Angiotensin II regulates the cell cycle of vascular smooth muscle cells from SHR

Abstract

We have demonstrated that spontaneously hypertensive rats (SHR)-derived vascular smooth muscle cells (VSMC) show the exaggerated growth and produce angiotensin II (Ang II). In the current study, we investigated the role of endogenous Ang II in the regulation of the cell cycle in VSMC from SHR. Levels of Ang II in conditioned medium from SHR-derived VSMC cultured without serum were significantly higher than levels in conditioned medium from Wistar-Kyoto (WKY) rat-derived VSMC. Basal DNA synthesis was higher in quiescent VSMC from SHR than that in cells from WKY rats. An Ang II type 1 receptor antagonist, CV11974, significantly inhibited the elevation in DNA synthesis in quiescent VSMC from SHR but did not affect it in cells from WKY rats. Cellular DNA content analysis by flow cytometry revealed that the proportion of cells in S phase was higher, whereas the proportion of cells in G₁+G₀ phase was lower in VSMC from SHR than those in cells from WKY rats. CV11974 significantly decreased the proportion of cells in S phase and correspondingly increased the proportion of cells in G₁+G₀ phase in VSMC from SHR, but it did not affect the proportion in cells from WKY rats. Cyclin-dependent kinase 2 (CDK2) activity, which is known to induce the progression from G₁ to S phase, was higher in VSMC from SHR than in cells from WKY rats. Expression of CDK2 inhibitor p27^kip1 mRNA was markedly higher in VSMC from SHR than in cells from WKY rats. CV11974 decreased expression of p27^kip1 mRNA in VSMC from SHR, whereas CV11974 increased it in cells from WKY rats. These findings indicate that enhanced production of endogenous Ang II regulates the cell cycle especially in the progression from G₁ to S phase, and increases CDK2 activity, which is independent of p27^kip1 in VSMC from SHR. Am J Hypertens 2000;13:1117–1124 © 2000 American Journal of Hypertension, Ltd.

Angiotensin II, receptor, vascular smooth muscle, cell cycle, CDK, p27^kip1

Cultured vascular smooth muscle cells (VSMC) from spontaneously hypertensive rats (SHR) show exaggerated growth compared to cells from normotensive Wistar-Kyoto (WKY) rats.^1,2 Angiotensin II (Ang II) is a potent vasoconstrictor as well as a promoter of VSMC growth. The growth-promoting effect is mediated via the Ang II type 1 (AT1) receptor by protein kinase C, and activated mitogen-activated protein kinases^3,4 promote VSMC growth with induction of protooncogenes c-fos and c-myc.^5,6 Ang II has been shown to induce cell hypertrophy⁷ and a hyperplastic effect has been described for some cells including VSMC from WKY rats and SHR in vitro.^8,9 In addition, Ang II induces expression of basic fibroblast growth factor (bFGF) and transforming growth factor-β (TGF-β), which then induces the expression of platelet-derived growth factor (PDGF) A-chain.^10–12 We have demonstrated that a nonpeptide antagonist of AT1 receptor inhibited basal growth in VSMC from SHR, but that it had no effect on cells from WKY rats.¹³ We recently demonstrated that Ang II is produced by VSMC from SHR but not in cells from WKY rats, and that there are increases in angiotensinogen, cathepsin D, and angiotensin-converting enzyme.¹⁴ This was the first report to show that VSMC have the complete system to produce Ang II. In addition, we demonstrated that the mechanisms of the production of Ang II in VSMC from SHR is associated with the changes in VSMC from the contractile to the synthetic phenotype, which expresses greater levels of cathepsin D and angiotensin converting enzyme.¹⁵

We have observed that TGF-β₁,¹⁶ PDGF A-chain,¹⁷ and bFGF¹⁸ mRNAs accumulate to a greater extent in VSMC from SHR when compared to levels in cells from WKY rats and that an antagonist of AT1 receptors considerably inhibits expression of growth factor mRNAs in VSMC from SHR.¹⁹ These findings indicate that endogenous production of Ang II is involved in the exaggerated growth by activating growth-promoting signals as well as in the increased expression of growth factors in VSMC from SHR.

Spontaneously hypertensive rats-derived VSMC have been reported to have shortened cell cycle with accelerated entry into S phase.^20,21 It is possible that the increase in endogenous Ang II levels is associated with the shortened cell cycle of VSMC from SHR. Progression of the eukaryotic cell cycle is regulated by cyclins and their catalytic subunits cyclin-dependent kinases (CDK).²² CDK activities are regulated positively by cyclin and negatively by CDK inhibitor p27^kip1.²³ The current study was undertaken to investigate roles of endogenous Ang II in the regulation of the cell cycle, CDK2 activity, and expression of p27^kip1 in VSMC from SHR.

Methods

Cell culture

VSMC were obtained by an explant method²⁴ from aortas of 8-week-old male SHR/Izumo and WKY/Izumo rats (SHR Corporation, Funabashi, Chiba, Japan) as described previously.¹ They were seeded and grown in Dulbecco’s modified Eagle’s medium (DMEM) with 10% calf serum (Gibco Life Technologies, Gaithersburg, MD), penicillin (100 U/mL), and streptomycin (100 mg/mL). The cells achieved confluency after 7 to 10 days, at which time they exhibited the hill-and-valley pattern characteristic of smooth muscle cells in culture. They were passaged by trypsinization with 0.05% trypsin (Gibco) in Ca²⁺- and Mg²⁺-free Dulbecco’s phosphate-buffered saline (PBS) and incubated in 75-cm² tissue culture flasks at a density of 10⁵ cells/mL. Experiments were performed after three to seven passages. No contamination with other cell types was apparent by light microscopy.

Establishment of quiescence

Trypsinized cells were transferred to 24-well culture plates or 25-cm² tissue culture flasks and incubated first for 24 h in DMEM containing 10% calf serum and then serum starved for 48 h in DMEM with 0.2% calf serum.

Preparation of conditioned medium

VSMC (10⁶) from SHR and WKY rats were inoculated in 10-cm² wells with DMEM containing 10% calf serum and were incubated for 24 h. The cells were then washed twice with PBS and incubated for 24 h with DMEM without serum. The culture medium was then collected and centrifuged at 600 g for 10 min, and the resulting supernatant (conditioned medium) was collected. The conditioned medium was treated with 1 mmol/L each of aprotinin, leupeptin, and pepstatin A and 0.1 mmol/L phenylmethylsulfonyl fluoride, and then stored at −80°C until analysis for Ang II by radioimmunoassay (RIA).

Measurement of angiotensin peptides

Determination of angiotensin peptides in collected conditioned media was performed as described previously.¹⁴ Samples were applied to a Sep-Pak C18 cartridge (Waters Associates, Milford, MA), and peptides were eluted with 3 mL of methanol–water–trifluoroacetic acid (80:19.9:0.1, v/v). The eluate was dried in a vacuum centrifuge, and the angiotensin peptides were separated by reversed-phase high-performance liquid chromatography (RP-HPLC). Samples were then loaded on a Shodex column (OPD-50; Showa Denko, Tokyo, Japan), and the peptides were eluted with an exponential gradient of acetonitrile from 20% to 50% (v/v) in 0.05% trifluoroacetic acid at a flow rate of 1 mL/min. Fractions (0.5 mL) were collected and dried in a vacuum centrifuge, and the residues were resuspended in 0.1 mol/L Tris-HCl (pH 7.4) and subjected to RIA for Ang II.

The Ang II antiserum (Amersham, Little Chalfont, UK) 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 for 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.

Determination of DNA synthesis

[³H]Thymidine incorporation into newly synthesized DNA was performed as described previously²⁴ First, 24-well cluster dishes holding quiescent VSMC were replenished with fresh DMEM containing 100 U/mL of penicillin and 100 mg/mL of streptomycin. An AT1 receptor antagonist, CV11974 (0.1 mmol/L (Takeda Pharmaceutical, Osaka, Japan) was added for 0, 2, 6, 12, and 24 h. The medium was then changed to DMEM containing 0.5 mCi/mL of [³H]thymidine (NEN, Wilmington, DE). [³H]Thymidine incorporation was terminated after 2 h by removal of the labeled medium. Each well was washed with 1 mL of isoosmotic solution (150 mmol/L NaCl) to eliminate excess [³H]thymidine, and the cells were fixed with 1 mL of ethanol:acetic acid (3:1) for 10 min. The cells were washed with 1 mL of H₂O, acid-insoluble material was precipitated with 1 mL of 0.5 N ice-cold perchloric acid, and DNA was extracted into 1.5 mL of perchloric acid by heating at 90°C for 20 min. The perchloric acid containing solubilized DNA was transferred to scintillation vials, and the radioactivity was measured in a liquid scintillation spectrometer.

Cellular DNA content analysis by flow cytometry

Quiescent VSMC were incubated with 0.1 mmol/L CV11974 (Takeda Pharmaceutical) for 2, 6, 12, and 24 h and compared to untreated (0 h) control cells. They were trypsinized with 0.05% trypsin (Gibco) in Ca²⁺- and Mg²⁺-free Dulbecco’s PBS. VSMC were washed with ice-cold PBS, fixed with 50% ice-cold methanol and stored at −20°C overnight. Cells were then washed two times with ice-cold PBS, centrifuged, and resuspended in 1 mg/mL RNase at 37°C for 30 min to prevent staining of RNA by propidium iodide. DNA was stained with propidium iodide (20 μg/mL), a dye specific for double-stranded nucleic acids (Sigma, St. Louis, MO). After a 30-min incubation at 37°C, the cells were analyzed in a FACScan flow cytometer (Cyto ACE 150, Nihon Bunko, Tokyo, Japan) with an argon laser adjusted to emit 15 mW at 488-nm wavelength. From each cell sample, 10⁴ events were accumulated for each histogram. The proportion of cells in each phase of the cell cycle was determined from each histogram as described previously.²⁵ VSMC sizes were estimated simultaneously from the same samples by analysis of forward light scatter.

Measurement of CDK2 activity in VSMC

Quiescent VSMC were incubated with 0.1 mmol/L CV11974 for 2, 6, 12, and 24 h and compared to untreated (0 h) control cells. The whole cell lysate was prepared by adding lysis buffer [50 mmol/L Tris-HCl (pH 7.4), 1 mmol/L EDTA, 100 mmol/L NaCl, 1 mmol/L dithiothreitol, 0.1 mmol/L phenylmethyl sulfonyl fluoride]. The cell lysates were centrifuged at 15,000 g for 15 min and cleared with protein A Sepharose 6MB (Pharmacia Biotech AB, Uppsala, Sweden). The cleared supernatants were incubated with 2 μg of anti-CDK2 antibody (Upstate Biotechnology Inc. Lake Placid, NY) and precipitated with protein A Sepharose 6MB. The immunoprecipitates were washed with lysis buffer. H1 kinase assays of the immunoprecipitates were performed at 30°C for 20 min in kinase buffer [50 mmol/L HEPES (pH 8.0), 10 mmol/L MgCl₂, 20 μmol/L ATP, 1 mmol/L dithiothreitol, 2 mmol/L glutathione, 10 mmol/L β-glycerophosphate, 1 mmol/L sodium fluoride, 0.1 mmol/L Na₃VO₄] containing 0.1 mg/mL of H1 histone (Sigma Chemical, St Louis, MO), 50 μmol/L ATP, and 5 μCi [³²P] γ-ATP. Phosphorylated H1 histone was analyzed by 10% polyacrylamide gel electrophoresis and autoradiography.

Reverse transcription and polymerase chain reaction (RT-PCR) analysis for mRNA of p27^kip1

Quiescent VSMC were incubated with 0.1 μmol/L CV11974 for 2, 6, 12, and 24 h and compared to untreated (0 h) control cells. Cells were washed with PBS and lysed in 800 mL of RNAzol B (Biotex, Houston, TX). Cell lysates were mixed with 80 μL of chloroform, incubated at 4°C for 15 min, and centrifuged at 12,000 g for 15 min to extract total RNA. A portion (300 μL) of each aqueous phase was mixed with an equal volume of isopropanol, incubated at −20°C for 45 min, and centrifuged at 12,000 g for 15 min at 4°C to precipitate the RNA. The RNA pellet was washed twice with 500 μL of 75% (v/v) ethanol by vortex mixing and centrifugation at 7500 g for 8 min at 4°C. The pellet was dried and dissolved in a 10 μL-solution containing 10 mmol/L Tris-HCl (pH 8.0) and 1 mmol/L EDTA by incubation for 15 min at 65°C. Each sample was then treated with 0.5 U of DNase (Gibco) in 0.5 μL 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 min, after which the DNase was inactivated by adding 0.5 μL of 20 mmol/L EDTA and heating at 98°C for 10 min.

Reverse transcription and polymerase chain reaction was performed as described previously.²⁶ Briefly, aliquots of RNA (1 μg/20 mL) were reverse-transcribed into single-stranded cDNA by incubation for 10 min at 30°C, 30 min at 42°C, and 5 min at 99°C in a final volume of 20 μL containing 5 U of avian myeloblastoma virus reverse transcriptase (Life Sciences, St. Petersburg, FL), 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 μmol/L random hexamers. The diluted cDNA products (5 μL) were then subjected to PCR in a final volume of 25 μL 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, Osaka, Japan), and 0.2 μmol/L each of upstream sense (5′-GTCAAACGTGAGAGTGTCTA-3′) and downstream antisense primers (5′-CAAATGCCGGTCCTCAGAGT-3′) were used for PCR amplification of p27^kip1 mRNA. Sense primer (5′-TCAAG- AACGAAAGTCGGAGG-3′) and antisense primer (5′-GGACATCTAAGGGCATCACA-3′) for human 18S ribosomal RNA were used as an internal control. To confirm that no genomic DNA was coamplified during PCR, control RT-PCR experiments without reverse transcriptase using every set of primers were performed. No product was amplified. For semiquantative analysis of mRNA, the kinetics of the PCR reaction were monitored; the number of cycles at which the PCR products became detectable on the gel was compared between the different samples.²⁷ Serial 10-fold dilutions of cDNA (100, 10, and 1 ng) were amplified. PCR products became detectable at earlier cycles with increasing amounts of cDNA. PCR was performed using a DNA Thermal Cycler (Perkin-Elmer Cetus, Norwalk, CT). After an initial denaturation at 94°C for 10 min, PCR amplifications of p27^kip1 mRNA and human 18S ribosomal RNA were performed using 30 cycles of denaturation at 94°C for 30 sec, annealing at 55°C for 30 sec, primer extension at 72°C for 30 sec, and a final extension at 72°C for 10 min. PCR products were separated by electrophoresis through 1.5% agarose gels 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, or by two-way analysis of variance (ANOVA) followed by Duncan’s multiple range test.

Results

Ang II levels in conditioned medium of VSMC from WKY rats and SHR

Ang II levels were 37.5 ± 2.8 and 2.5 ± 1.3 fmol/mL in conditioned medium of VSMC from SHR and WKY rats, respectively, indicating that Ang II levels were significantly (P < .01) greater for VSMC from SHR than for VSMC from WKY rats.

Effect of CV11974 on basal DNA synthesis in VSMC from WKY rats and SHR

Basal DNA synthesis was significantly (P < .01) higher in VSMC from SHR than that in cells from WKY rats. A dose of 0.1 μmol/L CV11974 significantly (P < .05) decreased basal DNA synthesis in VSMC from SHR at 12 and 24 h, but CV11974 did not affect basal DNA synthesis in cells from WKY rats (Fig. 1).

Time course of CV11974 treatment on basal DNA synthesis in vascular smooth muscle cells (VSMC) from Wistar-Kyoto (WKY) rats and spontaneously hypertensive rats (SHR). Quiescent VSMC from WKY rats (○) and SHR (•) were incubated with 0.1 μmol/L CV11974 for 2, 6, 12, and 24 h before determination of [³H]thymidine incorporation and compared to untreated (0 h) control cells. Data are mean ± SEM (n = 4). *P < .05 v without CV11974 (0 h).

Fig. 1.

Effect of CV11974 on the proportion of DNA in the cell cycle in VSMC from WKY rats and SHR

Fig. 2A shows a typical flow cytometry DNA histogram in quiescent VSMC from WKY rats and SHR. The proportion of DNA in S phase cells was greater, and that in G₁+G₀ phase cells was less in VSMC from SHR rats than it was in cells from WKY rats. Figure 2B shows the time course of CV11974 treatment on the proportion of cells in each cell cycle phase of VSMC from WKY rats and SHR. A dose of 0.1 μmol/L CV11974 significantly (P < .05) decreased the proportion of cells in S phase in VSMC from SHR at 12 and 24 h. In contrast CV11974 did not affect the proportion of cells in S phase in VSMC from WKY rats. Corresponding to the changes in proportion of S phase in VSMC from WKY rats and SHR, the proportion of cells in G₁+G₀ phase in VSMC from SHR was less than that in WKY rats. This value increased significantly (P < .05) with 0.1 μmol/L CV11974 from 2 to 24 h in a time-dependent manner in VSMC from SHR.

A) Typical flow cytometry DNA histogram of quiescent vascular smooth muscle cells (VSMC) from Wistar-Kyoto (WKY) rats and spontaneously hypertensive rats (SHR). B) Time course of CV11974 treatment on the proportion of cells in each phase of the cell cycle in VSMC from WKY rats and SHR. Quiescent VSMC were incubated with 0.1 μmol/L CV11974 for 2, 6, 12, and 24 h and compared to untreated (0 h) control cells. VSMC were trypsinized, fixed with 50% ice-cold methanol, and treated with RNase. DNA was stained with propidium iodide. Cells were analyzed in a flow cytometer. Data are mean ± SEM (n = 4). *P < .05, **P < .01 v without CV11974 (0 h).

Fig. 2.

Effect of CV11974 on CDK2 activity in VSMC from WKY rats and SHR

Fig. 3 shows the time course of CV11974 treatment on CDK2 activity in VSMC from WKY rats and SHR. A dose of 0.1 μmol/L CV11974 significantly (P < .05) inhibited CDK2 activity in VSMC from SHR at 24 h, whereas CV11974 did not affect CDK2 activity in VSMC from WKY rats.

Time course of CV11974 treatment on cyclin-dependent kinase 2 (CDK2) activity in vascular smooth muscle cells (VSMC) from Wistar-Kyoto (WKY) rats and spontaneously hypertensive rats (SHR). Quiescent VSMC were incubated with 0.1 μmol/L CV11974 for 2, 6, 12, and 24 h and compared to untreated (0 h) control cells. VSMC lysates were incubated with anti-CDK2 antibody and precipitated with protein A Sepharose. H1 kinase assays on the immunoprecipitates were performed with kinase containing H1 histone and [³²P]γ-ATP. Phosphorylated H1 histone was analyzed by polyacrylamide gel electrophoresis and autoradiography. Each value was corrected relative to the mean value at 0 h from WKY rats as 1.0. Data are mean ± SEM (n = 4). *P < .05 v without CV11974 (0 h). The arrow indicates phosphorylated H1 histone.

Fig. 3.

Effect of CV11974 on expression of p27^kip1 mRNA in VSMC from WKY rats and SHR

Abundances of p27^kip1 mRNA in VSMC from SHR was significantly (P < .01) higher than those in cells from WKY rats. CV11974 significantly (P < .05) decreased the abundance of p27^kip1 mRNA from 6 to 24 h in VSMC from SHR. In contrast, CV11974 significantly (P < .05) but transiently increased the abundance of p27^kip1 mRNA at 6 and 12 h in VSMC from WKY rats (Fig. 4).

Time course of CV11974 treatment on expression of p27^kip1 mRNA in vascular smooth muscle cells (VSMC) from Wistar-Kyoto (WKY) rats and spontaneously hypertensive rats (SHR). Quiescent VSMC were incubated with 0.1 μmol/L CV11974 for 2, 6, 12, and 24 h and compared to untreated (0 h) control cells. A) p27^kip1 mRNA in VSMC from WKY rats and SHR was analyzed in side by side RT-PCR. B) The ratio of the abundance of p27^kip1 mRNA to that of 18S mRNA was determineds by densitometric analysis. Data are mean ± SEM (n = 4). *P < .05, **P < .01 v without CV11974 (0 h).

Fig. 4.

Discussion

DNA synthesis in quiescent VSMC is thought to be maintained by production of endogenous growth promoting factors such as epidermal growth factor (EGF), insulin-like growth factor (IGF), PDGF, FGF, and Ang II. Both EGF and IGF act as progression factors, whereas PDGF and FGF act as competence factors during the cell cycle of VSMC.^28,29 Ang II is thought to act as a competence factor in the cell cycle, especially during the transition from G₁ to S phase.²⁹ In the present experiments, the proportion of cell in S phase and CDK2 activity were greater in VSMC from SHR than in cells from WKY rats. The AT1 receptor antagonist CV11974 reduced the proportion of cells in S phase and the CDK2 activity in VSMC from SHR. These findings suggest that endogenous Ang II is involved in the accelerated entry into S phase during the cell cycle in VSMC from SHR.

Active cyclin-CDK complexes appear to phosphorylate the retinoblastoma susceptibility gene products, thereby promoting cell cycle progression toward DNA replication.³⁰ G₁ phase is a major point of control for cell proliferation in mammalian cells, and CDC2 family kinases (CDK2 and CDC2) play pivotal roles in cell cycle regulation.³¹ Recently, the CDK inhibitor p27^kip1 was cloned and found to be an inhibitory protein involved in mediating cell cycle arrest at G₁/S phase with interference of cyclin E-CDK2 and cyclin D1–CDK4 activities in response to TGF-β.^31,32

Angiotensin II has been reported to cause only VSMC hypertrophy.⁷ Rao³³ examined the effects of Ang II on the modulation of G₁/S transition molecules and reported that Ang II has no significant effect on steady-state levels of p27^kip1 or on CDK2 activity, whereby Ang II induces only the hypertrophy. However, hyperplastic effects of Ang II have been described in VSMC from SHR.³⁴ In the present experiments, CV11974 inhibited DNA synthesis and reduced the proportion of cells in S phase in VSMC from SHR. In addition, we also observed that CV11974 inhibits the proliferation of VSMC from SHR.¹³ These findings suggest that endogenous Ang II induces hyperplasia of VSMC from SHR. The discrepancy between the hypertrophic and hyperplastic effects of Ang II may be accounted by the different effects of TGF-β on the growth of VSMC.

Transforming growth factor-β acts as a growth inhibitor on most cell types,³⁵ but has a dual effect on VSMC growth, suppressing it at low cell density and stimulating it at high cell density in vitro.³⁶ This dual effect on VSMC growth results from the interaction of TGF-β with a distinct receptor subtype.³⁷ Previously, we demonstrated that VSMC from WKY rats and SHR appear to differ in their expression of TGF-β receptor subtypes.³⁸ Moreover, we recently observed abnormal regulation of TGF-β receptors by Ang II on VSMC from SHR by which endogenous TGF-β induced by Ang II could not counteract the growth-promoting action of Ang II in VSMC from SHR.³⁹ This abnormal regulation of TGF-β receptors seems to be associated with the hyperplastic effects and the accelerated entry of cells into S phase caused by Ang II in VSMC from SHR.

In the present experiments, expression of the CDK2 inhibitor p27^kip1 mRNA was markedly higher in VSMC from SHR than in cells from WKY rats, in which the expression of these mRNAs was significantly inhibited by CV11974. Expression of p27^kip1 is known to be induced by Ang II⁴⁰ and TGF-β.⁴¹ These findings indicate that increased endogenous Ang II maintains the high levels of p27^kip1 in VSMC from SHR. Despite the increased levels of p27^kip1 mRNA, CDK2 activity was higher in VSMC from SHR, suggesting that the elevated CDK2 activity is insensitive to p27^kip1 in VSMC from SHR. Cipriano and Chen⁴² recently reported an insensitivity to growth inhibition to TGF-β known to induce p27^kip1, which is correlated with a lack of inhibition of CDK2 activity in prostate carcinoma cells, suggesting that the insensitivity of CDK2 activity to p27^kip1 is involved in growth of transformed cells. It is considered that the insensitivity of CDK2 activity to p27^kip1 like transformed cells might be associated with the accelerated entry in S phase in the cell cycle and the exaggerated growth in VSMC from SHR. CV11974 slightly increased the expression of p27^kip1 mRNA in VSMC from WKY rats. This differed from the findings in VSMC from SHR. This difference may be explained by the distinct expression of TGF-β receptor subtypes in these VSMC. Further studies are necessary to clarify these points.

Our results indicate that enhanced production of endogenous Ang II regulates the cell cycle, especially during the progression from G₁ phase to S phase, and increases CDK2 activity, which is independent of p27^kip1, in VSMC from SHR.