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Differential Effects of Imidapril and Candesartan Cilexetil on Plasminogen Activator Inhibitor-1 Expression Induced by Prolonged Inhibition of Nitric Oxide Synthesis in Rat Hearts

Katoh, Makoto*†; Egashira, Kensuke; Mitsui, Takashi*; Takeshita, Akira; Narita, Hiroshi*

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Journal of Cardiovascular Pharmacology: June 2000 - Volume 35 - Issue 6 - p 932-936
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Abstract

Endothelium-derived niric oxide (NO) may play an important role in preventing the development of arteriosclerotic and atherosclerotic changes of the blood vessels. We (1-6) and others (7) have reported that long-term administration of Nω-nitro-L-arginine methyl ester (L-NAME), an inhibitor of NO synthesis for 4-8 weeks, produces systemic arterial hypertension and causes vascular remodeling (medial thickening and fibrosis) and myocardial remodeling (fibrosis and hypertrophy) in rats. The prolonged administration of L-NAME increases cardiac tissue angiotensin-converting enzyme (ACE) activity and the number of angiotensin type 1 (AT1) receptors (1,5), and treatment with an ACE inhibitor or an AT1 antagonist prevents the L-NAME-induced vascular and myocardial remodeling (4-6). These observations support the hypothesis that a defect in endothelial NO synthesis may lead to activation of the local renin-angiotensin system, which may in turn contribute to vascular remodeling.

Recent experimental and clinical studies have suggested that the renin-angiotensin system may be an important regulator of plasminogen activator inhibitor-1 (PAI-1) expression in vivo (8,9). PAI-1 is a major physiologic inhibitor of fibrinolysis in vivo (10). Increased PAI-1 expression has been demonstrated in atherosclerotic lesions (11,12). Several reports have shown that angiotensin (Ang) II-induced PAI-1 expression in cultured endothelial and vascular smooth muscle cells is mediated through the AT1 receptor (13-15). Conversely, recent reports suggest that the effect of an Ang II hexapeptide metabolite, Ang IV, acting through the AT4 receptor may also be involved in PAI-1 expression in cultured endothelial (16) and proximal tubular epithelial cells (17). We recently showed that the ACE inhibitor imidapril inhibits cardiac and aorta PAI-1 expression induced by long-term administration of L-NAME in rats (18). Because treatment with the ACE inhibitor is expected to attenuate both receptor pathways in vivo, it is unclear whether the Ang II or IV receptor is responsible for the increased vascular PAI-1 expression in vivo.

Therefore this study was undertaken to elucidate whether the AT1 receptor is involved in the expression of PAI-1 in the rats with hypertension by long-term inhibition of NO synthesis. For this purpose, we compared the effects of the ACE inhibitor imidapril and the AT1-receptor antagonist candesartan cilexetil on cardiac PAI-1 expression in this model in vivo.

METHODS

Drugs

Imidapril, an ACE inhibitor, and candesartan cilexetil, a selective AT1 antagonist, were synthesized in Tanabe Seiyaku Co., Ltd. (Saitama, Japan). L-NAME was purchased from Sigma Chemical Co., Ltd. (St. Louis, MO, U.S.A.).

Animal model of prolonged inhibition of NO synthesis

All experiments were reviewed and approved by the Committee on Ethics of Animal Experiments, Kyushu University Faculty of Medicine, and conducted according to the Guidelines for Animal Experiments of Kyushu University Faculty of Medicine. Twenty-week-old male Wistar-Kyoto rats, obtained from an established colony at the Animal Research Institution of Kyushu University Faculty of Medicine, housed individually under control conditions (temperature, humidity, and light periods) and fed normal rat chow, were randomly divided into four groups of eight rats. For a treatment period of 7 days, the first (control) group received plain drinking water; the second group (L-NAME) received L-NAME in drinking water (1 mg/ml, ∼30-40 mg/rat/day); the third group (L+ACEI), L-NAME plus imidapril (0.2 mg/ml) in drinking water; and the fourth group (L+AT1RA), L-NAME plus candesartan cilexetil, which was given in 0.5% methyl cellulose by oral gavage (3 mg/kg/day). The systolic arterial pressure of each rat were measured by the tail-cuff method (UR-5000; Ueda, Tokyo, Japan) before and after the 7-day period of treatment.

RNA isolation and Northern blot analysis

Five rats randomly selected from each group were anesthetized with intraperitoneal pentobarbital on day 7. The chest was opened, and the heart was rapidly removed. The left ventricle (LV) was separated from the atria, great vessels, and right ventricular free wall. The LV was snap-frozen in liquid nitrogen and stored at −80°C. Total RNAs were extracted from the LV by the acid guanidinium thiocyanate-phenol-chloroform extraction method (ISOGEN; Nippon Gene, Tokyo, Japan), and Poly(A)+RNA was purified on oligo (dT)-cellulose columns (Takara Shuzo, Otsu, Japan). Two μg of each Poly(A)+RNA sample of the LV was electrophoretically fractionated on agarose gel, transferred to a nylon membrane (Hybond N+; Amersham, U.K.), and immobilized by UV irradiation. The membrane was hybridized overnight with specific cDNA probes for bovine PAI-1 (19), human type I collagen (American Type Culture Collection, Rockville, MD, U.S.A.), human skeletal α-actin (20), and mouse glyceraldehyde-3-phosphate dehydrogenase (GAPDH; American Type Culture Collection), labeled with [32P]dCTP by a random primer labeling kit (Takara Shuzo). After hybridization and washing, autoradiography was performed with Kodak XAR5 films at −80°C with intensifying screens for 24 h. Relative amounts of PAI-1, type I collagen, and skeletal α-actin mRNA were normalized against GAPDH mRNA.

Immunohistochemical localization of PAI-1

The hearts of three rats from each group, killed on day 7, were perfused with oxygenated phosphate-buffered saline without fixation. The tissues were immediately embedded in the OCT compound, frozen, cut into 5-μm-thick slices, and mounted on slides. For immunohistochemistry, the slices were fixed in acetone and then incubated with 0.3% H2O2 in methanol to quench endogenous peroxidase. The sections were preincubated with 10% goat serum to decrease nonspecific binding and incubated with a 1 μg/ml of the anti-rat PAI-1 polyclonal antibody (American Diagnostica, Greenwich, CT, U.S.A.) overnight at 4°C. The slides were washed and incubated with biotinylated, affinity-purified goat anti-rabbit immunoglobulin G (IgG; Nitirei, Tokyo, Japan). Samples were visualized with 3′,3′-diaminobenzidine, and counterstained with hematoxylin.

Statistical analysis

Data are expressed as mean ± SEM. Serial time-related changes in parameters of each group were compared by two-way analysis of variance (ANOVA) and Bonferroni's multiple comparison test. Differences between groups were analyzed by one-way ANOVA and a multiple comparison test. Values of p < 0.05 were considered statistically significant.

RESULTS

Blood pressure, body weight, and cardiac hypertrophy

The L-NAME group showed a progressive increase in systolic arterial pressure. The systolic arterial pressure showed no significant change in the control and L+ACEI groups, but it was significantly reduced in the L+AT1RA group compared with the control group (Table 1). Body weights did not differ significantly among the groups before and after 1 week of treatment. LV weight-to-body weight ratios did not change significantly in the control, L-NAME, L+ACEI, and L+AT1RA groups (Table 1).

TABLE 1
TABLE 1:
Body weight (BW), systolic blood pressure (SBP) and left ventricular weight (LVW)/BW ratio

Cardiac mRNA levels for PAI-1, type I collagen, and skeletal α-actin

The cardiac mRNA levels for PAI-1, type I collagen, and skeletal α-actin were significantly higher in the L-NAME group than in the control group (Fig. 1). The increases in gene expression of type I collagen and skeletal α-actin were significantly reduced by treatment with imidapril as well as with candesartan cilexetil. The increase in PAI-1 mRNA expression was prevented by treatment with imidapril, but not with candesartan cilexetil (Fig. 1).

FIG. 1
FIG. 1:
Plasminogen activator inhibitor-1 (PAI-1) transcript levels in the control, N ω-nitro-L-arginine methyl ester (L-NAME), L+ACEI, and L+AT1RA groups on day 7 of treatment. A: Typical autoradiograms of Northern blot analysis of left ventricular mRNAs for PAI-1, type I collagen, skeletal α-actin, and glyceraldehyde-3-phosphate dehydrogenase (GAPDH). B: Summary of densitometric analysis of data in A. Data are expressed as ratios of designated mRNA to GAPDH mRNA relative to the control group, which was given an arbitrary value of 1. Each bar has n = 5. *p < 0.01 versus control group.

Immunohistochemical analysis of PAI-1

In the control rats, PAI-1 immunoreactivity was hardly present in the coronary vessels and myocardium (Fig. 2A). In the L-NAME group at 7 days, intense immunoreactivity for PAI-1 was observed in the part of endothelium and media (vascular smooth muscle cells) of coronary arteries (Fig. 2B). Treatment with imidapril prevented the increase in PAI-1 immunoreactivity, but candesartan cilexetil showed no such effect (Fig. 2C and D). No immunoreactivity was noted when the PAI-1 antibody was replaced with nonimmune serum (negative control).

FIG. 2
FIG. 2:
Immunohistochemical staining of coronary arterial sections with antibodies against plasminogen activator inhibitor-1 (PAI-1) in the control (A), N ω-nitro-L-arginine methyl ester (L-NAME) (B), L+ACEI (C), and L+AT1RA (D) groups on day 7 of treatment. Bar indicates 50 μm.

DISCUSSION

We recently showed that imidapril inhibits cardiac and aortic PAI-1 expression induced by long-term administration of L-NAME in rats (18). Treatment with an ACE inhibitor is expected to attenuate the whole angiotens-inreceptor pathways in vivo. This study sought to determine whether the AT1 receptor is responsible for the increased vascular PAI-1 expression in vivo. We found that imidapril inhibited the cardiac PAI-1 expression induced by inhibition of NO synthesis, but candesartan cilexetil did not. This finding suggests that the increased PAI-1 level is caused by activation of receptor subtypes other than AT1 receptor, and treatment with imidapril would prevent this activation.

In this study, the cardiac hypertrophy did not occur by day 7 of L-NAME treatment, whereas the increases in gene expression of skeletal α-actin were evident at day 7. These findings are consistent with previous reports (1,3,5), indicating that the increase in the gene expression preceded the development of structural changes. The increases in cardiac gene expression of type I collagen and skeletal α-actin induced by L-NAME were markedly inhibited by treatment with candesartan cilexetil, as well as with imidapril. Our result agrees with prior observations that infusion of Ang II induces increases in the levels of type I collagen and skeletal α-actin mRNA in rat hearts in vivo and that the increases in these mRNA levels during the process of cardiac fibrosis/hypertrophy in vivo were prevented by AT1 antagonists (23,24). In addition, the blood pressure lowering by candesartan cilexetil was greater than that by imidapril. Therefore it is unlikely that both the change in systolic arterial pressure and the AT1-mediated effects were responsible for the increase in PAI-1 expression in this model.

There are two possible mechanisms by which imidapril exerts its beneficial effects: inhibition of bradykinin breakdown and inhibition of AngII formation. We previously reported that the beneficial effects of the ACE inhibitor in combination with HOE140, a specific bradykinin B2-receptor antagonist, did not alter the effects of the ACE inhibitor on systolic arterial pressure, vascular remodeling, or ACE activation induced by long-term inhibition of NO synthesis in rats (4). Recently, AT1 antagonists have been shown to activate the bradykinin/NO system through the AT2 receptor (25,26). Therefore these findings suggest that bradykinin may not participate in the effect of imidapril on the increased PAI-1 expression in this model. One explanation for the differential effects of imidapril and candesartan cilexetil on PAI-1 is the hypothesis that Ang II increases PAI-1 expression through its hexapeptide metabolite, Ang IV, and the AT4 receptor, which has been observed in cultured endothelial and proximal tubular epithelial cells (16,17). The AT4 receptor has been found in several cardiovascular tissues and cells (27,28).

The cardiovascular events accompanied by the L-NAME-induced upregulation of PAI-1 were not explored in this study. Our previous report has shown that treatment with imidapril ameliorates the development of thrombosis in stenosed carotid arteries of rats with hypertension by prolonged inhibition of NO synthesis (29). Recent reports have shown that the effects of ACE inhibition on PAI-1 levels are different from those of AT1 antagonism. Mugellini et al. (21) reported that ACE inhibition with trandolapril lowered plasma PAI-1 antigen concentrations in postmenopausal hypertensive women during ad libitum salt intake, but AT1 antagonism with losartan did not. Brown et al. (22) reported that the effects of losartan were less than those of the ACE inhibitor quinapril on plasma PAI-1 antigen and activity during salt depletion in normotensive humans. The results of this study in rats are consistent with these reports. Thus the differential effects of ACE inhibition and AT1 antagonism on PAI-1 expression could be observed irrespective of species. Imidapril may be beneficial to prevent thrombus formation in patients with ischemic heart disease. Further studies are needed to elucidate the roles of cardiac PAI-1 expression in this model.

In conclusion, we have demonstrated that treatment with imidapril prevented the increased gene expression and immunoreactivity of PAI-1 in the rat model of hypertension by long-term inhibition of NO synthesis, but candesartan cilexetil showed no such effects. These findings deserve further careful scrutiny.

Acknowledgment: We thank Dr. Takeyama for his appropriate suggestions to improve the manuscript.

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Keywords:

Angiotensin-converting enzyme inhibitor; Angiotensin II type 1 receptor antagonist; Plasminogen activator inhibitor-1; Nitric oxide

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