Published online before print July 7, 2003, doi: 10.1161/01.HYP.0000082360.65547.7C
(Hypertension. 2003;42:184.)
© 2003 American Heart Association, Inc.
Scientific Contributions |
Different Cell Cycle Regulation of Vascular Smooth Muscle in Genetic Hypertension
From Cardiovascular Research, Physiology Institute, University Zürich-Irchel, and the Division of Cardiology, University Hospital, Zürich (F.C.T., H.G., K.F., T.F.L.); and the Physiology Institute, University of Fribourg (C.B.), Fribourg, Switzerland.
Correspondence to Felix C. Tanner, MD, Cardiovascular Research, Physiology Institute, University Zürich-Irchel, Winterthurerstrasse 190, CH-8057 Zürich, Switzerland. E-mail felix.tanner{at}access.unizh.ch
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Abstract |
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Vascular smooth muscle cells (VSMC) from spontaneously hypertensive rats (SHR) proliferate faster than those from Wistar-Kyoto rats (WKY). Therefore regulation of cell cycle progression was examined in VSMC from both strains. Analysis of G1 progression was performed in VSMC synchronized by serum starvation. Double staining for propidium iodide and bromodeoxyuridine revealed that G1 progression was faster in SHR as compared with WKY. Indeed, 59±6% of VSMC from SHR but only 14±10% of those from WKY had left G1 phase after 24 hours of mitogenic stimulation. Moreover, 15±2% of SHR cells had already completed the cycle at this time point. Western blot analysis demonstrated that the level of cyclin D, cyclin E, and cyclin A was higher in SHR cells progressing through G1 phase, whereas expression of cyclin-dependent kinase 2 as well as the cyclin-dependent kinase inhibitors p21 and p27 were similar in the two groups. Consistent with a higher level of cyclins, the activity of cyclin-dependent kinase 2 was more pronounced in SHR cells. Analysis of G2 progression was performed in VSMC synchronized by treatment with aphidicolin and revealed an additional difference in cell cycle regulation between SHR and WKY. Indeed, the level of cell division cycle kinase 2 was higher in cells from SHR, whereas that of its catalytic partner cyclin B was similar. Consistent with this pattern of expression, the activity of cell division cycle kinase 2 was more pronounced in VSMC from SHR as compared with WKY. Thus, these data demonstrate that the different proliferation of VSMC from SHR and WKY is related to a different progression in G1 phase as the result of the expression of cyclin D, cyclin A, and cyclin E as well as a different progression in G2 phase caused by expression of cell division cycle kinase 2.
Key Words: rats, spontaneously hypertensive • muscle, smooth, vascular • hypertension, genetic • molecular biology
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Introduction |
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Vascular smooth muscle cell (VSMC) proliferation contributes to the arterial remodeling occurring in hypertension.1 Indeed, VSMC from spontaneously hypertensive rats (SHR) proliferate faster as compared with normotensive control rats (Wistar-Kyoto rats; WKY).2 This difference has been attributed to cyclin-dependent kinase II activity.3,4 A detailed analysis of cell cycle regulation in SHR as compared with WKY, however, has not been performed yet.
Cell cycle progression is determined by formation of protein complexes between cyclins and cyclin-dependent kinases (cdk), which depends on the cell cycle–regulated expression of cyclins assembling with preexisting cdk.5 Cyclin D associates with cdk2, cdk4, and cdk6 and is important for early G1 progression, whereas cyclin E associates primarily with cdk2 and promotes late G1 progression.6 Once the cell progresses toward the G1-S border, cyclin E is degraded and cyclin A enters into a complex with cdk2, hence triggering S-phase entry.7 Several potent protein inhibitors of cyclin-cdk complexes are implicated in regulating kinase activity. p21 is expressed during all phases of the cell cycle and may be induced when quiescent cells are stimulated to proliferate. This apparent paradox is resolved by the observation that conversion of active to inactive cyclin-cdk complexes is achieved by altering the ratio of cyclin-cdk to p21. Thus, p21 is involved in control of cyclin-cdk activity during all phases of the cell cycle.8 p27 is present mainly during the G1 phase of the cell cycle, although it continues to be synthesized at low levels during the remaining phases of the cycle. p27 protein levels are elevated in quiescent cells and decrease on mitogenic stimulation.9 As downregulation of p27 permits proliferation, this protein is of major importance for restriction point control.10 Apart from its downregulation, sequestration of p27 by cyclin D complexes is an important mechanism for activation of cdk2 kinase and G1 progression toward the restriction point.5
Growth-promoting and growth-inhibitory extracellular signals are integrated in G1 phase. We therefore hypothesized that G1 progression differs in SHR as compared with WKY. To analyze regulation of G1 progression, we examined expression and activity of cdk2, its catalytic partners, and its endogenous inhibitors. To exclude an additional defect in G2 phase, a similar analysis was performed with the proteins regulating G2 progression.
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Methods |
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Cell Culture and Proliferation
VSMC were isolated from aortas of SHR and WKY as described.11 Cells were characterized by immunofluorescent staining with anti–smooth muscle-
actin antibody (No. 1148818, Boehringer Mannheim) and cultured as described.11 Cells from passage 3 to 10 were used for experiments. To measure proliferation, cell number was determined after 0, 2, and 4 days of mitogenic stimulation with an improved Neubauer hematocytometer. To synchronize cells in G0, they were kept in serum-free medium containing 0.1% BSA (Sigma) for 48 hours; G1 progression was examined for up to 48 hours after stimulation with 10% FCS.12 To synchronize cells in the S phase, they were kept in medium with 10% FCS and 30 µg/mL aphidicolin (Sigma); G2 progression was studied for up to 24 hours after removal of aphidicolin.12
FACS Analysis
Growth-arrested cells were stimulated with 10% FCS for up to 48 hours. When cell cycle distribution after 24 hours of stimulation was examined, BrdU (10 µmol/L) was added after 12 hours of stimulation, resulting in a 12-hour incorporation time. Similarly, when cell cycle distribution after 48 hours of stimulation was examined, BrdU was added after 36 hours of stimulation. Cells were harvested, washed twice in PBS (4°), fixed in 70% ethanol for 30 minutes on ice, resuspended in 2 mol/L HCl for 30 minutes at room temperature, and permeabilized in 0.5% Tween 20. Anti-BrdU Alexa-Fluor 488 antibody (Molecular Probes) was added at a 1:10 dilution and incubated for 30 minutes at room temperature. After 2 washes in PBS (4°), 50 µg/mL propidium iodide and 0.1 µg/mL RNase were added and the cells were analyzed through the use of the FACScalibur (Becton Dickinson) cytometer.13
Western Blot Analysis and H1 Kinase Assay
VSMC were lysed as described.14 Sixty micrograms of protein was loaded per lane, resolved by SDS-PAGE under reducing conditions, and blotted on Immobilon-P transfer membranes (Millipore). Equal loading was confirmed by Ponceau S staining for all experiments. Membranes were incubated with antibodies against cyclin D (No. 06–137, Upstate Inc), cyclin E (No. sc-481), cyclin A (No. sc-751), cyclin B (sc-245), cdk2 (No. sc-163), p21Cip1 (No. sc-6246; all from Santa Cruz Biotechnology Inc); cdc2 (No. PC25, Oncogene Research Products); p27Kip1 (No. K25020, Transduction Laboratories); and acetylated tubulin (No. T-6793, Sigma). Proteins were detected with a horseradish peroxidase–coupled secondary antibody by means of the ECL system (Amersham Pharmacia Biotech). Immunoprecipitations for kinase assays were performed as described.15 Labeled proteins were resolved on 12% SDS-PAGE and subjected to autoradiography.
DAPI Staining
VSMC on tissue culture slides were incubated with 2 µg/mL DAPI (Boehringer Mannheim) in methanol for 20 minutes at 4° in the dark and then washed in PBS. The percentage of nuclei with mitotic figures was determined with the use of a fluorescence microscope (Leitz).16
Statistics
Results represent the mean value of 5 experiments. Data are expressed as mean±SEM, and statistical comparisons were performed with the use of a Student unpaired t test or repeated ANOVA as appropriate.
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Results |
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Proliferation
VSMC proliferation was assessed by determining cell number in response to stimulation with 10% FCS over a period of 4 days. VSMC from SHR proliferated faster than those from WKY (Figure 1). Cells with fragmented DNA, as determined by propidium iodide staining and flow cytometric analysis, were found to a similar percentage in VSMC from SHR (0.76±0.76%) and WKY (0.42±0.32%; P=NS; n=5), indicating that the different growth rates are not affected by cell death.
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Cell Cycle Progression
VSMC were growth-arrested and then stimulated with 10% FCS. Cell cycle progression was examined by double staining with propidium iodide and BrdU. After 24 hours of mitogenic stimulation, 59±6% of VSMC from SHR but only 14±10% of those from WKY had incorporated BrdU (P<0.05; n=5; Figure 2). After 48 hours of stimulation, 92±2% of VSMC from SHR as compared with 65±23% of those from WKY were BrdU-positive (P=NS; n=5; Figure 2). These data indicate that VSMC from SHR progress faster in the cell cycle as compared with those from WKY. Consistent with this interpretation, 25±9% of SHR VSMC in the S phase, 20±5% in the G2/M phase, and 15±2% in the G0/G1 phase had incorporated BrdU after 24 hours of stimulation, as opposed to 14±10% of WKY VSMC in all cell cycle phases together (Figure 3). In line with this observation, the number of mitotic figures in a randomly proliferating population at a given time point was higher for SHR as compared with WKY (SHR, 2.0±0.1%; WKY, 0.7±0.6%).
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Cell Cycle Regulation
Regulation of the cell cycle was examined by Western blot analysis for cell cycle proteins as well as by the respective kinase assays. To assess G1 progression, VSMC were growth-arrested by serum starvation and then stimulated with 10% FCS. The level of cyclin D in arrested VSMC from SHR was higher as compared with those from WKY. Moreover, induction of cyclin D occurred faster and reached a higher level in SHR as compared with WKY. Indeed, induction started within 6 hours in SHR VSMC, whereas a weak effect on protein level was only observed at 12 hours in WKY (Figure 4). In SHR VSMC, induction of cyclin E and cyclin A started within 12 hours and reached a maximum at 36 hours, with the level of both proteins beginning to decline after 48 hours of stimulation. In contrast, in WKY VSMC, induction of both cyclins started within 24 hours and was still ongoing after 48 hours of stimulation (Figure 5). Regulation of the catalytic partner of these cyclins, cyclin-dependent kinase 2 (cdk2), in SHR was identical to that seen in WKY; the protein level started to rise within 12 hours and reached a plateau after 36 hours of stimulation. This pattern of regulation was observed for both nonphosphorylated (upper band) and threonine-phosphorylated (lower band) cdk2 (Figure 5). The cyclin-dependent kinase inhibitor (cki) p21 was induced in proliferating VSMC. The degree of induction was similar in SHR and WKY, with a tendency toward a lower p21 level in both arrested and proliferating cells from SHR (Figure 5). The latter observation, however, was not made in all experiments; in some blots, the p21 level was comparable between SHR and WKY. The cki p27 was downregulated over 36 hours of stimulation and remained at this low level at 48 hours. This pattern was identical in VSMC from SHR and WKY (Figure 5). Finally, cdk2 kinase activity was assessed by using histone H1 as substrate. Consistent with the expression pattern of cdk2 and its catalytic partners, kinase activity was barely detectable in arrested cells and increased after mitogenic stimulation; this activation occurred faster and reached a higher level in SHR as compared with WKY (Figure 5).
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To assess G2 progression, VSMC were synchronized in the S phase with the use of 10 µg/mL aphidicolin and then released from this block by removal of the drug. In both SHR and WKY, cyclin B expression was not altered during G2 progression; moreover, expression was similar to that in randomly proliferating cells (Figure 6). In contrast, the catalytic partner of cyclin B, cell division cycle kinase 2 (cdc2), was upregulated during G2 progression. The level at the beginning of G2 as well as during progression of the latter was higher in VSMC from SHR as compared with WKY. A similar difference was observed in randomly proliferating cells. Consistent with this pattern of expression, cdc2 kinase activity increased during G2 progression, and activity was higher in cells from SHR as compared with WKY (Figure 6).
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Discussion |
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TopAbstractIntroductionMethodsResultsDiscussionReferences |
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This study demonstrates that the different proliferation rates of VSMC from SHR as compared with WKY are related to faster G1 progression in the SHR cells; this effect is caused by a higher level of cyclin D, cyclin E, and cyclin A, whereas cdk2, p21, and p27 show a similar regulation. An additional difference in cell cycle progression of SHR as compared with WKY was detected in theG2 phase; this effect is related to the cdc2 level.
The primary regulator of cdk activity is the cyclin subunit. The cyclin level oscillates during the cell cycle, whereas cdk expression is much less subjected to periodic alterations.17 Hence, cyclin expression increases during specific phases of the cycle leading to association of the cyclin with the corresponding cdk. Activation of cdk2 is related to induction of cyclin D in the early G1 phase, cyclin E in the late G1 phase, and cyclin A toward the G1/S-border.18,19 Therefore, the faster and higher induction of cyclin D as well as the higher level of cyclin E and cyclin A in VSMC from SHR is consistent with both the higher cdk2 activity and the faster G1 progression in these cells. Moreover, the higher level of cyclin D induces sequestration of p27 into cyclin D complexes, leading to a shift in kinase activity from cyclin D toward cyclin E and cyclin A. This shift promotes G1 progression independent of cdk2 protein level; hence, the similar cdk2 level but higher cdk2 activity in SHR as compared with WKY is explained by the higher level of cyclin D, cyclin E, and cyclin A as well as by sequestration of p27.
The cki p21 and p27 are regulated in a typical manner in both SHR and WKY; p21 is slightly induced when cells progress in G1 and enter the S phase, whereas p27 is downregulated during this part of the cell cycle.20,21 Because both proteins are regulated by extracellular signals and represent potent inhibitors of G1 progression,22 it is somewhat surprising that there is no differential regulation between VSMC from SHR and WKY. In a recent report, such a differential regulation of p27 has been observed at the mRNA level.4 This finding, however, is not necessarily inconsistent with our observations, as p27 expression is regulated both at the mRNA and the protein level; hence, definitive conclusions regarding the effect of p27 can only be drawn from an analysis of protein expression. Thus, the conclusion that neither p21 nor p27 mediates the differences in G1 progression in VSMC from SHR as compared with WKY seems well justified.
Complexes of cyclin B with cdc2 assemble during the early G2 phase; when these complexes are activated, they signal G2 progression and entry into mitosis.23 Both expression and activity of cdc2 were higher in VSMC from SHR as compared with WKY, indicating that regulation of G2 phase differs in the two strains. The difference was observed in both randomly proliferating and synchronized cells. A higher cdc2 activity in randomly proliferating cells may occur secondary to the higher proliferation rates in this group. Indeed, in a population with faster proliferation, a higher number of cycling cells is present at a given time point, leading to a higher activity of cell cycle proteins in all phases. In contrast, in synchronized cells, a real difference in protein activity must be present, as the cells were arrested in the S phase by treatment with aphidicolin and then released by removal of the drug; differences in the number of cycling cells are largely eliminated under these conditions. In contrast to the G1 phase, the higher activity of G2 regulatory proteins in SHR is not related to a cyclin, as the cyclin B level was identical in VSMC from SHR and WKY. The difference is rather caused by cdc2, which was expressed at a higher level in SHR. Thus, cell cycle progression in VSMC from SHR and WKY differs in G1 phase as well as in G2 phase, and different types of proteins mediate these effects.
Hypertrophy and polyploidization contribute to the medial thickening in hypertensive arteries.24 Polyploidization of VSMC from SHR has been attributed to a defective mitotic spindle cell cycle checkpoint. Indeed, VSMC from SHR express high levels of Akt/PKB and hence fail to keep cyclin B levels elevated in the presence of colcemid, resulting in polyploidization.25 These observations have been made in VSMC exposed to colcemid only, indicating that the mitotic checkpoint control but not regulation of G2 progression per se is defective in SHR. Hence, these data are complementary to our observations regarding the regulation of G2 progression. Indeed, a different mitotic spindle checkpoint control and a different regulation of G2 progression may well exist in parallel.
Perspectives
Our data reveal that the higher proliferation of VSMC from SHR as compared with WKY is due to differences in regulation of G1 and G2 progression. Indeed, VSMC from SHR have a higher level of cyclin D, cyclin E, and cyclin A, whereas the level of cdk2 does not differ from that of WKY. As a consequence, the higher cdk2 activity in VSMC from SHR is determined by the higher cyclin level as well as by enhanced sequestration of p27. These observations improve our understanding of cell cycle regulation in VSMC. Furthermore, they demonstrate that VSMC exhibit intrinsic differences of cell cycle regulation in genetic hypertension. Hence, the current study raises the question of whether such differences may contribute to the development of medial thickening in hypertensive subjects. Moreover, such differences may be involved in the pathogenesis of atherosclerosis in hypertensive individuals. Therefore, the cell cycle may be a potential therapeutic target for the prevention of vascular disease in hypertensive patients.
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Acknowledgments |
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This study was supported by the Swiss National Science Foundation (grants 31-47119.96 and 32-51069.97), the Swiss Heart Foundation, and the Hartmann Müller Foundation.
Received November 22, 2002; first decision December 12, 2002; accepted June 9, 2003.
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