Long-term ACE Inhibition Doubles Lifespan of Hypertensive Rats
Abstract
Background We compared the outcome of lifelong treatment with the ACE inhibitor ramipril in young prehypertensive stroke-prone spontaneously hypertensive rats (SHR-SP) and age-matched normotensive Wistar-Kyoto (WKY) rats. Ramipril was given in an antihypertensive and subantihypertensive dose. In addition to the primary end point, lifespan, surrogate parameters such as cardiac left ventricular hypertrophy, cardiac function and metabolism, and endothelial function were studied.
Methods and Results One-month-old SHR-SP and WKY rats, 135 of each, were randomized into 3 groups. Each group was treated via drinking water with an antihypertensive high dose of ramipril (HRA, 1 mg · kg−1 · d−1), a nonantihypertensive low dose of ramipril (LRA, 10 μg · kg−1 · d−1), or placebo. Body weight and blood pressure were determined every 3 months. Molecular, biochemical, and functional data were assessed in SHR-SP and WKY rats after 15 and 30 months, respectively. These were the times when ≈80% of the corresponding placebo group had died. Early-onset long-term ACE inhibition with HRA doubled lifespan to 30 months in SHR-SP, which was identical to the lifespan of placebo-treated normotensive WKY rats. LRA treatment prolonged lifespan from 15 to 18 months. In SHR-SP, left ventricular hypertrophy was completely prevented by HRA but not by LRA treatment. Cardiac function and metabolism as well as endothelial function were significantly improved by both doses of ramipril. Carotid expression of endothelial NO synthase was moderately enhanced, whereas cardiac ACE expression and activity were decreased to values of placebo-treated WKY rats.
Conclusions Lifelong ACE inhibition doubles lifespan in SHR-SP, matching that of normotensive WKY rats. This effect correlated with preservation of endothelial function, cardiac function/size, and metabolism. Thus, these data predict a beneficial outcome on survival in high-risk patients with hypertension and associated cardiovascular diseases by ACE inhibition.
Malignant long-lasting hypertension causes structural and functional modifications in the cardiovascular system associated with an increase in mortality and morbidity.1 The SHR-SP is an experimental model of malignant hypertension in which the animals develop severe cerebral and cardiac dysfunction/damage and die of stroke.23 Such complications were also found in high-risk patients with long-lasting hypertension.
Several clinical studies with long-term inhibition of ACE exhibited (1) increases in life expectancy of patients with advanced heart failure,4 (2) delays in the progression to heart failure in patients with asymptomatic left ventricular dysfunction,5 and (3) mitigations in left ventricular dilatation and dysfunction in patients surviving myocardial infarction.6
However, no data exist at present concerning prevention of morbidity and mortality with preventive long-term ACE inhibition in high-risk patients with severe cardiovascular diseases and stroke. Therefore, a large clinical study7 was started in 1995 in patients with at least 4 years of ramipril treatment.
Some data in genetically hypertensive rats point to a prolongation of survival through ACE inhibition. In young (6-week-old) SHR, antihypertensive treatment with the ACE inhibitor perindopril for 4 weeks attenuated the development of hypertension throughout their life, and the lifespan of these animals appeared to be extended.8 Adult (15-week-old) SHR treated with perindopril in an antihypertensive dose for 12 weeks showed a significant increase in lifespan.9 Moreover, in salt-loaded SHR-SP, which are at even greater risk for stroke, mortality was suppressed after treatment from the 5th to the 20th week of age by an antihypertensive as well as a nonantihypertensive dose of the ACE inhibitor trandolapril.2 However, none of these studies revealed the potential maximal lifespan extension of hypertensive rats because of the relatively short-term treatment.
Therefore, we investigated the effect of early-onset (1 month after birth) lifelong ACE inhibitor treatment with ramipril in an antihypertensive as well as a nonantihypertensive dose on maximal lifespan extension in SHR-SP and normotensive WKY rats. Factors that may be involved in lifespan extension, such as cardiac function/size and metabolism, endothelial function, expression of ecNOS, and ACE expression and activity, were also studied.
Methods
Animals
Male SHR-SP and WKY rats were purchased from Møllegrad Denmark at the age of 1 month with equal body weights and systolic blood pressures. They were housed 3 per cage under standardized conditions of temperature, humidity, and light. The rats had free access to standard diet (Altromin Maintenance Diet 1320; sodium content, 0.2%) and drinking water ad libitum. All experiments were performed in accordance with the German animal protection law.
Study Design
One hundred thirty-five animals of each strain were randomly allotted to three groups such that each group had 45 animals from each strain. All groups were treated via drinking water with placebo, HRA (1 mg · kg−1 · d−1), or LRA (10 μg · kg−1 · d−1), commencing immediately after randomization and adjusted to the actual consumption. Body weights and systolic blood pressures, by tail plethysmography, were determined every 3 months; deaths were recorded as they occurred.
Interim Analyses
Interim analyses were scheduled when ≈80% of the corresponding placebo-treated animals had died, which was after 15 months in SHR-SP and 30 months in WKY rats. Ten animals of each strain were randomly selected and anesthetized (hexobarbitone 80 mg/kg IP) for direct recording of mean arterial blood pressure in the left carotid artery. Thereafter, blood samples, thoracic aortas, carotid arteries, and hearts were removed for molecular, biochemical, and/or functional analyses. Renin activity (Renin-MAIA, Serono Diagnostika GmbH) and concentrations of aldosterone (RIA-Kit, Coat-A-Count, Aldosterone, Diagnostic Products Corp) and Ang II (RIA-Kit, Nichols Institute Diagnostika GmbH) were determined in plasma. Quantification of the latter was performed after separation through high-performance liquid chromatography. ACE activities in plasma, thoracic aorta, and right cardiac ventricle were radioenzymatically measured with [3H]Gly-Gly as substrate (Hycor ACE activity test).
Expression of ecNOS in the Carotid Artery (Western Blot)
Frozen (−70°C) vessels were thawed and extracted with guanidinium isothiocyanate/phenol/chloroform.10 Crude protein fractions were obtained by alcohol precipitation of the phenol phase. A total of 100 μg of the protein extracts was subjected to SDS-PAGE and transferred to nitrocellulose membranes (Bio-Rad).11 Ponceau staining was performed to verify the quality of the transfer and the equipartition of protein in each lane. ecNOS protein was detected with a specific antibody (mouse NOS III, Transduction Laboratories) and visualized by enhanced chemiluminescence with a commercially available kit (Amersham). The autoradiographs were analyzed by scanning densitometry.
Expression of mRNA ACE in the Left Cardiac Ventricle (RNAse Protection Assay)
Primer ACE I (5′-ATATCTAGAGGCGCTGGAGGGTCTTTGACGGAAGCATCA-3′) and ACE II (5′-TATGGATCCTATCACACTTGTACAGGGGGCCGGTGTGCC-3′) were used to amplify a 255-nucleotide piece of the rat ACE gene by reverse transcriptase–polymerase chain reaction (rat lung total RNA), and the product was cloned into Xba I–and BamHI-cleaved pGEM-11Zf+ (Promega). The antisense RNA probe was transcribed from the T7 promoter for 20 minutes at 37°C with [α-32P]CTP (800 Ci/mmol). After DNAse digestion, the probe was purified via G50 Quick spin columns and quantified by β-counting. Usually, a 500 000 cpm probe was used in a single RNAse protection experiment using 20 mg total RNA from the left cardiac ventricle. The protection assay was performed according to the manufacturer’s guidelines (Boehringer Mannheim); dsRNA was ethanol-precipitated, washed, dissolved in sample buffer, and applied to a 5% denaturing polyacrylamide gel. The radioactive signals were quantified by a phosphoimager, and rat γ-actin was used as an internal standard.
Isolated Working Heart
Hearts were perfused according to the Langendorff technique with a constant perfusion pressure of 65 mm Hg.12 LVP, left ventricular dP/dtmax, and heart rate were measured via a balloon catheter. Coronary flow was determined with an electromagnetic flow probe placed above the cannula that connected the heart with the perfusion apparatus. After a washout phase of 5 minutes, 3 times 1 mL of the coronary effluent was sampled for measuring lactate concentration, the activities of LDH and CK,12 and concentrations of kinins. The antibody used in the kinin radioimmunoassay did not distinguish among bradykinin, lysyl-bradykinin, and methionyl-lysyl-bradykinin.13 Thereafter, the hearts were gently blotted to dryness, and the weights of the total heart and the left and right ventricles were determined.
Apex sections of the left ventricles were taken for hydroxyproline and proline measurements,14 and the ratio of hydroxyproline/proline was calculated.
The rest of the left ventricle was stored in liquid nitrogen for determination of ACE expression (see above).
Isolated Rings of Thoracic Aorta
The excised aorta was immersed in Tyrode’s solution and cleaned of adhering fat and connective tissue. Then a small segment of each aorta (5 mm behind the aortic arch) was carefully cut into rings (2 mm) and transferred to a temperature-regulated (37°C) 10-mL organ bath with modified Tyrode’s solution (composition in mol/L: NaCl 136.9, NaHCO3 11.9, KCl 2.7, CaCl2 0.5, MgCl2 2.0, NaHPO4 0.4, glucose 5.5 [pH 7.4]) gassed with 95% O2/5% CO2. Each strip was mounted vertically between two fine stainless steel pins. The upper pin was connected to an isometric strain-gauge transducer. The transducer signal was recorded with a computer-assisted biosignal analyzer. Aortic strips were suspended under a passive tension of 4.9 mN. After an equilibration period of 1 hour, the strips were contracted by KCl 20 mmol/L. At the plateau of KCl-induced contraction, acetylcholine was added in concentrations of 10−8, 10−7, 10−6, and 10−5 mol/L in a cumulative manner to relax the vessel strips. Acetylcholine-induced relaxations were related to the respective KCl-induced contractions, called 0% relaxation.
Statistical Analysis
The data are given as mean±SEM. Cumulative survival was analyzed for differences according to Kaplan-Meier followed by Cox-Mantel log-rank test. ANOVA or ANOVA on ranks, as appropriate, followed by multiple pairwise comparisons according to Student-Newman-Keuls, was used. Null hypotheses were rejected at P<.05.
Results
Measurements of Body Weight and Systolic Blood Pressure
Body weights increased from 72±3 g (1 month old) to 385±6 g (15 months old) in placebo-treated SHR-SP and from 75±4 g (1 month old) to 587±7 g (30 months old) in placebo-treated WKY rats. Ramipril treatments did not significantly affect the body weights of either strain. Systolic blood pressure of 98±5 mm Hg in young (1-month-old) placebo-treated SHR-SP was significantly increased after 3 months and reached the highest value after 12 months (Fig 1A). After that time, systolic blood pressure showed a slight decrease in placebo-treated SHR-SP, most likely due to a development of heart failure in senescent animals. Only HRA treatment completely prevented the rise in blood pressure in SHR-SP (Fig 1A). Systolic blood pressure remained unaltered between 95 and 105 mm Hg in all WKY rats (Fig 1B).
Cumulative Survival
All placebo-treated SHR-SP survived for the first 9 months of age. Between 9 and 15 months, these animals died successively, revealing a maximal lifespan of 15 months. LRA treatment significantly extended maximal lifespan to 18 months, and HRA treatment doubled the life expectancy to 30 months (Fig 2A). Placebo-treated WKY rats died successively only between 18 and 32 months, revealing a maximal lifespan of 32 months, which was not influenced by either treatment regimen with ramipril (Fig 2B). It is worth noting that the survival curve of HRA-treated SHR-SP concurred with that of placebo-treated normotensive WKY rats.
Interim Analyses
Mean arterial blood pressures measured in the carotid arteries of SHR-SP were 150±6 mm Hg (placebo), 109±5 mm Hg (HRA), and 156±6 mm Hg (LRA), and in WKY rats, the average of all groups was 90±5 mm Hg. These data agreed with those found by plethysmographic tail-cuff determination (Fig 1A and 1B).
Total and left and right ventricular heart weights per 100 g body weight were significantly reduced only by HRA treatment in SHR-SP compared with placebo (Fig 3A). These values were similar to those of placebo-treated WKY rats (Fig 3B). The slight but significant reduction of total and left ventricular weights in WKY rats after HRA treatment may reflect a beneficial influence on physiological undefined aging processes by long-term ACE inhibition.
Similarly, hydroxyproline/proline ratios (index for fibrosis) were significantly reduced only by HRA treatments in both rat strains (SHR-SP, 12.8±0.9 versus placebo 26.3±1.6; WKY rats, 15.8±1.3 versus placebo 11.1±1.0).
Markers of the RAS were not significantly different in the plasma of placebo-treated animals of both rat strains. As expected, only HRA treatment significantly increased plasma renin activities (ng Ang I · mL−1 · 10 min−1) (SHR-SP, 3.1±0.3 to 18.0±4.0; WKY rats, 2.7±0.4 to 22.9±4.9) and significantly decreased plasma Ang II concentrations (pg · mL−1) (SHR-SP, 69.2±9.1 to 29.9±7.8; WKY rats, 60.7±9.0 to 18.7±3.7). Plasma aldosterone concentrations (pg · mL−1) were significantly and equally reduced by both doses of ramipril (SHR-SP: placebo 379.5±29.0, HRA 235.2±15.1, LRA 253.5±19.9; WKY rats: placebo 310.3±28.7, HRA 226.8±10.5, LRA 274.6±16.7). Plasma ACE activities (nmol · min−1 · mL−1) were significantly and dose-dependently reduced by ramipril in both rat strains (SHR-SP: placebo 166.2±7.7, HRA 10.8±2.0, LRA 68.4±5.6; WKY rats: placebo 142.4±7.7, HRA 4.6±2.1, LRA 69.8±10.5).
Unlike in the plasma, ACE activities in the thoracic aorta and right cardiac ventricle were significantly higher in placebo-treated SHR-SP than in matching WKY rats (Table 1). The reduction of ACE activity by ramipril treatments was significant and dose-dependent in SHR-SP and dose-independent in WKY rats. A significant and dose-dependent reduction of mRNA ACE expression by ramipril treatments was also observed in SHR-SP but not in WKY rats (Table 1).
Isolated Working Heart Preparation
Hearts from placebo-treated SHR-SP revealed a significant decrease in LVP and left ventricular contraction rate (dP/dtmax) compared with hearts from matching WKY rats. Both parameters increased after HRA and LRA treatment in hearts from SHR-SP, whereas in hearts from WKY rats, only HRA treatment was effective (Table 2, Fig 4).
The heart rate of 133±9 bpm in placebo-treated SHR-SP was significantly increased by HRA treatment (167±4 bpm) and LRA treatment (158±6 bpm). The significantly higher heart rate in placebo-treated WKY rats (180±8 bpm) compared with matching SHR-SP was not changed by either ramipril treatment.
The coronary flow in hearts from placebo-treated SHR-SP was significantly lower than in matching WKY rats. In both rat strains, ramipril treatment significantly and dose-dependently increased coronary flow (Fig 5A and 5B).
The release of kinins into the coronary effluent was significantly enhanced in both rat strains only by HRA treatment compared with placebo treatments (Fig 5A and 5B).
The activities of cytosolic enzymes (CK, LDH) as well as lactate release into the coronary effluent were higher in hearts from placebo-treated SHR-SP than in matching WKY rats (Table 2). This increased release of markers for ischemia was significantly and dose-dependently reduced by ramipril in hearts of SHR-SP. In hearts from normotensive WKY rats, ramipril was also able to reduce these markers, probably reflecting impaired metabolism in senescent rats.
Isolated Thoracic Aorta
tk;4Endothelium-dependent relaxation in response to acetylcholine was strongly impaired in KCl-precontracted aortic strips from placebo-treated SHR-SP compared with matching WKY rats. This vasodilator hyporesponsiveness was dose-dependently prevented by ramipril treatment (Fig 6A). No relevant influence on acetylcholine-induced relaxation could be observed in strips from WKY rats treated with HRA and LRA compared with placebo-treated animals (Fig 6B). KCl induced a contraction of 528±53 mN in thoracic aortic strips from placebo-treated SHR-SP. In strips from ramipril-treated SHR-SP, KCl-induced contractions were significantly reduced (234±34 mN with HRA and 316±29 mN with LRA) in a dose-dependent manner. Aortic strips from placebo-treated WKY rats revealed a less pronounced contraction (321±48 mN) in response to KCl, which was not influenced by ramipril treatments.
Expression of ecNOS in the Carotid Artery
Densitometric analysis of Western blots showed significant increases of the expression of ecNOS in ramipril-treated rats of both strains. The relative increases of ecNOS expression in both SHR-SP and WKY rats were moderately enhanced, threefold and fivefold by HRA and LRA treatment, respectively (Fig 7).
Discussion
Effects of ACE Inhibition on Lifespan Extension
We showed that lifelong administration of the ACE inhibitor ramipril in a blood pressure–lowering dose (1 mg · kg−1 · d−1) doubled life expectancy in SHR-SP. This extended lifespan corresponded to the lifespan of normotensive placebo-treated WKY rats. Effects that may contribute to this lifespan extension by ACE inhibition were (1) inhibition of left ventricular hypertrophy, (2) preservation of heart function and metabolism, and (3) preservation of endothelial function.
Even at a dose that did not affect high blood pressure (10 μg · kg−1 · d−1), ramipril was able to prolong the lifespan of SHR-SP significantly, but to a lesser extent. This suggests that a minor part of the effect of ramipril treatment is linked to the preservation of endothelial function, independent of the suppression of high blood pressure. These observations are in line with data from Richer et al,2 showing that trandolapril, also in a nonantihypertensive dose, reduced the mortality of young salt-loaded SHR-SP. In this study, however, the analysis of survival was confined to a treatment of 15 weeks. Thus, it is not known whether the striking survival benefit would be sustained during the very late follow-up.
Effects of ACE Inhibition on Cardiac Left Ventricular Hypertrophy
The RAS has been implicated in the development and maintenance of hypertension and cardiac hypertrophy.15 However, there is circumstantial evidence that RAS components in the tissue rather than in the plasma are involved in the pathogenesis of these processes. This is in line with our data, which did not reveal a stimulation of RAS components in the plasma of hypertensive SHR-SP with cardiac hypertrophy: renin activity, Ang II, and aldosterone concentrations as well as ACE activity in the plasma were not significantly elevated in these rats. This agrees with other investigations showing no significant changes of RAS components in the plasma of WKY rats with cardiac pressure overload.16 The contribution of activated RAS components in rat tissues was shown for several hypertensive heart diseases, such as left ventricular hypertrophy, heart failure, and remodeling. In WKY rats with pressure-overload hypertrophy, an enhanced ACE mRNA expression and activity in the left and right ventricles was demonstrated,1718 which was reflected by an accelerated intracardiac conversion of Ang I to Ang II in isolated perfused rat hearts.1719 Also in our study, senescent SHR-SP with cardiac hypertrophy showed significant increases in cardiac ACE mRNA expression and activity compared with age-matched normotensive WKY rats (Table 1). Consistent with these findings are data in failing human hearts in which ACE expression of cardiac left ventricle was upregulated.20
Decreased local Ang II generation via ACE inhibition was shown to improve cardiac function and hypertrophy in hypertensive rats1819 and humans.21 In our study, ramipril dose-dependently decreased myocardial ACE mRNA expression and activity in SHR-SP, reaching values of normotensive placebo-treated WKY rats. However, in SHR-SP, only HRA treatment prevented the development of myocardial hypertrophy and fibrosis, indicating an additional role of high blood pressure in these processes. In contrast, in rat carotid arteries, the vascular remodeling process of hypertension seems to be directly mediated by an activated RAS independent of its effect on blood pressure.22
Effect of ACE Inhibition on Heart Function and Metabolism
The isolated heart preparation is free of confounding factors of systemic neurohumoral activation and pericardial constraint, therefore permitting easy investigations of both cardiodynamics and myocardial metabolism. Because these hearts were reperfused after removal, the influence of ischemia is also present. Hence, isolated hearts from placebo-treated SHR-SP showed an impaired cardiac function (LVP, dP/dtmax, and heart rate) compared with hearts from placebo-treated WKY rats. This suggests that hypertrophic hearts from SHR-SP were less able to compensate for reperfusion-related stress. This is also reflected by increased metabolic markers for ischemia (CK, LDH, and lactate, Table 2) in placebo-treated SHR-SP. The impaired cardiac function in placebo-treated SHR-SP with left ventricular hypertrophy was related to a higher cardiac Ang II content mediated by increased ACE mRNA expression and activity.
Ramipril treatment (HRA and/or LRA) prevented the impairment of myocardial function and metabolism in both rat strains. These cardioprotective effects may be due to (1) prevention of left ventricular hypertrophy and (2) locally increased kinin synthesis and release, especially after HRA treatment. ACE inhibition prevents the degradation of endothelium-derived kinins in the rat heart.2324 Kinins, in turn, increased coronary flow, improved myocardial metabolism, and prevented postischemic reperfusion injuries in isolated normoxic and ischemic working hearts of the rat and guinea pig.23 In our study, both HRA and LRA treatment dose-dependently increased coronary flow; however, the associated increased kinin release was present only after HRA treatment. Therefore, we suggest that the increased coronary flow by LRA treatment is probably due to an enhanced formation of myocardial capillaries and collaterals observed after nonantihypertensive ACE inhibitor treatment in SHR-SP.25 In the human coronary circulation, a contribution of endogenous kinins to vasomotor control was reported.26 Moreover, in patients with end-stage heart failure, a contribution of local kinins was shown in coronary microvessels.27
Effect of ACE Inhibition on Isolated Thoracic Aorta
The impairment of endothelial function is subject to heterogeneous mechanisms depending on the vascular bed and/or the model of hypertension as well as aging. Recently, it was shown by direct measurement of NO and superoxide in the aorta28 and mesenteric artery29 of SHR-SP that an excess of superoxide scavenges endothelial NO, thus contributing to an increased vascular smooth muscle contraction in these blood vessels.
In our study, the reduction in potassium chloride–induced force development probably results from a greater production of endothelial NO in the aortas of SHR-SP treated with ramipril. Similarly, the lesser contraction in aortas from untreated WKY rats compared with untreated SHR-SP may be caused by a greater availability of NO.
In animals with genetic hypertension, subchronic ACE inhibition was found to attenuate endothelial dysfunction in the aorta3031 and mesenteric32 and coronary arteries.33 Moreover, this effect seems to be partly independent of the hypotensive action of ACE inhibitors. Not only an antihypertensive but also a nonantihypertensive dose of ramipril prevented the development of endothelial dysfunction in SHR and SHR-SP treated in utero and subsequently up to 20 weeks of age.34 Furthermore, this effect was associated with a dose-dependently increased content of aortic cGMP. Increases in cGMP were strongly correlated to endothelial NO synthesis and release by acute ACE inhibition.35 A clinical study documented that ACE inhibition with quinapril for 6 months in normotensive patients with coronary heart disease in part restores endothelium-mediated coronary vasodilation.36 A prevention study in humans is still lacking.
In our study, the development of endothelial dysfunction in aortas of placebo-treated SHR-SP was dose-dependently prevented by lifelong ramipril treatment. Such a protective effect was also observed in aortas of senescent SHR after lifelong ramipril treatment.37 In addition, this study revealed that endothelial preservation after lifelong ramipril treatment was positively correlated with an increased availability of NO, which in turn seems to be due to an enhanced ecNOS expression and resultant activity as well as a strongly decreased basal and calcium ionophore–stimulated superoxide production in carotid arteries and aortas. These results in SHR are in line with the present data in SHR-SP showing moderately increased ecNOS expression in carotid arteries after lifelong ramipril treatment (Fig 7). Like the effect of ramipril on aortic ACE inhibition (Table 1) and on endothelial function (Fig 6), the upregulation of ecNOS expression was also already present after nonantihypertensive treatment with ramipril. In normotensive WKY rats, a similar increase in ecNOS expression after lifelong ramipril treatment was observed. However, unlike in hypertensive rats, after lifelong ramipril treatment in normotensive rats, a significant amount of the increased ecNOS enzyme was disarranged, probably by insufficient substrate, causing markedly enhanced basal and calcium ionophore superoxide production.37 Nevertheless, the availability of NO in these animals was enhanced by lifelong ramipril treatment.
Thus, it can be assumed that in SHR-SP, as in SHR,37 lifelong ramipril treatment upregulates ecNOS, leading to a higher NO availability, which will improve vasodilation of the cardiovascular system, and lower superoxide production, which may significantly decrease oxidative stress and extend the efficiency of the endothelium. Presumably, both effects are major contributors to the lifespan extension in hypertensive rats.
Alternatively, it might be considered that ACE inhibition may enhance NO availability by an additional mechanism. It was shown that specific stimulation of NADH and NADPH oxidase activity by Ang II leads to enhanced superoxide production in rat blood vessels.3839 The strongly reduced plasma Ang II concentrations after lifelong HRA treatment in SHR and WKY rats, therefore, points to the possibility of a decreased oxidative stress (and subsequent enhanced NO availability) under these conditions.
The underlying mechanism(s) responsible for ecNOS upregulation as well as decreased superoxide production after lifelong ramipril treatment requires further studies. It might be conceivable that ramipril-induced inhibition of endogenous breakdown of kinins mediates this effect. Data from Gohlke et al34 showed that cotreatment with the bradykinin receptor antagonist icatibant suppressed the enhanced aortic cGMP content after high- and low-dose treatment with ramipril. Furthermore, in cultured endothelial cells incubated for 48 hours with 8-bromo-cGMP, an upregulated ecNOS was found.40 Recently, in humans, an upregulation of ecNOS by ACE inhibition in atrial myocardium was also described.41
Conclusions and Clinical Implications
The present results show that different beneficial effects of lifelong antihypertensive ACE inhibitor treatment on major cardiovascular functions seem to contribute to the doubling of lifespan extension of rats with a genetic form of hypertension. These major beneficial effects of ACE inhibition are (1) prevention of the development of hypertension, (2) cardiac left ventricular hypertrophy most likely associated with improvement of myocardial functions, and (3) preservation of endothelial function. The latter was already observed after nonantihypertensive ACE inhibitor treatment. Various molecular/biochemical mechanisms can be attributed to these effects. Suppression of tissue ACE expression and activity (1) decreases local Ang II concentrations, which are mainly related to the antihypertensive and antihypertrophic actions, and (2) increases local concentrations of kinins, which may preserve cardiac functions. In addition, (3) the upregulation of ecNOS most likely associated with subsequently enhanced NO availability provides preservation of the endothelium. The latter plays an important role in the maintenance of normotension and the prevention of cardiovascular diseases. These ACE inhibitor–mediated beneficial effects and especially the extension of life expectancy in high-risk rats with hypertension foretell a favorable outcome in high-risk patients with myocardial infarction, coronary artery disease, and stroke after long-term ACE inhibition.7
Selected Abbreviations and Acronyms
| Ang II | = | angiotensin II |
| CK | = | creatine kinase |
| ecNOS | = | endothelial constitutive nitric oxide synthase |
| HRA | = | high (antihypertensive) dose of ramipril (1 mg · kg−1 · d−1) |
| LDH | = | lactate dehydrogenase |
| LRA | = | low (nonantihypertensive) dose of ramipril (10 μg · kg−1 · d−1) |
| LVP | = | left ventricular pressure |
| RAS | = | renin-angiotensin system |
| SHR-SP | = | stroke-prone spontaneously hypertensive rats |
| WKY | = | Wistar-Kyoto |
Figure 1.
Changes of systolic blood pressure in SHR-SP (A) and WKY rats (B) during lifespan of animals. •, Placebo; ▴, HRA; ▿, LRA. *P<.05 vs placebo treatment.
Figure 2.
Effect of early-onset lifelong treatment with ramipril on cumulative percent survival in SHR-SP (A) and WKY rats (B). •, Placebo; ▴, HRA; ▿, LRA. Percent survival with either treatment was significantly enhanced vs placebo in SHR-SP (Kaplan-Meier analysis followed by Cox-Mantel log-rank test, HRA: χ2=41.88, P<.05; LRA: χ2=18.16, P<.05) but not in WKY rats.
Figure 3.
Total and left and right ventricular heart weights at interim analyses: SHR-SP after 15 months (A), WKY rats after 30 months (B). *P<.05 vs matching placebo, #P<.05 vs matching WKY rats, n=10 per group.
Figure 4.
Changes in maximal developed LVP rate (dP/dtmax) of isolated working hearts at interim analyses: SHR-SP after 15 months (A), WKY rats after 30 months (B). *P<.05 vs matching placebo, #P<.05 vs matching WKY rats, n=10 per group.
Figure 5.
Changes in coronary flow (CF, mL · min−1 · g heart wet wt−1, left ordinate) and kinins in coronary effluent (fmol · min−1 · g heart wet wt−1, right ordinate) of isolated working hearts at interim analyses: SHR-SP after 15 months (A), WKY rats after 30 months (B). *P<.05 vs matching placebo, #P<.05 vs matching WKY rats, n=10 per group.
Figure 6.
Endothelium-dependent relaxations by acetylcholine expressed as % reversal of KCl (20 mmol/L)–elicited contractions in aortic rings at interim analyses: SHR-SP after 15 months (A), WKY rats after 30 months (B). •, Placebo; ▴, HRA; ▿, LRA. *P<.05 vs matching placebo, n=10 per group.
Figure 7.
Expression of ecNOS (Western blot) in % changes versus respective placebo treatments (100%: 75.7±36.3 and 35.8±4.7 intensity OD/mm2 for SHR-SP and WKY rats, respectively) at interim analyses (SHR-SP after 15 months, WKY rats after 30 months). Western blots were performed in separate experiments for SHR-SP and WKY rats. *P<.05 vs matching placebo, n=6 per group.
| SHR-SP | WKY Rats | |
|---|---|---|
| ACE activity in the thoracic aorta, nmoL · mg protein−1 · h−1 | ||
| Placebo | 1646.1 ±58.93 | 361.0±36.5 |
| HRA | 88.2±9.912 | 90.1±13.71 |
| LRA | 399.9±19.013 | 112.3±9.91 |
| ACE activity in the right cardiac ventricle, nmoL · mg protein−1 · h−1 | ||
| Placebo | 82.7±11.63 | 23.2±2.1 |
| HRA | 11.8±1.912 | 5.1±0.81 |
| LRA | 29.8 ±4.813 | 3.4±0.81 |
| ACE expression in the left cardiac ventricle, mRNA signal intensity−ACE/γ-actin · 10−4 | ||
| Placebo | 258.2±31.83 | 194.7±8.6 |
| HRA | 149.3±5.6123 | 188.0±6.9 |
| LRA | 201.2 ±16.11 | 195.2±4.1 |
| SHR-SP | WKY Rats | |
|---|---|---|
| LVP, mm Hg | ||
| Placebo | 66.2±4.33 | 91.9±4.1 |
| HRA | 107.4±5.21 | 109.8±4.21 |
| LRA | 84.7 ±3.91 | 92.9±5.3 |
| CK activities in coronary effluent, mU · min−1 · g heart wet wt−1 | ||
| Placebo | 1.03±0.13 | 0.77 ±0.08 |
| HRA | 0.42±0.08123 | 0.23±0.031 |
| LRA | 0.62 ±0.0913 | 0.34±0.041 |
| LDH activities in the coronary effluent, mU · min−1 · g heart wet wt−1 | ||
| Placebo | 1.48 ±0.123 | 0.94±0.11 |
| HRA | 0.55±0.0912 | 0.48±0.031 |
| LRA | 0.88±0.0713 | 0.54±0.041 |
| Lactate content in the coronary effluent, μmol · min−1 · g heart wet wt−1 | ||
| Placebo | 25.2±1.13 | 14.3±1.3 |
| HRA | 3.0±0.0912 | 3.9±0.1512 |
| LRA | 14.0 ±0.513 | 10.3±0.41 |
We kindly thank Prof Edwin Fink (München) for determination of kinins, Dr Anne Bouloumier (Frankfurt/Main) for measurement of ecNOS expression, Dr Wolfgang Ulmer (Hoechst Marion Roussel) for determination of hydroxyproline/proline, and Dr Dieter Ruppert (Hoechst Marion Roussel) for determination of plasma renin activity, angiotensin II, and aldosterone. We also thank Peter Pleines for excellent management of the study and Ute Fürst, Marion Jorge, Albin Köhler, Monika Platil, Heinz Otto Stern, Thomas Ulshöfer, and Wolfgang Jung for expert technical assistance.
Footnotes
References
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