The renin-angiotensin system is considered to be a main determinant of vascular structure. Angiotensin II has been demonstrated to stimulate protein synthesis in cultured vascular smooth-muscle cells, resulting in cellular hypertrophy (1,2). Local stimulation of the angiotensin-converting enzyme (ACE) has been reported to increase media thickness in the rat carotid artery (3). Systemic inhibition of ACE reduces media thickness in hypertensive rats and decreases intima proliferation in animal models of restenosis (4-6). ACE inhibition also has been demonstrated to inhibit the development of atherosclerosis in Watanabe rabbits and to restore disturbed endothelial function in hypertensive and cyclosporin A-treated rats (7-9). However, the trophic effects of angiotensin II or ACE inhibition are mainly studied in vascular smooth-muscle cell cultures or in animal models for cardiovascular disease. In diseased animals, systemic or local stimulation of the renin-angiotensin system may sensitize the organism to respond to these drugs. Little information is available concerning the role of the renin-angiotensin system in the physiologic regulation of vascular structure. Because ACE has several biologically active substrates (e.g., bradykinin), a part of the effects of ACE inhibition may be mediated by these mediators rather than by reduced angiotensin II generation (10-13).
In this study, the effects of long-term inhibition of the renin-angiotensin system on aortic media thickness were determined in normal Wistar rats to study the physiologic regulation of vascular smooth-muscle cell growth by the renin-angiotensin system in vivo. To distinguish cellular hypotrophy from tissue hypoplasia, smooth-muscle cell density was determined. The functional consequences of altered media thickness regarding the vasoconstrictor responses were investigated by measuring the maximal contraction to different vasoconstrictors. Furthermore, the effects on the intima were assessed and heart weight was determined as an independent parameter for cellular growth, as compared with the vascular smooth-muscle effects.
The long-term effects of two different ACE inhibitors were compared with those of two different angiotensin II-type 1 (AT1)-receptor antagonists to differentiate the effects of reduced angiotensin II generation from those of bradykinin potentiation or other subtypes of AT receptors. The role of angiotensin II and its receptor function was further elucidated by combining the treatments with cyclosporin A, which selectively amplifies rat aortic contraction to angiotensin II (9,14,15). Whether cyclosporin A also potentiates the trophic effects of angiotensin II, which may contribute to the serious vascular side effects of the drug, is not known. Additionally, the effects of long-term calcium-entry blockade as a vasodilator not directly affecting the renin-angiotensin system were investigated in parallel and compared with the effects of the inhibitors of the renin-angiotensin system.
Treatment and tissue preparation
The investigation was performed in conformation with the Guide for the Care and Use of Laboratory Animals, published by the U.S. National Institutes of Health. A total of 210 male Wistar rats, aged 6-8 weeks, with a body weight of 200-250 g was obtained from the Bundesgesundheitsamt, Berlin, Germany. The rats were randomly assigned to treatment groups of 15 rats each. The study was performed in two large experimental series. In each series, the effects of an ACE inhibitor were compared with those of an AT1-receptor antagonist. Lisinopril and D 8731 (both provided by Zeneca, Plankstadt, Germany) were used in the first experiment, and fosinopril (Bristol Myers Squibb, Munich, Germany) and losartan (Merck Sharp Dohme, Rahway, NY, U.S.A.) in the second. Each drug was given orally in the dose of 10 mg/kg/day, which was demonstrated to cause maximal inhibition of ACE activities or blockade of AT1 receptors. In the second series, an additional group was treated with the calcium antagonist isradipine, 60 mg/kg/day (Sandoz, Nuernberg, Germany). All agents were given alone and in combination with cyclosporin A, 15 mg/kg/day (Sandoz). In each series, a cyclosporin A-treated group and a control group were included. The treatments were prepared daily in liquid solutions; they were dissolved either in water (lisinopril, fosinopril, losartan, isradipine, D 8731) or in 1 ml olive oil (cyclosporin A). The control groups received olive oil solely. Rats were brought up under standard conditions. The animals had free access to tap water and standardized chow (Ssniff M/R 15; Special Diets, Soest, Germany). Once a week, body weight was measured. After 6 weeks of treatment, the rats were narcotized by an intraperitoneal injection of sodium pentobarbital, 50 mg/kg (Nembutal; Abbott Laboratories, Chicago, IL, U.S.A.). The peritoneal cavity was opened, the aortic bifurcation cannulated, and a blood specimen aspirated for cyclosporin A determination. The heart wet weight was determined immediately after excision, and the descending thoracic aorta harvested for contraction experiments. The abdominal aorta was rinsed with buffered saline, fixed in situ by perfusion with 60 ml buffered 10% formalin solution with a hydrostatic pressure of 100 cm H2O for 3 min, and thereafter harvested and additionally immersed in formalin solution for several hours. This procedure resulted in an appropriate conservation of the tissue with adherent endothelium and distended elastic lamellas. Segmental rings of each aorta were embedded in paraffin and processed to determine media thickness, smooth-muscle cell density, and the extent of intimal lesions. All measurements were performed by an observer blinded to the treatment of the animals.
Media thickness was determined in 3-μm-thick resorcin-fuchsin-stained histologic cross-sections of the suprarenal part of the abdominal aorta. The entire circumference was visualized at magnifications between ×25 and ×32.5. The digitized images were analyzed by a newly developed morphometry software (Morpho; DHZB, Berlin, Germany). In detail, innermost and outermost elastic lamella were defined as outlines of media thickness and interactively plotted on screen (16,17). The determination of media thickness was based on an algorithm constructing an auxiliary line running circularly in the middle of the media. Perpendicular to that line, media thickness was directly measured at every fourth pixel. For each rat, two aortic slices were measured for calculating mean media thickness (m). The radius (r) of the aortic lumen was determined from the length of the innermost lamella (length = 2 πr) and the ratio m/r calculated. Further media thickness was standardized to body weight by calculation of body weight-corrected media thickness. The coefficient of variation for the entire processing including histologic and anatomic variation (difference between media thickness of the first and of the second ring divided by mean media thickness) was 2.2% of the mean media thickness. The coefficient of variation for the image processing (difference between two measurements of the same ring divided by mean media thickness) was 0.5% as assessed in 30 images, which were plotted and measured twice by the same investigator.
Smooth-muscle cell density
Aortic media smooth-muscle cell density was determined in 3-μm-thick hematoxylin-eosin-stained sections by using an ocular reticle and a magnification of ×400. Longitudinal cut profiles of nuclei of media myocytes were counted in eight fields distributed over the whole circumference with an overall area of 100,000 μm2.
Intima thickness was determined in immunohistologic stainings with an antimuscle-actin monoclonal antibody (Clone HHF 35; Enzo Diagnostics, New York, NY, U.S.A.) to achieve a maximal demarcation of the intima to aberrant media protuberances of smooth-muscle cells covered with small elastic lamellas. For that purpose, 3-μm-thick sections were incubated with the antibody solution at room temperature for 30 min. For detection, the alkaline phosphatase-antialkaline phosphatase (APAAP) method was applied with Fast Red TR (Sigma, Munich, Germany) as chromogen (18). A counterstaining with hematoxylin was performed. Sections with branching sites were excluded from the analysis. The unaltered aortic intima consists of a thin endothelial layer directly adherent to the basement membrane and is nearly invisible by bright-field light microscopy. Subendothelial space was assessed as enlarged when the intima was clearly visible and thickness > 1.5 μm (16,17). By using an eyepiece and a magnification of ×1,000, we classified intima thickness semiquantitatively into minimal (≤ 5 μm), obvious (5-10 μm), and markedly (>10 μm) enlarged (16). The cumulative length of these sites with increased intima thickness was morphologically measured in two aortic sections, and the mean of both measurements was expressed in percentage of the circumference.
Smooth-muscle cell contraction
Vasomotor studies were performed as described (19). In brief, aortic rings of 4-mm length were mounted between a clip and a force transducer in organ chambers, which were filled with modified Krebs-Henseleit bicarbonate solution and aerated at 37°C with 95% O2/5% CO2. Endogenous nitric oxide synthase was inhibited by nitro-L-arginine, 10−4M, to exclude endothelium-mediated effects on smooth-muscle contraction. The rings were stepwise set at 5 g of passive tension and isometric contractions in response to phenylephrine, 10−10-10−6M, serotonin, 10−8-10−4.5M, and angiotensin II, 10−9−10-6 M, were measured in grams. In our study, the correlation between media thickness and vasoconstrictor-induced maximal contraction was analyzed. Detailed studies regarding the modulation of the vasomotor function by inhibition of the renin-angiotensin system and cyclosporin A and angiotensin-receptor binding studies under these conditions have been published elsewhere (13,14,19).
Statistical significance of differences was tested by analysis of variance by using the ANOVA test and Scheffé F test. Significance was accepted at p < 0.05. If not mentioned otherwise, significant differences were computed with regard to the related control group in each experimental series. Coefficients of correlation and regression were calculated for description of the dependence of two variables on each other. All data are presented as means ± SEM.
Rat body-weight gain and body weight at death were comparable in the control groups and in the groups solely treated with cyclosporin A or one of the other drugs (Table 1). Body-weight gain was moderately decreased during combined treatment with lisinopril plus cyclosporin A, or fosinopril plus cyclosporin A, or losartan plus cyclosporin A. At the time of death, body weight was comparable in all groups of series one and slightly reduced in the groups treated with a vasodilator and cyclosporin A in series two (fosinopril plus cyclosporin A, losartan plus cyclosporin A, isradipine plus cyclosporin A; each p < 0.05).
Media thickness in untreated animals was comparable in the control groups of series one and two. Treatment with ACE inhibitors resulted in a reduced media thickness (−16.2% with lisinopril, p < 0.0001; −7.8% with fosinopril, p < 0.01; absolute values in Table 2). After treatment with AT1 antagonists, a significant decrease in media thickness was found with D 8731 (−8.2%, p < 0.01) but not with losartan (−4.2%; NS). The effect of ACE inhibition was more pronounced than that of AT1 antagonism. This was demonstrated in series one by a stronger effect of lisinopril as compared with D 8731 (p < 0.01; in the presence of cyclosporin A, p < 0.001) and in series two by a reduction of media thickness by fosinopril, but not by losartan. Calcium-entry blockade with isradipine also decreased media thickness (−10.8%, p < 0.001). Single treatment with cyclosporin A had no effect (−1.4%, respectively, −2.9%, each NS), whereas a combined treatment with cyclosporin A and one of the vasodilators led to a reduction of media thickness comparable to the vasodilator alone (cyclosporin plus lisinopril, −20.5%, p < 0.0001; plus fosinopril, −14.2%, p < 0.0001; plus D 8731, −11.6%, p < 0.0001; plus losartan, −12.5%, p < 0.0001; plus isradipine, −13.4%, p < 0.001). Aortic luminal radius itself was not statistically different between the treatment groups (range of the mean luminal radius, 645-696 μm). The ratio of media thickness to luminal radius was altered by treatment according to the changes in media thickness. Further, all changes in media thickness were confirmed to be statistically significant after standardization of media thickness to body weight (data not shown).
Smooth-muscle cell density
Aortic smooth-muscle cell density was comparable in both control groups. After treatment with ACE inhibitors, cell density was increased (+14.7% with lisinopril, +13.9% with fosinopril; both p < 0.001; Table 2). Whereas AT1-receptor blockade by D 8731 resulted in an increased smooth-muscle cell density (+9.8%, p < 0.001), there was no significant effect of losartan (+4.8%, NS). Calcium-entry blockade with isradipine increased smooth-muscle cell density (+12.7%, p < 0.01). Cyclosporin A had no significant effect in series one (+6.1%, NS) but showed a moderate increase in cell density in series two (+8.5%, p < 0.05). Combined treatment with cyclosporin A and one of the vasodilators resulted in an increased smooth-muscle cell density in all groups (cyclosporin A plus lisinopril, +20.2%, p < 0.0001; plus fosinopril, +14.5%, p < 0.0001; plus D 8731, +11.0%, p < 0.001; plus losartan, +15.1%, p < 0.001; plus isradipine, +18.8%, p < 0.0001). A moderate negative correlation between cell density (dependent variable) and media thickness was found (r = −0.40; p < 0.0001; n = 210).
Smooth-muscle cell contraction
Long-term treatment with the ACE inhibitors lisinopril and fosinopril significantly reduced maximal aortic contractions to phenylephrine (Fig. 1) and serotonin (data not shown). The statistical comparison of wall thickness and vasomotor response revealed a significant positive correlation between media thickness and the maximal aortic contraction in response to phenylephrine (r = 0.57; p < 0.0001) and serotonin (r = 0.58; p < 0.0001). In contrast, there was no significant interdependency between media thickness and the contraction to angiotensin II (r = 0.07, NS).
In the major parts of the vessels, the endothelial layer was directly adherent to the internal elastic lamina. When the subendothelial space was visible, it was localized coherently or distributed over the whole circumference of the abdominal aorta. Enlarged subendothelial space was almost exclusively observed in the range of 1.5-5 μm thickness, which was termed minimal lesion. In the untreated animals, the cumulative percentage of the circumference showing these minimal lesions was 18.4 ± 2.4% and 14.8 ± 1.4% for the control groups in series one and two, respectively. Long-term ACE inhibition resulted in a reduction of the extent of these intimal lesions (−65% in the lisinopril group, p < 0.0001; −35% in the fosinopril group, p < 0.05; Table 2). Single treatment with AT1-receptor blockers or isradipine or cyclosporin A had no significant effect (Table 2). In contrast, combined treatment with D 8731 plus cyclosporin A (−45%; p < 0.01), or losartan plus cyclosporin A (−60%; p < 0.0001), or isradipine plus cyclosporin A (−30%; p < 0.05) significantly reduced intimal lesions.
Heart weight expressed as heart weight/body weight ratio (HW/BW) in the control groups was 3.37 ± 0.16 mg/g in series one and 3.00 ± 0.08 mg/g in series two (Table 2). ACE inhibition by lisinopril reduced the HW/BW ratio (−16.3%; p < 0.01), whereas fosinopril had no significant effect (+3.3%). After treatment with AT1 antagonists, a reduction in HW/BW was seen for D 8731 (−12.8%; p < 0.01) but not for losartan (−4.0%; NS). The comparison of ACE inhibition and AT1 antagonism within both series did not show any significant differences in the effects of single treatment. In contrast, calcium-entry blockade with isradipine led to an increase in the HW/BW ratio (+9.0%; p < 0.05). Cyclosporin A itself had no significant effect (−4.5% and +4.3%, respectively; NS). After cotreatment with cyclosporin A, HW/BW was constantly decreased by ACE inhibitors and AT1 antagonists in both series compared with the control groups and cyclosporin A alone (cyclosporin A plus lisinopril, −22.2%, p < 0.0001; plus fosinopril, −9.3%, p < 0.05; plus D 8731, −13.6%, p < 0.01; plus losartan, −7.7%, p < 0.05). In series one, treatment with lisinopril plus cyclosporin A resulted in a more pronounced reduction in the HW/BW ratio in comparison to D 8731 plus cyclosporin A (p < 0.05). In series two, fosinopril and losartan did not show any differences from one another. The combination isradipine plus cyclosporin A significantly increased HW/BW (+17.3%; p < 0.001).
This study shows that long-term inhibition of the renin-angiotensin system has significant effects on vascular structure and heart weight in the normal Wistar rat, indicating its physiologic role in the trophic control of the cardiovascular system. The observed reduction of media thickness is caused by cellular smooth-muscle hypotrophy and is associated with reduced effects to physiologic vasoconstrictors such as serotoninergic and α-adrenergic stimulation. For the first time, a hypotrophic effect of ACE inhibition and AT1-receptor blockade on media thickness in normotensive rats could be documented. The modulation of AT1-receptor function by long-term treatment with cyclosporin A was not accompanied by any effects on the morphologic parameters measured (9,14,15).
Aortic media thickness is reduced after long-term ACE inhibition with lisinopril or fosinopril. Whereas a reduction in media thickness by ACE inhibition is well documented in hypertensive rats, there was no significant effect in normotensive rats reported so far (5,21,22). Because the effect could be demonstrated with two different ACE inhibitors, it is unlikely to be a substance-specific effect. In this study, the large number of animals as well as the precision of measurement may have provided the statistical power to detect these changes. A reduction was also achieved by AT1-receptor blockade with D 8731 and the calcium antagonist isradipine, whereas there was no significant effect after treatment with losartan. Head-to-head comparison of the two different forms of inhibition of the renin-angiotensin system revealed larger effects of the ACE inhibitors in both studies; lisinopril is more effective than D 8731 and fosinopril reduces media thickness, whereas losartan does not. Assuming that both manipulations, ACE inhibition and AT1-receptor blockade, were effective, the results raise the question whether ACE inhibitors act by mechanisms other than by preventing the effects of angiotensin II at the AT1 receptor. In the first study, lisinopril and D 8731 were applied in doses that prevent blood-pressure responses in rats to angiotensin I or angiotensin II, respectively, for > 6 h without accumulation of the compound during long-term treatment (23). Thus we hypothesized that these dosages induce the maximal effective blockade, which can be achieved in a long-term study without toxic side effects, and therefore these treatments will elucidate the regulatory role of ACE and the AT1 receptor, respectively. In the second comparison, the same dosages were chosen because the drug effects are similar in acute applications; however, the long-term pharmacokinetics have not been compared. Nevertheless, losartan, 10 mg/kg/day, has been shown to modulate hemodynamics and renal function and to reverse cardiac hypertrophy without affecting blood pressure in rats with heart failure, although some investigators used higher doses to achieve hypotrophic effects in long-term animal models (24). Thus the data indicate that both ACE inhibition and AT1-receptor blockade can reduce media thickness, suggesting that at least a part of the effect of the ACE inhibitors is mediated by reduced stimulation of AT1 receptors. The results are compatible but cannot prove the concept that ACE inhibitors may exert a part of their effects by other mechanisms such as bradykinin potentiation or reduced stimulation of other AT-receptor subtypes (10-13,20). It remains to be determined whether the failure of losartan in this regard is the result of the dosage or whether drug-specific effects of losartan or D 8731 are responsible for the different effects.
Smooth-muscle cell hypotrophy
The drug-induced reduction of media thickness was accompanied by an increase in media smooth-muscle cell density, indicating that media thickness was reduced mainly by a decrease in mean cellular volume. The concordant effects of ACE inhibitors and AT1-receptor antagonists suggest that angiotensin II is a determinant of vascular smooth-muscle cell volume in normotensive rats. Our data are in line with the reported effects of some ACE inhibitors on vascular smooth-muscle cell density in spontaneously hypertensive rats, in which aortic smooth-muscle cell hypertrophy develops during the onset of chronic hypertension in young rats (25,26). In these studies, the ACE inhibitor quinalapril increased smooth-muscle cell density, whereas perindopril had no significant effect (4,21). However, the effect of drug-induced reduction in vascular smooth-muscle cell volume in non-hypertensive rats demonstrates that this parameter is under physiologic control by the renin-angiotensin system and can be reduced by appropriate intervention.
Vascular smooth-muscle contraction
Maximal vasoconstrictor responses to serotonin and the α-adrenergic agonist phenylephrine showed a significant positive correlation to aortic media thickness. Because two different vasoconstrictors are affected and an approximately linear relation between maximal aortic contraction and aortic wall thickness is observed, the reduced force development after vasodilator treatment may reflect the smaller quantities of contractile material available to produce contractions. In contrast, the responses to angiotensin II failed to show any correlation between maximal contraction and media thickness, thus demonstrating no dependence on the vascular smooth-muscle mass. This effect must be the result of the specific changes of the AT1-receptor function or density after long-term modulation of the renin-angiotensin system (9,14,19).
The heart wet weight was determined to compare the trophic regulation of the myocardium with the changes in vascular structure during long-term vasodilator treatment. In series one, heart weight was decreased by ACE inhibition with lisinopril and AT1 antagonism with D 8731; in series two, fosinopril and losartan significantly reduced heart weight only during combined treatment with cyclosporin A. In contrast, treatment with isradipine increased heart weight. Thereby calcium antagonism with isradipine demonstrates opposing effects on heart weight and similar effects concerning media thickness as compared with the inhibitors of the angiotensin pathway. These data support the view of a direct trophic effect of angiotensin II on the heart rather than a secondary effect resulting from blood-pressure changes. A comparable failure in the regression of increased heart weight after abdominal aortic banding in Sprague-Dawley rats has been shown with the calcium antagonist nifedipine. In the same animal model, the ACE inhibitor ramipril reduced heart weight even in non-blood pressure-lowering dosages (27). In previous studies, ACE inhibitors and losartan constantly reduced heart weight in hypertensive rats, whereas in normotensive rats, heart weight was either not altered, as reported for lisinopril, or reduced, as reported for captopril (21,22,27-31). Our data support the hypothesis that modulation of media thickness, smooth-muscle cell density, and heart weight by inhibitors of the renin-angiotensin system is not restricted to hypertensive states and that there is a comparable trophic up- and downregulation even in normotensive animals.
Trophic regulatory mechanisms
There are two pathways to be considered in explanation of the antitrophic effects of ACE inhibitors, AT1-receptor blockers, and calcium-entry blockers. Cell volume might be directly regulated by alterations of the hormonal influences or it might be indirectly affected by reducing the blood pressure. With regard to direct mechanisms, there is experimental evidence from in vitro experiments that angiotensin II and calcium antagonist can directly influence vascular smooth-muscle cells. Angiotensin II stimulates protein synthesis and induces cellular hypertrophy in cultured aortic smooth-muscle cells (1,2). High concentrations of calcium-entry blockers inhibit the proliferation of rat smooth-muscle cell in culture and may inhibit DNA synthesis (30-32). Furthermore, ACE inhibitors and calcium-entry blockers inhibit the platelet-derived growth factor-induced transcription of the protooncogenes c-fos and c-jun, which are involved in the regulation of cardiovascular hypertrophy (33). The hypothesis of direct effects is further supported by the observation that equieffective doses of antihypertensive agents modulate media thickness and heart weight to a different extent in hypertensive rats (5,25,34,35). In hypertensive rats, blood pressure is constantly reduced under adequate therapy, but the effects on media thickness are different. The ACE inhibitor captopril was reported to reduce media thickness in spontaneously hypertensive rats much more than predicted by covariance and multiple-regression analysis of its blood pressure-reducing effect (36). In the same study, the β-adrenergic blocker propranolol reduced only blood pressure, whereas there was no effect on media hypertrophy (36). The vasodilator hydralazine reduced blood pressure in spontaneously hypertensive rats just beyond the values of the normotensive control group but also failed to reduce media thickness (37). Thus vascular smooth-muscle hypertrophy is not simply a determinant of blood pressure but also depends on other factors.
In normotensive rats, vasodilators showed inconsistent effects on blood pressure. Inhibitors of the angiotensin pathway were mostly reported to have no effect on blood pressure, as demonstrated for captopril and lisinopril, 15 mg/kg/day, and losartan, 10 mg/kg/day (29,30,38). In our study, continuous invasive blood-pressure measurement could not be applied for the large number of animals and the duration of the experiments; intermittent measurements by tail-cuff method were considered to be not precise enough to detect the small effects expected in normotensive animals. Thus a small reduction in blood pressure cannot be excluded to contribute to the structural changes in the aorta. Regarding changes of heart weight, an indirect mechanism by blood-pressure reduction is rather unlikely, because the calcium antagonist had opposing effects. Nevertheless, our study demonstrates that inhibitors of the renin-angiotensin system inhibitors can modulate myocardial and vascular structure even in normotensive animals. This effect demonstrates the regulatory role of the renin-angiotensin system on vascular structure, whether the effects of the inhibitors are caused by the direct trophic effects on the myocytes and vascular smooth-muscle cells or whether they are indirectly mediated by the reduction of vascular tone, which in the latter case, must be also attributed to the activity of the renin-angiotensin system under physiologic conditions.
Long-term cyclosporin A treatment enhances rat aortic contraction to angiotensin II (9,19). A possible effect of cyclosporin A on vascular smooth-muscle cells is of clinical interest because cyclosporin A has been suggested to contribute to the pathogenesis of accelerated atherosclerosis in transplanted organs (37,38). On the other hand, cyclosporin A inhibits media thickening after experimental injuries of rat carotid and common iliac arteries (39,40). In our study, single treatment with cyclosporin A has no effect on media thickness and heart weight. Thus the reported cyclosporin A-induced potentiation of functional effects of angiotensin II is not accompanied by a potentiation of the trophic effects of angiotensin II on rat vascular smooth-muscle cells (9,14).
Cotreatment with cyclosporin A moderately amplifies the effects of ACE inhibitors, AT1 antagonists, and isradipine. Concerning the vascular effects, the effects of the vasodilators appear to be augmented. Two explanations have to be considered but cannot be separated on the basis of our data. Cyclosporin may have an additional depressing effect on vascular smooth-muscle cell volume, as was observed in the rat carotid and iliac artery, which only becomes significant in combination with one of the vasodilators (41,42). Alternatively, cyclosporin may augment the effect of the vasodilators by pharmacokinetic interactions (e.g., reduced excretion of the drugs). The latter mechanism would also explain the augmentation of the effects of isradipine on heart weight in the combination with cyclosporin where opposing effects would be, based on the first hypothesis.
The subendothelial space was investigated particularly to seek for a morphologic substrate of the cyclosporin A-induced impairment of endothelial function (9). In both control and cyclosporin A-treated rats, we found only minimal intimal lesions ≤ 5-mm thick, demonstrating no difference in the circumferential extent of these lesions. Therefore the observed impaired endothelial function is not caused by morphologic changes, as detectable by bright-field microscopy.
In contrast, long-term ACE inhibition reduces the extent of intima lesions, demonstrating a regulation of the rat aortic intima by the renin-angiotensin system. This effect is in line with recent studies on ACE inhibitors reporting a protection of endothelial function in spontaneously hypertensive rats or an inhibition of atherosclerosis in hyperlipidemic rabbits (7,8). It remains unclear whether the AT1 receptor is essentially involved in this process, because D 8731 and losartan showed a reduction of intimal lesions only in combination with cyclosporin A, which was also the case for isradipine. These combined effects of AT1-receptor blockers, as well as isradipine with cyclosporin A, remain to be cleared up in further studies.
Acknowledgment: We thank Ms. Karin Riedel for her excellent technical assistance.
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