Vascular Smooth Muscle Polyploidy and Cardiac Hypertrophy in Genetic Hypertension
Abstract We studied the mechanisms responsible for vascular and cardiac hypertrophy in hypertension (pressure load and humoral and genetic factors) in two experimental approaches: (1) We carried out a cosegregation analysis to correlate cardiac and vascular hypertrophy with subphenotypes of blood pressure in an F2 generation of a cross between stroke-prone spontaneously hypertensive rats (SHRSP) and normotensive Wistar-Kyoto rats; (2) we treated 8-week-old SHRSP with perindopril, an angiotensin-converting enzyme inhibitor; losartan, an angiotensin type 1 receptor antagonist; or perindopril combined with a nitric oxide synthase inhibitor to investigate the relative contributions of blood pressure and angiotensin II to the pathogenesis of cardiac hypertrophy and vascular smooth muscle polyploidy. Vascular smooth muscle polyploidy was measured with flow cytometry DNA analysis. Cardiac hypertrophy was assessed by measuring the ratios of heart weight to body weight and left ventricle+septum weight to body weight. Blood pressure was measured with radiotelemetry in the F2 cosegregation experiment and with tail-cuff plethysmography in the pharmacological study. In the F2 rats, the best predictor of smooth muscle polyploidy by ANCOVA was systolic pressure (F=29.28, P<.0001). The ratio of left ventricle+septum weight to body weight had four major predictors: the male progenitor of the cross, sex, pulse pressure, and change in systolic pressure during salt (F=43.67, P<.0001; F=16.37, P<.0001; F=8.41, P=.0022; and F=12.39, P=.0003, respectively). The ratio of heart weight to body weight had similar predictors. In the pharmacological study, treatment with losartan alone, perindopril alone, or perindopril in combination with NG-nitro-l-arginine methyl ester prevented the development of smooth muscle polyploidy and cardiac hypertrophy. The prevention of cardiac hypertrophy was most marked in the SHRSP treated with perindopril plus NG-nitro-l-arginine methyl ester, despite blood pressure being higher in this group than in the two other treatment groups. We conclude that vascular and cardiac hypertrophy in this form of hypertension are regulated by different variables. However, suppression of the action of angiotensin II lessens hypertrophy of both types of muscle.
- angiotensin II
- muscle, smooth, vascular
- cell cycle
- hypertension, genetic
- rats, stroke-prone SHR
Hypertrophy of the capacitance arteries has recently been demonstrated in hypertensive patients by noninvasive, in vivo ultrasound techniques.1 2 Some studies suggest that hypertrophy of capacitance arteries parallels left ventricular hypertrophy, whereas other data suggest that vascular hypertrophy may occur earlier and/or be more prevalent than cardiac hypertrophy in hypertension.3 The mechanisms responsible for vascular and cardiac hypertrophy are a sustained pressure load and less-defined genetic and humoral factors. It is difficult to study the cellular effects of these factors directly in human hypertension. The SHRSP is a good model for studying the mechanisms of vascular and cardiac hypertrophy. Previous studies have documented significant vascular and cardiac hypertrophy in this model,4 and classic genetic crosses can be performed to test the cosegregation of each hypertrophy phenotype with BP.5 In addition, pharmacological interventions may be used to manipulate in a controlled fashion BP and Ang II, which appears to be one of the most important growth factors for VSMCs and cardiac myocytes.6 7 8
Ang II has been reported to induce hyperplasia (cell proliferation) or hypertrophy (enlargement of preexisting cells) in cultured aortic smooth muscle cells6 7 and hypertrophy in cardiomyocytes.8 These effects are mediated by the AT1 receptor, whereas a growth-inhibitory effect of Ang II in coronary endothelial cells is mediated via the AT2 receptor.9 10 Much less is known about the role of Ang II in vascular and cardiac hypertrophy in vivo. VSMCs in vivo may also undergo hypertrophy or hyperplasia. Hypertrophy occurs in capacitance arteries of chronically hypertensive animals or humans and is accompanied by the development of polyploidy; that is, the cells replicate their DNA but do not undergo cell division.4 11 It has been suggested that under some conditions, Ang II may act as an incomplete growth factor whereby the cell receives signals for the increased cell mass and DNA replication associated with cell cycle progression but not for cell division.6 However, these mechanisms have not been studied in vivo. DNA endoreduplication and resulting polyploidy often accompany cellular hypertrophy, occurring in terminally differentiated cardiac myocytes12 as well as in nonterminally differentiated hepatocytes13 and smooth muscle cells.4 11
Previous data from Owens and his collaborators11 14 15 16 showed that the percentage of polyploid aortic smooth muscle cells increases with age and duration of hypertension. Aortic smooth muscle polyploidy was demonstrated in the SHR14 15 16 ; in the two-kidney, one clip Goldblatt model17 ; and in deoxycorticosterone acetate–salt hypertension in the rat.18 Our studies showed that the highest percentage of polyploid cell populations was found in the SHRSP.4 This phenotype has never been subjected to a cosegregation analysis, which is the first step in any analysis of possible causal relationships between a phenotype of interest and hypertension.5 19
We have shown previously that treatment of mature animals with the ACE inhibitor perindopril for 4 weeks resulted in a significant regression of polyploidy in the SHRSP.4 This was associated with a significant reduction in Ang II concentration and significant regression of cardiac hypertrophy.4 Treatment with a combination of hydralazine and hydrochlorothiazide, which resulted in equivalent antihypertensive effect, and increased Ang II concentration had no effect on the percentage of polyploid aortic smooth muscle cells.4 Moreover, equivalent antihypertensive doses of the AT1 receptor antagonist losartan resulted in a significant reduction in VSMC polyploidy and cardiac hypertrophy that was indistinguishable from changes induced by perindopril.20 A recent study by Black et al21 confirms that Ang II might stimulate cardiac and vascular hypertrophy directly in the SHR, independent of its effect on BP.
Data are not conclusive on the effects of early treatment with ACE inhibitors on VSMC polyploidy. Such studies assume greater importance in light of recent data from Wu and Berecek22 which showed that captopril administration to pregnant dams prevented the development of hypertension in SHR up to the second filial generation. This study is an illustration of profound effects of ACE inhibition on the cardiovascular system, effects that may be attributable to central and/or peripheral reduction of Ang II concentration and its direct effect on vascular and cardiac function and structure.
It has been suggested that VSMC quiescence within the vessel wall requires a correct balance between growth-promoting Ang II and growth-inhibitory NO.23 Moreover, NO has been shown to inhibit VSMC proliferation in culture.24 A chronic, nonspecific blockade of NO synthesis with L-NAME raised BP without significant cardiac hypertrophy25 or produced a smaller degree of cardiac hypertrophy than expected from achieved BP.26 These data suggest that the relative contribution of Ang II and NO to growth processes may differ between VSMCs and cardiomyocytes.
The aims of the current study were (1) to perform a classic cosegregation analysis of cardiac hypertrophy and VSM polyploidy to evaluate the relationships between these phenotypes and BP in F2 segregating cohorts of SHRSP×WKY crosses; (2) to test the hypothesis that ACE inhibitor or AT1 receptor antagonist treatment of young rats, before hypertension is fully developed, would prevent VSM polyploidy and cardiac hypertrophy; and (3) to test the contribution of NO to the effects of the ACE inhibitor.
SHRSP and WKY were obtained from colonies established at the Department of Medicine and Therapeutics, University of Glasgow. The breeding stocks were obtained from the colonies maintained at the University of Michigan, which in turn had obtained their breeding stocks from the National Institutes of Health.27 28 Rats were housed under controlled conditions of temperature (21°C) and light (12-hour light/dark cycle; 7 am to 7 pm) and were maintained on normal rat chow (rat and mouse No. 1 maintenance diet, Special Diet Services) and water ad libitum.
For the two reciprocal genetic crosses, one male SHRSP was mated with two WKY females (cross 1), and one male WKY was mated with two SHRSP females (cross 2). These two crosses generated 143 F2 rats (60 in cross 1 and 83 in cross 2); a detailed description of breeding procedures has been previously published.28
For the experiments that examined the effects of pharmacological interventions in vivo, the following experimental groups were used: perindopril-treated SHRSP (n=6; 2 mg/kg per day), SHRSP treated with a combination of perindopril (2 mg/kg per day) and L-NAME (n=6; 20 mg/kg per day), losartan-treated SHRSP (n=8; 10 mg/kg per day), and two sex- and age-matched SHRSP control groups (n=6 in each group). Treatments were given for 7 weeks, starting at 8 weeks of age. Perindopril, L-NAME, and losartan were given in the drinking water. The experiments were approved by the Home Office according to regulations regarding experiments with animals in the United Kingdom. These regulations meet all the requirements of the American Physiological Society.
Indirect BP was measured by tail-cuff plethysmography in conscious, restrained rats as previously described.28 These measurements were undertaken before pharmacological intervention and then once a week during the experiment.
Direct BP was measured with the Dataquest IV telemetry system (Data Sciences Inc) as previously described.28 Briefly, rats at 16 weeks of age were anesthetized with halothane, the flexible catheter was surgically secured in the abdominal aorta, and the transmitter was sutured to the abdominal wall. Rats were housed in individual cages after the operation, and each cage was placed over a receiver panel connected to the personal computer for data acquisition. The hemodynamic data were sampled every 5 minutes for 10 seconds. BP and heart rate data were collected from day 12 to day 16 after surgery as baseline measurements. It has been previously suggested that salt-loaded BP may be used as a separate phenotype for genetic studies.28 In the evening of day 16, the rats received 1% NaCl in their drinking water, and this was continued until day 28 when they were killed. The measurements collected between day 25 and day 28 constituted salt-loaded measurements. Mean values were calculated for intervals of 60 minutes and exported from the Dataquest program in an ASCII format. The BP data used in all the cosegregation analyses represent means of 96 hours of direct telemetry recordings in the individual F2 hybrids at baseline and after salt loading.
Evaluation of Cardiac Hypertrophy
Immediately after exsanguination, the thorax was opened and the heart removed, blotted with tissue paper, and weighed. The atria and right ventricle were then removed, and the left ventricle and septum were weighed. The HW/BW and LV+S/BW ratios were determined.
Preparation of Nuclei and Flow Cytometric Analysis
VSMCs were obtained from enzymatically dissociated rat aortas as previously described.4 From each aorta, 105 primary VSMCs were used for flow cytometry. Nuclei were prepared and stained according to the method of Vindelov et al.29 The cells were resuspended in phosphate buffer (170 mmol/L NaCl, 3.4 mmol/L KCl, and 10 mmol/L Na2HPO4) to a final concentration of 1×106 cells per milliliter. In sequence, 100 μL of this suspension was treated with 450 μL of solution A (30 μg/mL trypsin, pH 7.6) for 10 minutes at room temperature (approximately 25°C) and 375 μL of solution B (0.5 mg/mL trypsin inhibitor and 0.1 mg/mL RNAse, pH 7.6) for a further 10-minute incubation at room temperature, and then 375 μL of solution C (0.4 mg/mL propidium iodide and 1.1 mg/mL spermine tetrahydrochloride, pH 7.6) was added directly; this was incubated in darkness at 0°C until analysis. Human peripheral blood lymphocytes were treated as above to provide a diploid profile for DNA peak standardization. DNA flow cytometry was carried out with a fluorescence-activated cell sorter (Becton-Dickinson UK Ltd) with a 15-mW argon air-cooled laser and emission wavelength of 488 nm. Analysis of DNA profiles obtained was carried out with the LYSYS software package (Becton-Dickinson).
Plasma Renin-Angiotensin System
In experiments that examined the effects of pharmacological interventions in vivo, rats were anesthetized with halothane at the end of the experimental period, and blood was drawn for terminal measurements of Ang II concentration and ACE activity. Plasma Ang II was measured by radioimmunoassay after Sep-Pak C28 (Waters Associates Inc) extraction, after which high-performance liquid chromatography of the Sep-Pak extract was performed as a check for its direct assay.4 20 30 Plasma ACE activity was measured directly with a color kit (Fujirebo Inc; distributed in the United Kingdom by Mast Diagnostics).
Drugs and Materials
Perindopril was a gift from Servier Laboratories; losartan was a gift from DuPont/Merck. Enzymes, trypsin inhibitor, spermine tetrahydrochloride, propidium iodide, and L-NAME were purchased from Sigma Chemical Co.
In experiment 1, associations between VSM polyploidy or the cardiac mass indexes and baseline or salt-loaded BP values were assessed with Pearson’s product-moment correlations. In view of possible confounding variables, these simple correlations were supplemented by ANCOVA, carried out in the statistical package (Minitab, release 10). A value of P<.05 was considered significant. In experiment 2, the analysis of BP and heart rate across time for the two control and three treatment groups was carried out with repeated measures ANOVA, based on Wald’s test, in the BMDP statistical package. For pairwise comparisons, the results for each rat were averaged across time, and Tukey’s correction for multiple comparisons was applied when necessary. For the two perindopril-based treatment groups and their related controls, one-way ANOVA was used to compare mean values of the cardiac mass indexes, polyploidy, and ACE and Ang II levels, followed by Tukey’s pairwise comparisons. Unpaired t tests were used to contrast means of the same measurements in the losartan treatment group and its control sample. The usual residuals plots were used to check assumptions of normality, and log transformations were used when appropriate (Minitab, release 10).
Experiment 1: Cosegregation Analysis
Detailed analysis of direct telemetry BP values in the F2 cohorts studied has been previously described.28 Fig 1⇓ shows plots of individual phenotypes studied in parental SHRSP and WKY strains, in F1 hybrids that inherit 50% of genetic material from either parent, and in F2 cosegregating cohorts. VSM polyploidy was significantly higher in SHRSP than WKY (Fig 1A⇓). The percentage of cells in the G2+M phase of the cell cycle in F1 hybrids showed some overlap with parental values but lesser variance, whereas VSM polyploidy in the F2 cosegregating generation spanned the entire variance from the lowest normotensive values to the highest hypertensive polyploidy (Fig 1A⇓). The HW/BW ratio had a pattern of distribution similar to that of VSM polyploidy in the parental strains and F1 and F2 cohorts (Fig 1B⇓). Fig 1C⇓ shows a plot of the LV+S/BW ratio. The pattern of distribution of this phenotype in the F1 hybrids appears different from that of the other two phenotypes studied. The LV+S/BW ratios in F1 hybrids cluster around the SHRSP values, suggesting a dominant inheritance of this trait.
In the F2 segregating cohorts, there were significant positive correlations between VSM polyploidy and direct (obtained by radiotelemetry) baseline and salt-loaded systolic, diastolic, and pulse pressures (Fig 1D⇑ and Table 1⇓). The HW/BW and LV+S/BW ratios were significantly correlated with baseline systolic and pulse pressures and with salt-loaded systolic, diastolic, and pulse pressures (Table 1⇓). There was no significant correlation between HW/BW ratio and baseline diastolic pressure, but the LV+S/BW ratio showed a significant positive relationship with baseline diastolic pressure (Table 1⇓). Moreover, VSM polyploidy had no significant relationship with either HW/BW or LV+S/BW ratio (n=99; r=.15, P=NS and r=.13, P=NS, respectively). In view of possible confounding variables such as sex or the strain of the male progenitor of the cross, we used ANCOVA, selecting variables for inclusion in the model by a stepwise procedure. We used seven putative predictors for the ANCOVA: sex, male progenitor of the cross, the origin of the Y chromosome, baseline systolic pressure, change of systolic pressure during salt loading, baseline pulse pressure, and change of pulse pressure during salt loading, the latter four predictors being determined by direct radiotelemetry measurements. The only significant predictor of VSM polyploidy was baseline systolic BP (P<.0001, Table 2⇓). Furthermore, the ANCOVA showed that the HW/BW ratio had four major predictors: male progenitor of the cross, sex, change of systolic BP during salt loading, and baseline pulse pressure (P=.004, P=.0001, P=.0017, and P=.0068, respectively; Table 2⇓). The LV+S/BW ratio had the same four predictors (P<.0001, P<.0001, P=.0022, and P=.0003, respectively; Table 2⇓).
Experiment 2: Pharmacological Interventions
BP and Heart Rate
Systolic BPs for each control group and their respective treated groups are shown in Fig 2⇓. There were significant differences between the three groups (control, perindopril-treated, and perindopril+L-NAME–treated; F=42.4, P<.001), and there was a significant group×time interaction (F=121.5, P<.001; Fig 2A⇓). Pairwise comparisons showed that average BPs of both treatment groups were significantly different from those in the control group (95% CI, 23.9 to 56.6 and 5.6 to 38.4 mm Hg; P=.02 each for perindopril-treated and perindopril+L-NAME–treated groups, respectively). Moreover, average BPs in the perindopril-treated group were significantly lower than in the perindopril+L-NAME–treated group (95% CI, −34.6 to −1.9 mm Hg, P=.02). The comparison of losartan-treated rats with their control group showed a group difference (F=16.4, P<.001), and again, there was a significant group×time interaction (F=136, P<.001; Fig 2B⇓). Pairwise comparisons of heart rates for each control group and their respective treated groups showed no statistically significant differences (data not shown).
Cardiac Mass Indexes
The measurements of heart weight and left ventricular weight are expressed as the ratios HW/BW (milligrams per gram) and LV+S/BW (milligrams per gram) and are shown in Fig 3⇓. The HW/BW ratio was significantly lower in the perindopril+L-NAME–treated group than in the control group (3.03±0.21 and 4.04±0.18 mg/g; F=7.5; P=.02; 95% CI, 0.32 to 1.69; Fig 3A⇓), but there was no significant difference in HW/BW ratio between the control and perindopril-treated groups (Fig 3A⇓). The LV+S/BW ratios were significantly lower in the perindopril+L-NAME–treated and perindopril-treated groups compared with untreated SHRSP controls (2.08±0.10, 2.61±0.11, and 3.13±0.18 mg/g, respectively; F=23.1; P<.001; 95% CI, 0.64 to 1.45 and 0.14 to 0.91, respectively; Fig 3A⇓). There was also a significant difference between the perindopril-treated and perindopril+L-NAME–treated groups (P=.02; 95% CI, 0.12 to 0.93; Fig 3A⇓).
In the losartan-treated group, HW/BW and LV+S/BW ratios were significantly lower than in the control group (3.89±0.07 and 4.68±0.07 mg/g; t=7.9; P<.001; 95% CI, 0.57 to 1.01; and 3.16±0.07 and 4.01±0.06 mg/g; t=8.5; P<.001; 95% CI, 0.63 to 1.08, respectively; Fig 3B⇑).
Flow Cytometry Analysis
The percentage of cells in the G2+M phase of the cell cycle was significantly lower in the perindopril-treated and perindopril+L-NAME–treated groups than in untreated SHRSP controls (18.8±1.8%, 22.8±1.8%, and 31.9±1.7%, respectively; F=15.2; P<.001; 95% CI, 6.7% to 19.5% and 2.4% to 15.7%, respectively; Fig 4A⇓). There was no significant difference between the perindopril-treated and perindopril+L-NAME–treated groups. The percentage of aortic cells in the G2+M phase of the cell cycle was significantly lower in the losartan-treated group compared with control SHRSP (13.2±1.04% and 27.8±2.7%, respectively; t=5; P=.0025; 95% CI, 7.4% to 21.8%; Fig 4B⇓).
Plasma Renin-Angiotensin System
ACE activity was lower in the perindopril-treated and perindopril+L-NAME–treated groups than in untreated SHRSP controls (2.89±0.9, 2.4±0.2, and 16.2±1.3 nmol/mL per minute, respectively; F=25.1; P<.001; 95% CI, 1.17 to 2.8 and 1.04 to 2.77, respectively; Fig 5A⇓). There was no significant difference between the perindopril-treated and perindopril+L-NAME–treated groups. Losartan treatment had no effect on ACE activity (Fig 5B⇓). Ang II concentrations were significantly lower in the perindopril-treated and perindopril+L-NAME–treated groups compared with untreated SHRSP (2.7±0.6, 11.8±2.6, and 174.3±19 pg/mL, respectively; F=117.3; P<.001; 95% CI, 3.53 to 5.0 and 2.03 to 3.56, respectively; Fig 5A⇓). There was also a small but significant difference between the perindopril-treated and perindopril+L-NAME–treated groups (P=.02; 95% CI, −2.25 to −0.7; Fig 5A⇓). In the losartan-treated group, Ang II concentration was significantly higher than in the control group (846.7±87.6 and 160.3±39.4 pg/mL; t=6.9; P<.001; 95% CI, 1.6 to 2.37; Fig 5B⇓).
To test the hypothesis that cardiac hypertrophy and/or VSM polyploidy traits are under the control of the same genetic mechanism that regulates BP, we measured these parameters in the F2 hybrids derived from two reciprocal crosses of SHRSP and WKY. The F2 progeny is called segregating because it is in these rats that the genes controlling BP and the putative genes controlling the other phenotypes had a chance to recombine.19 31 Among phenotypic traits that have been shown previously to cosegregate with BP are VSM responses to cations, oscillatory activity of mesenteric or tail arteries in SHR and SHRSP, increased lymphocyte potassium efflux in SHRSP, and several other traits (summarized in Reference 31). The current study is the first to describe a systematic cosegregation analysis of HW/BW and LV+S/BW ratios and VSM polyploidy with all subphenotypes of BP measured with a radiotelemetry system (baseline and salt-loaded systolic, diastolic, and pulse pressures). An additional strength of this cosegregation analysis comes from the reproducibility and fidelity of the phenotypic measurements performed with the use of radiotelemetry.28
It is apparent that all three phenotypes—VSM polyploidy, HW/BW ratio, and LV+S/BW ratio—showed the expected distribution in the F2 segregating cohorts. Moreover, all three phenotypes showed a significant positive relationship with the BP subphenotypes, the only exception being the HW/BW ratio and baseline diastolic pressure. Because of the interdependence of variables studied, ANCOVA was applied to these data, and major predictors were calculated for each of the phenotypes studied. VSM polyploidy was strongly predicted by systolic BP but not by other putative predictors examined in the analysis. In contrast, HW/BW and LV+S/BW ratios had four significant predictors: male progenitor of the cross, sex, baseline pulse pressure, and change of systolic pressure during salt loading. The presence of four predictors suggests more complex influences on cardiac than vascular hypertrophy. Previous studies reported that pulse pressure is a better predictor of vascular hypertrophy than either systolic or diastolic pressure.32 33 The current cosegregation analysis does not support this contention, with pulse pressure being a good predictor of cardiac hypertrophy but not of VSM polyploidy. It has to be noted that a positive cosegregation analysis indicates that BP and the phenotype tested are causally related but is not able to distinguish between the cause and effect.19 31 It is likely that more-complex relationships found by ANCOVA for the determination of cardiac mass indexes than the VSM polyploidy indicate that cardiac and left ventricular hypertrophy are significantly influenced by other trophic factors, a view that has been previously suggested in animal models of genetic hypertension and in essential hypertension in humans.34 35
These complex relationships between cardiac and vascular hypertrophy, BP, and other trophic factors are further shown by our pharmacological studies. Treatment of young SHRSP with either perindopril or losartan prevented the development of hypertension. The group treated with perindopril and L-NAME had BP values that were lower than those in untreated SHRSP controls but higher than in SHRSP treated with perindopril alone. However, the prevention of cardiac and left ventricular hypertrophy was most marked in the perindopril+L-NAME group. These data are in agreement with previous studies that showed that a nonspecific NO synthase inhibitor, such as L-NAME, when given alone causes lesser cardiac hypertrophy than would be expected from BP elevation.25 26 It has been suggested that L-NAME may compete with ribosomal enzymes involved in the incorporation of l-arginine into protein.26 This effect combined with reduced coronary blood flow may contribute to reduced protein synthesis in cardiomyocytes. In the current experiment, the prevention of left ventricular hypertrophy by treatment with losartan was highly significant and similar to the effects of perindopril. Therefore, prevention of the development of myocardial hypertrophy during perindopril treatment could be attributed to a decrease in BP and Ang II generation.26 Ang II is a stimulus for protein synthesis and thus increased cardiac mass.8 There was also a highly significant effect of losartan on cardiac hypertrophy expressed as HW/BW ratio, whereas this effect in the perindopril-treated group did not reach statistical significance. This may suggest that the effects of specific inhibition of AT1 receptors are also important in the right ventricle and atria under our experimental conditions. AT1 receptors have been demonstrated within all four cardiac chambers, and their density may be increased in hypertension.36 37
The analysis of VSM polyploidy contrasts with cardiac hypertrophy data. Treatments with perindopril, perindopril+L-NAME, and losartan all caused significant prevention of VSM polyploidy. The magnitude of difference was similar to those differences observed in previous studies performed in mature animals.4 20 38 39 After treatment, the percentage of aortic VSMCs in the G2+M phase of the cell cycle was comparable to the percentage of such cells observed in the normotensive WKY reference strain.4 20 39 Moreover, in contrast to cardiac and left ventricular hypertrophy, SHRSP treated with perindopril and L-NAME did not differ from rats treated with perindopril alone. The current results may support the existence of different regulatory mechanisms in the cell cycle of cardiac and vascular myocytes. We have shown previously that treatment with perindopril of mature SHRSP resulted in a significant reduction in circulating Ang II and ACE activity.4 Similar results have been shown in the current study. Campbell et al40 showed that the effects of perindopril on Ang II levels in aortic tissue were similar to those described for plasma. The combination treatment with perindopril and L-NAME resulted in Ang II levels that were slightly but significantly higher than levels in SHRSP treated with perindopril alone.
It has been suggested that VSM hypertrophy and polyploidy, which affect large capacitance arteries in hypertension, are associated with decreased arterial compliance.38 Safar et al41 showed that ACE inhibitors markedly reduced the local concentrations of the components of the renin-angiotensin system in the wall of large arteries in patients with essential hypertension. These changes were associated with improved arterial compliance.41 More recently, Bentos et al42 showed that AT1 receptor gene polymorphism is associated with the development of aortic stiffness in hypertensive patients. This finding reinforces the implication of the renin-angiotensin system in structural and functional changes of large conduit arteries. Two recent articles by Ichiki et al43 and Hein et al44 reported that targeted disruption of the mouse AT2 gene resulted in an increased sensitivity to the pressor action of Ang II. The study by Ichiki et al also demonstrated elevated BP in AT2 knockout mice at baseline compared with their wild-type controls. These results indicate that AT2 receptor mediates a depressor effect and antagonizes the AT1-mediated pressor action of Ang II.43 It is possible that these results are relevant to our pharmacological study, particularly to the losartan-treated group, in which Ang II acts unopposed on the AT2 receptor. The use of specific AT2 receptor antagonists will be necessary to further investigate these relationships.
We conclude that systolic BP and Ang II play a major role in VSM polyploidy in the SHRSP. Cardiac hypertrophy is regulated by a more-complex interaction of BP, sex, Ang II, and the l-arginine–NO pathway. These results suggest that the prevention of cardiac and vascular hypertrophy may require targeting of somewhat different growth-promoting mechanisms. Nevertheless, suppression of the action of Ang II has a growth-inhibitory effect in both tissues.
Selected Abbreviations and Acronyms
|Ang II||=||angiotensin II|
|AT1,AT2||=||angiotensin II receptor type 1, type 2|
|HW/BW||=||ratio of heart weight to body weight|
|L-NAME||=||NG-nitro-l-arginine methyl ester|
|LV+S/BW||=||ratio of left ventricle plus septum weight to body weight|
|SHR||=||spontaneously hypertensive rat(s)|
|SHRSP||=||stroke-prone spontaneously hypertensive rat(s)|
|VSM||=||vascular smooth muscle|
|VSMC||=||vascular smooth muscle cell|
This work was supported by British Heart Foundation grants 92100 and 95123. A.F.D. is a British Heart Foundation Senior Research Fellow. The authors thank the Institut de Recherches Internationales Servier for the supply of perindopril, DuPont/Merck for the supply of losartan, and Angela McKay for typing the manuscript. We also thank Dr Nicholas Schork for his advice on statistical analysis and its interpretation.
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