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(Hypertension. 1998;31:672-677.)
© 1998 American Heart Association, Inc.

Scientific Contributions

Abnormal Regulation of Transforming Growth Factor-ß Receptors on Vascular Smooth Muscle Cells From Spontaneously Hypertensive Rats by Angiotensin II

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

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

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

	Abstract

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Abstract—The effects of angiotensin II (Ang II) on the expression and characteristics of transforming growth factor-ß (TGF-ß) receptors on vascular smooth muscle cells (VSMC) from Wistar-Kyoto (WKY) rats and spontaneously hypertensive rats (SHR) were investigated. TGF-ß–induced stimulation of DNA synthesis by VSMC from WKY rats was abolished with Ang II, whereas basal and TGF-ß–stimulated DNA synthesis by VSMC from SHR was increased with Ang II. Ang II stimulated DNA synthesis by VSMC from WKY rats in the presence but not in the absence of neutralizing antibody to TGF-ß₁. Antibody to TGF-ß₁ enhanced the stimulatory effect of Ang II on DNA synthesis by VSMC from SHR. Ang II increased the specific binding of TGF-ß to VSMC from WKY rats by increasing both the expression of the lower-affinity of TGF-ß receptors as well as the total number of TGF-ß binding sites. In contrast, VSMC from SHR showed a higher affinity and number of TGF-ß receptors in the absence of Ang II than did cells from WKY rats, and these parameters were not affected by Ang II. Ang II increased the expression of TGF-ß type I receptor mRNA in VSMC from WKY rats but had no effect of TGF-ß receptor type I or II mRNA in VSMC from SHR, which predominantly express the type II receptor. These results indicate that an increase in the expression of the TGF-ß type I receptor by Ang II may facilitate the ability of endogenous TGF-ß to counteract the stimulatory effect of Ang II on growth in VSMC from WKY rats, whereas endogenous TGF-ß induced by Ang II cannot counteract the growth-promoting action of Ang II in VSMC from SHR. The abnormal regulation of TGF-ß receptors by Ang II may be associated with the exaggerated growth of VSMC from SHR.

Key Words: angiotensin II • transforming growth factors • receptors • vascular smooth muscle • rats, inbred SHR

	Introduction

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Vascular smooth muscle cells from normotensive WKY rats and SHR exhibit distinct growth phenotypes in culture.¹ Thus the growth of VSMC from SHR both under basal conditions as well as in response to calf serum, epidermal growth factor, or platelet-derived growth factor² is more pronounced than that of VSMC from WKY rats. Previous studies demonstrated that TGF-ß₁³ and platelet-derived growth factor A-chain⁴ mRNAs accumulate to a greater extent in VSMC from SHR than in those from WKY rats.

TGF-ß inhibits the growth of most cell types⁵ but has a dual effect on VSMC, suppressing growth at low cell densities and stimulating growth at high cell densities.⁶ This dual effect on the growth of VSMC has been reported to result from an interaction of TGF-ß with distinct receptor phenotypes at different cell densities.⁷ Several TGF-ß receptors or binding proteins have been identified.^{8 9} Three types of TGF-ß receptors (types I, II, and III) have been cloned.^{10 11 12} The type III receptor is a proteoglycan with a short cytoplasmic domain and is unlikely to mediate biological activities of TGF-ß.¹⁰ The predicted amino acid sequence of the type II receptor indicates that it is a serine-threonine kinase.¹² Both type I and type II receptors have been implicated in mediating the biological activities of TGF-ß.^{11 12}

VSMC from SHR and WKY rats show distinct growth responses to TGF-ß. Whereas exogenous TGF-ß inhibits DNA synthesis by VSMC from WKY rats, it stimulates DNA synthesis by those from SHR, which are less susceptible to growth inhibition by TGF-ß than are cells from WKY rats.^{3 13 14} We recently showed that VSMC from SHR show increased expression of TGF-ß type II receptor compared with cells from WKY rats and that this difference may contribute to the exaggerated growth of SHR-derived VSMC.¹⁴

Ang II is a potent vasoconstrictor as well as a promoter of VSMC growth. The growth-promoting effect is mediated by the Ang II type 1 receptor by protein kinase C, which activates the proto-oncogenes c-fos and c-myc.^{15 16} In addition, Ang II induces the expression of TGF-ß, which, in turn, induces the expression of PDGF A-chain.^{17 18} Ang II has been shown to induce cell hypertrophy,¹⁹ and a hyperplastic effect has been described for some cells including VSMC from WKY rats and SHR^{20 21} in vitro. The Ang II–induced hyperplasia of VSMC occurs without activation of TGF-ß.²² In addition, the blockade of endogenous TGF-ß synthesis or action with antisense oligonucleotides²³ or specific antisera¹⁷ enhances Ang II–induced DNA synthesis by VSMC, suggesting that TGF-ß inhibits VSMC growth in the presence of Ang II. These data also suggest that Ang II regulates the expression or function of TGF-ß receptors to modulate cell growth in normal VSMC; defective regulation of TGF-ß receptors by Ang II in VSMC from SHR may contribute to the exaggerated growth.

	Methods

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Cell Culture
VSMC were obtained by an explant method from aortas of 10-week-old male WKY rats and SHR (Charles River Japan, Atsugi, Japan) as described previously.²⁴ They were seeded and grown in DMEM supplemented with 10% calf serum (Gibco Life Technologies), 100 U/mL of penicillin (Gibco), and 100 µg/mL of streptomycin (Gibco). When the cells reached confluency in 7 to 10 days, they exhibited a hill-and-valley pattern that is typical of smooth muscle cells in culture. Indirect immunofluorescence to detect -smooth muscle–specific actin was performed on methanol-permeabilized 10th passage VSMC from WKY rats and SHR as previously described,²⁵ with a monoclonal antibody specific for the VSMC -actin isoform (DAKO A/S, Glostrup, Denmark). They were passaged by trypsinization with 0.05% trypsin (Gibco) in Ca²⁺- and Mg²⁺-free phosphate-buffered saline (Gibco) and incubated in 80-cm² tissue culture flasks at a density of 10⁵ cells/mL. Experiments were performed on the cells after 5 to 15 passages.

Establishment of Quiescence
Trypsinized cells were plated at a density of 5x10⁴ cells/cm² in culture medium in 24-well culture dishes. They were allowed to grow in DMEM containing 10% calf serum for 24 hours, and the culture medium was then changed to DMEM with 0.2% calf serum. The cells were incubated in this medium for 48 to 72 hours to establish quiescence.

Determination of DNA Synthesis
[³H]Thymidine incorporation into newly synthesized DNA was determined as described previously.²⁴ Quiescent VSMC in 24-well plates were incubated for 24 hours with DMEM containing penicillin (100 U/mL), streptomycin (100 µg/mL), and various concentrations of Ang II (Peptide Institute), TGF-ß₁ (R and D Systems) and neutralizing immunoglobulin G to human TGF-ß₁ (anti–TGF-ß₁) (R and D Systems). The medium was then changed to DMEM containing [³H]thymidine (0.5 µCi/mL) (New England Nuclear); after incubation for 2 hours, each well was washed with 1 mL of 150 mmol/L NaCl to eliminate excess [³H]thymidine and the cells were fixed in 1 mL of ethanol:acetic acid (3:1) solution for 10 minutes. After washing with 1 mL of H₂O, acid-insoluble material was precipitated with 1 mL of ice-cold perchloric acid and DNA was extracted into 1.5 mL of perchloric acid by heating at 90°C for 20 minutes. The perchloric acid containing solubilized DNA was transferred to a scintillation vial, and the radioactivity was measured with a liquid scintillation spectrometer.

TGF-ß Binding Assay
Quiescent VSMC were incubated for 20 hours in the absence or presence of 0.1 µmol/L Ang II in DMEM without serum, washed with 1 mL of binding buffer (DMEM, 1% bovine serum albumin, 20 nmol/L HEPES, pH 7.4), and incubated for 2 hours at 37°C to allow dissociation of bound endogenous TGF-ß. After washing, cells were incubated at 4°C for 4 hours in 0.2 mL of binding buffer containing 40 pmol/L ¹²⁵I–TGF-ß₁ (New England Nuclear) and unlabeled TGF-ß₁ (10 to 2000 pmol/L). The cells were then washed three times at 4°C with 0.5 mL of binding buffer, and the washings were combined to determine the amount of free ¹²⁵I–TGF-ß₁. Cell-associated radioactivity was solubilized by the addition of 0.5 mL of 1 mol/L NaOH. ¹²⁵I radioactivity was counted in a gamma-counter (ARC 300; Aloka). Binding data were analyzed according to the method of Scatchard.²⁶

RT-PCR Analysis for TGF-ß Type I and Type II Receptor mRNAs
Quiescent VSMC were incubated for 12 hours in the absence or presence of 0.1 µmol/L Ang II or 0.1 µmol/L CV-11974 (Takeda Pharmaceutical), washed with phosphate-buffered saline, and lysed in 800 µL of RNAzol B (Biotecx). Each sample was mixed with 80 µL of chloroform, incubated at 4°C for 15 minutes, and centrifuged at 12 000g for 15 minutes. An aliquot (300 µL) of the 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. The resulting RNA pellet was washed twice with 500 µL of 75% (vol/vol) ethanol by vortemixing and centrifugation at 7500g for 8 minutes at 4°C, dried, dissolved in 10 µL of 10 mmol/L Tris-HCl (pH 8.0) containing 1 mmol/L EDTA, and incubated for 15 minutes at 65°C. Each sample was treated at room temperature for 45 minutes with 0.5 U of deoxylibonuclease (Gibco) in 0.5 µL of 20 mmol/L Tris-HCl (pH 8.3), 50 mmol/L KCl and 2.5 mmol/L MgCl₂. The deoxylibonuclease was then inactivated by adding 0.5 µL of 20 mmol/L EDTA and heating at 98°C for 10 minutes.

RT-PCR was performed as described previously.²⁷ In brief, aliquots of RNA (1 µg) were reverse-transcribed into single-stranded cDNA by 5 U of avian myeloblastoma virus reverse transcriptase (Life Sciences) in a final volume of 20 µL of 10 mmol/L Tris-HCl (pH 8.3) containing 5 mmol/L MgCl₂, 50 mmol/L KCl, 1 mmol/L deoxy-NTPs (Takara Biochemicals), and 2.5 µmol/L random hexamer (Takara). An aliquot (5 µL) of the diluted cDNA product was mixed with 20 µL of 10 mmol/L Tris-HCl (pH 8.3) containing 50 mmol/L KCl, 4 mmol/L MgCl₂, 0.5 U of Taq DNA polymerase (Takara), and 0.2 µmol/L each of sense and antisense primers in a total volume of 25 µL. The 26-mer upstream sense primer (5'-GCTCTAGATTTCTGCCACCTCTGTAC-3') corresponding to bases 96 to 113 and the 26-mer downstream antisense primer (5'-GCGAATTCGACAGTGCGGTTATGGCA-3') complementary to bases 441 to 424 were derived from rat TGF-ß type I receptor cDNA. The 21-mer upstream sense primer (5'-AAGTCTTGCATGAGCAACTGC-3') corresponding to bases 251 to 271 and the 19-mer downstream antisense primer (5'-GACGTCAGAGAAGATGTCC-3') complementary to bases 931 to 949 were derived from rat TGF-ß type II receptor cDNA. A 21-mer sense primer (5'-CTGAAGGTCAAAGGGAATGTG-3') and a 21-mer antisense primer (5'-GGACAGAGTCTTGATGATCTC-3') for rat ribosomal protein L19 were included in the reaction as an internal control.²⁸ The amount of PCR product from TGF-ß type I and II receptor mRNAs increased linearly from 20 to 35 PCR cycles (data not shown). PCR therefore was performed for 30 cycles in a DNA Thermal Cycler (Perkin-Elmer Cetus) with the following thermal cycle profile: denaturing at 96°C for 45 seconds, primer annealing at 58°C for 45 seconds, and primer extension at 72°C for 2 minutes. PCR products were electrophoresed through a 1.5% agarose gel and stained with ethidium bromide.

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

	Results

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Expression of Smooth Muscle-Specific -Actin in VSMC
The cultures were uniformly composed of cells that stain positive for smooth muscle–specific -actin. There was no difference of the staining between VSMC from WKY rats (Fig 1A) and SHR (Fig 1B).

View larger version (96K): [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). Tenth passage VSMC were permialized and fixed in absolute methanol. Cells were then stained by indirect immunofluorescence with monoclonal antibody against the smooth muscle–specific isoform of -actin. Bar=50 µm.

Effect of Ang II on TGF-ß–Induced Growth of VSMC
In the absence of Ang II, DNA synthesis by VSMC from WKY rats and SHR showed the following response to TGF-ß: DNA synthesis was stimulated by TGF-ß₁ at 1.0 ng/mL and inhibited at 10 ng/mL. In the presence of 0.1 µmol/L Ang II, DNA synthesis by VSMC from WKY rats was not stimulated by TGF-ß₁ at 1.0 ng/mL, the value being significantly (P<.01) less than that in the absence of Ang II; at 10 ng/mL, TGF-ß₁ inhibited DNA synthesis to a greater extent (P<.05) in the presence than in the absence of Ang II (Fig 2, left). In contrast, DNA synthesis by VSMC from SHR was significantly higher (P<.01) in the presence of Ang II than in its absence at TGF-ß₁ concentrations of 0, 0.1, and 1.0 ng/mL. These data suggest that TGF-ß acts as a growth inhibitor in the presence of Ang II for VSMC from WKY rats but not for cells from SHR (Fig 2, right).

View larger version (37K): [in this window] [in a new window]   Figure 2. Effects of Ang II on TGF-ß–induced DNA synthesis by VSMC from WKY rats and SHR. Quiescent VSMC were exposed for 24 hours to various concentrations of TGF-ß1 in the absence (open columns) or presence (shaded columns) of 0.1 µmol/L Ang II, and then to [3H]thymidine for 2 hours. Radioactivity incorporated into DNA was measured. Data are mean+SEM of values from four experiments. P<.05, P<.01 vs without TGF-ß1. P<.05, P<.01 vs control.

To investigate the influence of endogenous TGF-ß on the action of Ang II, we determined the effect of anti–TGF-ß₁ on Ang II–induced DNA synthesis. Ang II at 0.01 to 1.0 µmol/L did not stimulate DNA synthesis by VSMC from WKY rats in the absence of anti–TGF-ß₁; however, in the presence of anti–TGF-ß₁, Ang II at 0.01 to 0.1 µmol/L significantly increased (P<.05) DNA synthesis by VSMC from WKY rats (Fig 3, left). DNA synthesis by VSMC from SHR was markedly stimulated by Ang II and, at 0.1 µmol/L Ang II, was significantly greater (P<.01) in the presence of anti-TGF-ß₁ than in the absence of Ang II (Fig 3, right).

View larger version (37K): [in this window] [in a new window]   Figure 3. Effects of neutralizing antibody to TGF-ß1 (anti–TGF-ß1) on stimulation of DNA synthesis by VSMC from WKY rats and SHR in the absence or presence of Ang II. Quiescent VSMC were exposed for 24 hours to various concentrations of Ang II in the absence (open columns) or presence (shaded columns) of 20 µg/mL of anti–TGF-ß1 and then to [3H]thymidine for 2 hours. Radioactivity incorporated into DNA was measured. Data are mean+SEM from four experiments and are expressed relative to control value for cells incubated without Ang II in the absence or presence of anti–TGF-ß1. P<.05, P<.01 vs without anti–TGF-ß1.

Effect of Ang II on TGF-ß Binding to VSMC
The specific binding of ¹²⁵I–TGF-ß₁ to VSMC from WKY rats showed saturation at 200 pmol/L TGF-ß₁ in the absence of Ang II but had not reached saturation at 800 pmol/L TGF-ß₁ in the presence of Ang II (Fig 4, left). Scatchard analysis revealed a single binding component [dissociation constant (K_d), 21 pmol/L; maximum number of binding sites (B_max), 4740] in the absence of Ang II, but two such components (K_d, 17 and 47 pmol/L) and an increased number of binding sites (B_max, 14 530) in the presence of Ang II (Fig 4, left, inset). Specific binding of ¹²⁵I–TGF-ß₁ to VSMC from SHR was increased relative to that for VSMC from WKY rats in the absence of Ang II and was not affected by the presence of Ang II (Fig 4, right). Scatchard analysis revealed the presence of high-affinity binding sites for TGF-ß₁ (K_d, 8.3 pmol/L; B_max, 7680) on VSMC from SHR, the kinetic properties of which were affected by Ang II (K_d, 7.2 pmol/L; B_max, 7672) (Fig 4, right, inset).

View larger version (17K): [in this window] [in a new window]   Figure 4. Effects of Ang II on the specific binding of 125I–TGF-ß1 to VSMC from WKY rats and SHR. VSMC were incubated with 40 pmol/L 125I–TGF-ß1 and the indicated concentrations of unlabeled TGF-ß1 in the absence () or presence () of 0.1 µmol/L Ang II, and specific binding was determined. Insets show Scatchard analysis of binding data. B indicates bound ligand; F, free ligand.

Effect of Ang II on the Amount of TGF-ß Receptor mRNAs in VSMC
Because examination of the time course of the effect of Ang II on expression of TGF-ß type I and type II receptor mRNAs in VSMC from WKY rats for 2, 4, 12, and 24 hours revealed that TGF-ß type I receptor mRNA was considerably increased by incubation with Ang II for 2 and 12 hours, and that TGF-ß type II receptor mRNA was increased by incubation with Ang II at 12 hours (data not shown), we used 12 hours as incubation time with Ang II in following experiments. The basal amount of TGF-ß type I receptor mRNA was significantly higher (P<.05) in VSMC from WKY rats than in cells from SHR (Fig 5). Incubation with 0.1 µmol/L Ang II increased the amount of TGF-ß type I receptor mRNA (P<.05) in VSMC from WKY rats but had no effect on that in cells from SHR (Fig 5). In contrast, the basal amount of TGF-ß type II receptor mRNA was significantly higher (P<.05) in VSMC from SHR than in those from WKY rats (Fig 6). The amount of the TGF-ß type II receptor mRNA tended to increase upon treatment with Ang II, but this difference had not reached statistical significance in VSMC from WKY rats, and it had no effect on mRNA in cells from SHR (Fig 6).

View larger version (38K): [in this window] [in a new window]   Figure 5. Effects of Ang II on the abundance of TGF-ß type I receptor (TGF-ß RI) mRNA in VSMC from WKY rats and SHR. A, Quiescent VSMC were incubated for 12 hours in the absence or presence of 0.1 µmol/L Ang II, after which the amounts of TGF-ß RI and ribosomal protein L19 mRNAs were determined by RT-PCR. B, The ratio of the amount of TGF-ß RI mRNA to that of L19 mRNA was evaluated by densitometric analysis. Open columns, absence of Ang II; shaded columns, presence of Ang II. Data are mean+SEM from four experiments. P<.05 between indicated columns.

View larger version (38K): [in this window] [in a new window]   Figure 6. Effects of Ang II on the abundance of TGF-ß type II receptor (TGF-ß RII) mRNA in VSMC from WKY rats and SHR. A, Quiescent VSMC were incubated for 12 hours in the absence or presence of 0.1 µmol/L Ang II, after which the amounts of TGF-ß RII and ribosomal protein L19 mRNAs were determined by RT-PCR. B, The ratio of the amount of TGF-ß RII mRNA to that of L19 mRNA was evaluated by densitometric analysis. Open columns, absence of Ang II; shaded columns, presence of Ang II. Data are mean+SEM from four experiments. P<.05 between indicated columns.

The Ang II–induced increase in the expression of TGF-ß type I receptor mRNA was abolished by the nonpeptide Ang II type 1 receptor antagonist CV-11974 in VSMC from WKY rats (Fig 7). CV-11974 did not affect the expression of TGF-ß type I receptor mRNA in VSMC from SHR in the presence of Ang II. The Ang II–induced increase in the expression of TGF-ß type II receptor mRNA was also reduced by CV-11974 in VSMC from WKY rats, whereas the expressions of TGF-ß type II receptor mRNA in the absence and presence of Ang II were reduced by CV-11974 in VSMC from SHR (Fig 7).

View larger version (47K): [in this window] [in a new window]   Figure 7. Effects of a nonpeptide Ang II type 1 receptor antagonist CV-11974 on Ang II–induced expression of TGF-ß type I receptor (TGF-ß RI) and type II receptor (TGF-ß RII) mRNAs in VSMC from WKY rats and SHR. Quiescent VSMC were incubated for 12 hours in the absence or presence of 0.1 µmol/L Ang II without or with 0.1 µmol/L CV-11974. The expression of TGF-ß RI, TGF-ß RII, and ribosomal protein L19 mRNAs were analyzed by RT-PCR.

	Discussion

Top Abstract Introduction Methods Results Discussion References

 
The effect of TGF-ß on the growth of VSMC depends on both cell density and TGF-ß concentration, which is associated with the expression of TGF-ß receptor subtypes.^{5 6 7} Goodman and Majack⁷ demonstrated that confluent VSMC expressed higher-affinity receptor subtypes with molecular sizes of 280 kD (type III receptor) and 85 kD (type II receptor), whereas cells at low density express lower-affinity receptor subtypes with molecular sizes of 75 kD (type I receptor) and 65 kD. In VSMC at high density, low concentrations of TGF-ß stimulate DNA synthesis through the higher-affinity TGF-ß receptor subtypes, whereas high concentrations of TGF-ß inhibit DNA synthesis through the lower-affinity receptor subtypes.²⁹

The TGF-ß type I receptor is a transmembrane protein kinase that associates with the type II receptor to generate diverse heteromeric serine-threonine kinase complexes with diverse signaling capacities.^{11 12} The type II receptor acts upstream of the type I receptor and only the type II receptor recognizes TGF-ß as a ligand; the type I receptor recognizes the ligand-bound type II receptor.³⁰ Subsequent signaling by these complexes induces the phosphorylation of the type I receptor, which appears to be catalyzed by the type II receptor kinase, the activity of which is not augmented by ligand binding.³¹ Thus biological responses to TGF-ß mediated by the type I receptor also require the type II receptor.

We recently showed that at high cell density, the expression of TGF-ß type II receptors is increased in VSMC from SHR compared with that in cells from WKY rats.¹⁴ At low cell density, expression of the type II receptor was not apparent, whereas expression of the type I receptor was greater at low cell density than at high cell density. Thus VSMC from WKY rats and SHR appear to differ in their expression of TGF-ß receptor subtypes, and the type II and type I receptors appear to mediate stimulation and inhibition of the growth of VSMC, respectively.

In the present study, TGF-ß–induced stimulation of DNA synthesis by VSMC from WKY rats was abolished with Ang II, whereas basal and TGF-ß–induced DNA synthesis by VSMC from SHR was increased with Ang II. Ang II is thought to promote the growth of VSMC through activation of protein kinase C, which also increases the expression of TGF-ß through a phorbor ester–responsive element in the promoter region of the TGF-ß gene.^{17 18} Ang II stimulated DNA synthesis by VSMC from WKY rats in the presence but not in the absence of anti–TGF-ß₁. Ang II markedly stimulated DNA synthesis by VSMC from SHR in the absence of anti–TGF-ß₁, and the effect was enhanced in the presence of anti-TGF-ß₁. In addition, there has been no report of differences of Ang II receptors on VSMC from WKY rats and SHR. These findings suggest that endogenous TGF-ß induced by Ang II counteracts the stimulatory effect of Ang II on growth in normal VSMC and that this phenomen is abnormal in VSMC from SHR.

Ang II increased the specific binding of TGF-ß to VSMC from WKY rats by increasing expression of lower-affinity TGF-ß receptors and increasing the total number of binding sites. VSMC from SHR showed a higher affinity and number of TGF-ß receptors in the absence of Ang II than did VSMC from WKY rats, but these parameters were not affected by Ang II. These data suggested that Ang II may increase the number of TGF-ß type I receptors on normal VSMC but that it does not affect the characteristics of TGF-ß receptors on VSMC from SHR, which appear mostly to express higher-affinity, type II receptors.

To confirm this possibility we investigated the effects of Ang II on the expression of mRNAs encoding TGF-ß type I and type II receptors in VSMC from WKY rats and SHR. The basal expression of the type I receptor mRNA was greater in VSMC from WKY rats than in cells from SHR, whereas the basal expression of the type II receptor mRNA was greater in those from SHR than cells from WKY rats. This latter observation is consistent with our previous affinity-labeling data showing that the expression of the type II receptor is increased on VSMC from SHR compared with that for cells from WKY rats.¹⁴ Thus under basal conditions, VSMC from SHR appear mainly to express the growth-promoting TGF-ß type II receptor, whereas VSMC from WKY rats mainly express the growth-inhibiting type I receptor.

The observation that Ang II also increased the expression of TGF-ß type I and type II receptor mRNAs, especially the type I receptor, in VSMC from WKY rats suggests that Ang II upregulates TGF-ß receptors on normal VSMC. Such an increase in the number of type I receptors in response to Ang II may facilitate the ability of endogenous TGF-ß to counteract the stimulatory effect of Ang II on cell growth. In contrast, Ang II did not affect the expression of TGF-ß receptor mRNAs in VSMC from SHR, which predominantly express the type II receptor, indicating that endogenous TGF-ß induced by Ang II cannot counteract the stimulatory effect of Ang II on the growth of VSMC from SHR and possibly explaining, at least in part, the exaggerated growth of these cells.

Our data showing that CV-11974 inhibited the Ang II–induced increase in the expression of TGF-ß type I receptor mRNA in VSMC from WKY rats indicate that this effect of Ang II may be mediated by Ang II type 1 receptor. CV-11974 also considerably reduced expression of type II receptor mRNA in VSMC from SHR in the presence of Ang II. Thus endogenous Ang II may upregulate TGF-ß type II receptors on VSMC from SHR. We recently showed that CV-11974, as well as the angiotensin-converting enzyme inhibitor delapril, inhibits basal DNA synthesis by VSMC from SHR but not in those from WKY rats, in the absence of serum,³² suggesting that VSMC from SHR may generate Ang II that promotes their basal growth in an autocrine/paracrine manner through the Ang II type 1 receptor. More recently, we demonstrated de novo synthesis of Ang II by radioimmunoassay in a homogenous culture of VSMC from SHR (but not in cells from WKY rats) as well as an Ang II–generating system with increases in expression of angiotensinogen, cathepsin D, and angiotensin-converting enzyme mRNAs, but not renin mRNA.³³ Thus endogenous Ang II generated by VSMC from SHR appears to contribute the abnormal regulation of TGF-ß receptors on these cells.

The renin-angiotensin system has been implicated as one of the causes of essential hypertension because of the antihypertensive effects of angiotensin-converting enzyme inhibitors. Several lines of evidence have demonstrated the presence of a local tissue renin-angiotensin system that is independent of the circulating renin-angiotensin system.³⁴ The renin-angiotensin system of the vascular wall acts to regulate vessel tone and blood flow as well as to induce vascular growth.³⁵ Our results suggest that hypertension is associated with the abnormal regulation of TGF-ß receptors on VSMC by Ang II generated by the vascular wall renin-angiotensin system and that this abnormality may facilitate vascular proliferation and underlie the development of hypertensive vascular disease.

	Selected Abbreviations and Acronyms

 

Ang II = angiotensin II DMEM = Dulbecco's modified Eagle's medium RT-PCR = reverse transcription and polymerase chain reaction SHR = spontaneously hypertensive rat(s) TGF-ß = transforming growth factor-ß VSMC = vascular smooth muscle cells WKY = Wistar-Kyoto

Received June 23, 1997; first decision July 10, 1997; accepted September 22, 1997.

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