is anchored in the cell membrane with its active site located outside the cell, which is thought to be the site of Ang II generation from Ang I.⁵² Nonett et al⁵³ investigated enzymatic and immunologic properties of ACE in gastric cells, and demonstrated that although the immunocytochemical localization mainly showed an intracellular localization, most of ACE activity was found to be ectoenzymatic, indicating that most of the intracellular ACE is immunologically active, but enzymatically inactive. On the other hand, Anderson et al⁵⁴ showed that exogenously administered Ang II-colloidal gold particles are taken up into the cytosol of VSMC. Ang I synthesized and secreted by VSMC may thus be converted to Ang II extracellularly, and the Ang II may then be taken up into the cells.

The AT1 receptor antagonist CV-11974 inhibited basal DNA synthesis in VSMC from SHR by 30% in the present study, similar to our previous results with SHR obtained from Charles River.²² This observation suggests that endogenously synthesized Ang II contributes to the basal growth of these cells. The growth of VSMC under basal conditions is thought to be maintained by the production of endogenous growth factors such as epidermal growth factor, insulin-like growth factor, PDGF, and fibroblast growth factor. Both epidermal growth factor and insulin-like growth factor act as progression factors, whereas PDGF and fibroblast growth factor act as competence factors, in the cell cycle of VSMC.^{55 56} Ang II and vasopressin are also thought to act as competence factors in the cell cycle, especially in the transition from G1 to S phase.⁵⁶ The increased production of Ang II by VSMC from SHR, relative to VSMC from WKY rats, may therefore contribute to the increased basal growth of these cells.

Both TGF-ß1 and PDGF A-chain mRNAs accumulate to a greater extent in VSMC from SHR than in cells from WKY rats.^{5 6} We have confirmed a role for these growth factors in the exaggerated growth of SHR-derived VSMC by demonstrating growth inhibition in response to corresponding antisense oligodeoxynucleotides.^{7 8} TGF-ß1 mRNA is induced by Ang II acting through a phorbol ester response element in the gene promoter, and then PDGF A-chain mRNA is induced by TGF-ß1 acting at TGF-ß type II receptors.⁹ Moreover, we have shown that expression of the TGF-ß type II receptor is increased in VSMC from SHR compared with VSMC from WKY rats, and that this increased expression contributes to the enhanced growth of these cells.⁵⁷ Endogenously generated Ang II may thus contribute to the growth of VSMC from SHR by increasing expression of TGF-ß1 and PDGF A-chain, and this growth-promoting effect may be enhanced by the increased expression of TGF-ß receptors on these cells.

It has been demonstrated that ACE activity is higher in endothelium than smooth muscle,⁵² and angiotensinogen production is greater in adventitial layers than in smooth muscle in vivo.⁵⁹ Whether the increased generation of Ang II in VSMC from SHR in vitro participates in the enhanced generation of Ang II in blood vessels of SHR in vivo is difficult to assess from this study. Recently we found that the generation of Ang II in VSMC from SHR is associated with changes in VSMC phenotype from contractile to synthetic, which induces nonspecific increases in cytosolic proteases including cathepsin D and ACE (N.F., unpublished data). If the phenotypic changes occurred in smooth muscle in blood vessels of SHR in vivo, the increased generation of Ang II in VSMC from SHR in vitro could contribute to the enhanced generation of Ang II in blood vessels of SHR in vivo.

The exaggerated growth characteristics of SHR-derived VSMC in culture, together with the observation that newborn SHR show hyperplasia of the heart and kidney before they develop high blood pressure,⁶⁰ indicates that the enhanced growth of cardiovascular organs in SHR is independent of high blood pressure. Our demonstration of an Ang II-generating system in homogeneous cultures of SHR-derived VSMC may contribute to our understanding of hypertensive vascular disease as well as, possibly, to the hypertension itself.

Received September 1, 1998; accepted November 4, 1998.

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