Cardioprotective Effects of Vasopeptidase Inhibition Vs

609

Original Article

Cardioprotective Effects of Vasopeptidase Inhibition vs. Angiotensin Type 1-Receptor Blockade in Spontaneously Hypertensive Rats on a High Salt Diet

Tina GRÖNHOLM＊1,2, Piet FINCKENBERG＊3, Eeva PALOJOKI＊1,2, Antti SARASTE＊4, Tom BÄCKLUND＊1,2, Anders ERIKSSON＊1,2, Mika LAINE＊1,2, Eero MERVAALA＊3, and Ilkka TIKKANEN＊1,2

The aim of our study was to compare the cardioprotective effects of vasopeptidase inhibition with those of

angiotensin type 1 (AT1)-receptor blockade, a diuretic and the combination of AT1-receptor blockade and a diuretic in an experimental rat model of essential hypertension on a high salt diet. Spontaneously hypertensive rats (SHR) (n＝73) were divided into 6 groups to receive the following diet and drug regimens for 8 weeks: 1) low salt controls (NaCl 0.5%); 2) high salt controls (NaCl 6%); 3) omapatrilat (40 mg/kg/d) on a high salt diet; 4) losartan (30 mg/kg/d) on a high salt diet; 5) hydrochlorothiazide (HCTZ; 10 mg/kg/d) on a high salt diet; and 6) losartan＋HCTZ (30＋10 mg/kg/d) on a high salt diet. Blood pressure was measured by tail- cuff plethysmography. The histological score of myocardial damage, myocardial collagen volume fraction (CVF), connective tissue growth factor (CTGF) expression and cardiomyocyte apoptosis were determined. As an antihypertensive, omapatrilat showed greater efﬁcacy than monotherapy with losartan or HCTZ, and was equally effective as the combination of losartan＋HCTZ. Assessed by myocardial damage score, omapatrilat and losartan protected cardiac morphology better than HCTZ or the drug combination. Omapatrilat decreased CVF to a greater extent than the other therapies, whereas losartan was most effective in decreasing CTGF expression. All drug treatments, except HCTZ, decreased cardiomyocyte apoptosis. Our ﬁndings

provide evidence that both vasopeptidase inhibition and AT1-receptor blockade exert cardioprotective properties beyond their blood pressure-lowering effects. Cardioprotection was associated with prevention of cardiomyocyte apoptosis and inhibition of extracellular matrix formation. (Hypertens Res 2004; 27: 609Ð618)

Key Words: hypertension, cardiac collagen, apoptosis, vasopeptidase inhibition, angiotensin type 1-receptor blockade

posed on the myocardium, endocrine, paracrine and au- Introduction tocrine factors such as cytokines, growth factors and vasoac- tive hormones are involved in the pathogenesis of cardiac In addition to hypertension-induced pressure overload im- hypertrophy and ﬁbrosis associated with hypertensive heart

From the ＊1 Minerva Foundation Institute for Medical Research, Biomedicum, Helsinki, Finland, ＊2 Department of Medicine, Helsinki University Central Hospital, Helsinki, Finland, ＊3 Institute of Biomedicine, Pharmacology, University of Helsinki, Biomedicum, Helsinki, Finland, and ＊4 Department of Anatomy, University of Turku, Turku, Finland. This study was supported by research grants from Helsinki University Central Hospital and the Sigrid Juselius Foundation and personal grants from the Finnish-Norwegian Medical Foundation (T.G.), the Finnish Medical Society (T.G.), the Paavo Nurmi Foundation (E.P. and M.L.) and the Finnish Foundation for Cardiovascular Research (E.P. and M.L.). Address for Reprints: Ilkka Tikkanen, M.D., Ph.D, Minerva Foundation for Medical Research, Biomedicum, Helsinki, Haartmaninkatu 8, FIN-00029 Helsinki, Finland. E-mail: ilkka.tikkanen@helsinki.fi Received February 6, 2004; Accepted in revised form May 11, 2004. 610 Hypertens Res Vol. 27, No. 8 (2004) disease (1–5). The main effector of the renin-angiotensin The aim of our study was to investigate whether we can system (RAS), angiotensin II (Ang II), has been shown to act achieve a better tissue protection with the VPI omapatrilat as a growth factor that regulates cell growth/apoptosis and than with the clinically established antihypertensive treat- collagen turnover (5). Locally synthesized Ang II may in- ments with the diuretic hydrochlorothiazide (HCTZ), the duce myocyte growth and fibrosis in the heart even without ARB losartan, or their combination. Furthermore, because the influence of hemodynamic changes (3, 6). high salt intake is a common problem in clinical practice, the The myocardial collagen synthesis is increased in adult cardiovascular effects of these drugs were investigated in spontaneously hypertensive rats (SHR) (7). Previous studies SHR on a high salt diet. We hypothesized that omapatrilat is in SHR have revealed that both angiotensin converting en- effective as an antihypertensive and has superior cardiopro- zyme (ACE) inhibitors and angiotensin type 1 (AT1)-recep- tective effects during conditions of sodium loading, when tor blockers (ARBs) prevent the development of left ventric- drugs inhibiting the RAS are usually not effective. ular hypertrophy (LVH) and cardiac fibrosis (8) and also induce regression of myocardial damage despite an incomplete Methods control of blood pressure (7, 9, 10). These findings suggest an important role for locally synthesized Ang II in the regu- Experimental Animals, Drug Treatments and Sample lation of cardiac cell growth and collagen synthesis in this Preparation genetic model of hypertension (11, 12). Previous studies indicate that Ang II indirectly regulates Male SHR were obtained from Harlan Sprague Dawley (In- cardiac fibroblast function via specific growth factors (13). dianapolis, USA). The rats were housed at 23–25¼C under a The cardiovascular and renal effects of Ang II are mediated 12-h light/dark cycle with free access to rat chow and normal primarily by AT1-receptors (14), and the Ang II-induced ex- tap water. All the experimental procedures were approved by tracellular matrix production seems to be mediated mainly the Animal Ethics Committee of the University of Helsinki. by transforming growth factor β (TGF-β) (5, 15, 16). We At the beginning of the experiment the rats were 8–9 weeks have recently shown in transgenic rats harboring human old and weighed 190–260 g. renin and angiotensinogen genes (17) that Ang II induces the The rats received powdered rat chow containing 6% sodi- myocardial expression of connective tissue growth factor um chloride (Altromin Gesellschaft fur Tierernährung mbH, (CTGF), a downstream effector of TGF-β profibrotic activi- Lage, Germany) for an acclimation period of 10 days. A ties (18–21). Expression of CTGF has been reported in a va- control group received powdered rat chow with low salt con- riety of fibrotic disorders, such as cardiac and renal fibrosis centration (0.5% NaCl). (17, 18, 21–23); moreover, the CTGF expression appears to After the acclimation period the SHR were divided into 6 correlate closely with the degree of fibrosis (22). groups (n＝11–13 animals in each group) to receive the fol- Continuous loss of cardiomyocytes by apoptosis has been lowing diet and drug regimens for 8 weeks: 1) low salt diet implicated in cardiac remodeling and development of heart controls (NaCl 0.5%); 2) high salt diet controls (NaCl 6%); failure. Increased cardiomyocyte apoptosis has been demon- 3) omapatrilat (40 mg/kg/d) on a high salt diet; 4) losartan strated in the hypertrophied left ventricle of SHR (24, 25) (30 mg/kg/d) on a high salt diet; 5) HCTZ (10 mg/kg/d) on a and in patients with essential hypertension (26, 27). Diez et high salt diet; and 6) losartan＋HCTZ (30＋10 mg/kg/d) on al. (28) have shown that increased cardiomyocyte apoptosis a high salt diet. in SHR is related in a temporal manner to local ACE activity Omapatrilat was provided by Bristol-Myers-Squibb Phar- independently of blood pressure level. Furthermore, treat- maceuticals (Princeton, USA) and losartan by Merck-Sharp- ment with an ACE-inhibitor or an ARB has been shown to Dohme (Rahway, USA), and HCTZ was obtained from Sig- prevent cardiomyocyte apoptosis (28, 29), indicating that ma Chemical Co. (St. Louis, USA). The doses of the drugs Ang II is involved in the pathogenesis of cardiomyocyte were chosen on the basis of our earlier study of omapatrilat apoptosis (30). (35) and other previously published experimental works (32, Vasopeptidase inhibitors (VPIs), which simultaneously in- 34, 36–38). The actual doses were calculated by measuring hibit ACE and neutral endopeptidase (NEP), represent a nov- the daily food consumption over the 5 days before the el class of antihypertensive drugs. The antihypertensive ef- medications started, and then adjusted during the treatment fect of VPI has been demonstrated in several models of hy- period by remeasuring the food consumption. pertension independently of the renin- and salt-status Systolic blood pressure (SBP) was measured biweekly in (31–34). The endogenous natriuretic peptides counteract the conscious lightly restrained rats by tail-cuff plethysmogra- detrimental effects of Ang II in the heart. The VPIs increase phy using a Doppler ultrasonic flowmeter to detect the pulse. the local effects of natriuretic peptides by blocking the NEP- SBP was measured an average of 3–5 times for each rat at mediated peptide metabolism and may therefore exert in- each measurement. The body weights were recorded weekly creased antiproliferative and antihypertrophic actions, and throughout the study period. thus improve end organ protection beyond their blood pres- After the 8-week treatment period the rats were sacrificed sure-lowering effect. by intraperitoneal administration of phenobarbital Grönholm et al: Cardioprotection in SHR on a High Salt Diet 611

(Mebunat¨, 55 mg/kg; Orion Pharma, Espoo, Finland), and oxynucleotide transferase-mediated ddUTP nick end labeling blood samples were collected into tubes containing Na2ED- (TUNEL) assay as described previously (40, 41). In brief, TA (150 mmol/l) and aprotinin (500 kallikrein inhibitor units nuclear DNA strand breaks were end-labeled with digoxi- per milliliter). The heart was excised, washed with ice-cold genin-conjugated dideoxy-UTP by terminal transferase and saline, blotted-dry, and weighed. Two 2-mm thick transverse visualized immunohistochemically with digoxigenin anti- sections from the midventricular level were fixed with 4% body conjugated to alkaline phosphatase. The assay was neutral buffered paraformaldehyde overnight and then standardized with the use of adjacent tissue sections treated processed in paraffin with routine techniques for further with DNase I to induce DNA fragmentation as a positive analysis of conventional morphology and determination of control of apoptosis. The percentage of TUNEL-positive car- apoptosis. Samples for immunohistochemistry were snap- diomyocytes was calculated in each animal (n＝9–12/group) frozen in isopentane (－35¼C) and the remaining apical and in a transverse left ventricle tissue section under a micro- basal parts were snap-frozen in liquid nitrogen. scope (×200 magnification) with an ocular grid. An average of 140 fields each containing an average of 125 myocyte nu- clei were studied. Cardiomyocytes were identified by the Cardiac Morphology presence of myofilaments surrounding the nucleus. Sections of the ventricular tissue were stained with Masson’s trichrome technique (n＝7–12 in each group). Arteries and Plasma Renin Activity (PRA) and Plasma Aldosterone fibrous tissue formation were evaluated. Each sample was scored from 0 to 3 according to morphological changes as PRA (42) and plasma aldosterone concentration (43) were described previously (39): measured by radioimmunoassay as described previously. 0: Epicardial vessels normal. No excessive ventricular connective tissue formation. Quantitative in Vitro Autoradiography of ACE 1: Normal intima. Media slightly thickened or normal. Slight increase of connective tissue around the epicardial and Quantitative in vitro autoradiography of cardiac ACE was intramuscular arteries. performed using the radioligand 125I-MK351A as described 2: Normal intima. Clear medial thickening and adventitial previously (35, 44, 45). The optical densities were quantified scarring in epicardial and intramuscular arteries. Patchy in- by an AIDA computer image analyzing system (AIDA 2D crease of slender connective tissue bundles. densitometry) coupled to the FUJIFILM BAS-5000 phos- 3: Normal intima. Clear medial thickening and adventitial phoimager (Tamro, Finland) (35). Specific binding was cal- scarring in epicardial and intramuscular arteries. Evident culated as total binding minus nonspecific binding. myocardial scars. Statistical Analyses Collagen Volume Fraction (CVF) Data were analyzed by ANOVA with the Statview 5 pro- CVF was determined by the picrosirius red method from gram. Comparisons of group means were performed by Fish- three different slices of Direct Red 80 (Fluka Chemie, Buchs, er’s post-hoc-analyses. When the data consisted of repeated Switzerland)-stained left ventricular sections under polarized measures at successive time points, ANOVA for repeated light. Areas of connective tissue network and myocytes were measures was used. Values of p<0.05 were considered to in- quantified by a semi-automated computer-based analysis dicate statistical significance. Data are shown as the mean± system as described by Brooks et al. (8). The results are pre- SEM unless otherwise indicated. sented as a percentage of collagen positive area per total myocardial area. Results

Immunohistochemistry SBP The expression of CTGF in the heart was determined by In the beginning of the experiment, SBP in the SHR aver- methods described elsewhere (12). Primary antibodies aged 154±3.1 mmHg. There were no statistically significant against rat CTGF (anti-rat CTGF; Abcam, Cambridge, UK) differences in SBP among the groups. After 10 days with dif- were used. The intensity of CTGF expression was evaluated ferent salt diets, the blood pressure-levels in the high salt by scoring the samples from 0 to 4 according to the amount groups were significantly elevated compared to those of the of antibody positive labeling (n＝6–8). low salt control group. A high salt diet for 10 weeks significantly increased the blood pressure (Fig. 1A). Before the medication started there still were no significant differences Apoptosis among the groups that included a high salt diet. All drug Cardiomyocyte apoptosis was assessed by the terminal de- treatments decreased the blood pressure significantly com- 612 Hypertens Res Vol. 27, No. 8 (2004)

Table 1. Body Weight and Relative Cardiac Weight (Heart Weight to Body Weight) Were Measured at the End of the Experiment

Body weight Relative cardiac Group (g) weight (mg/g) 0.5% NaCl control 330±7 3.8±0.07 6% NaCl control 328±4 4.3±0.07＊＊＊ 6% NaCl＋oma 315±4 3.9±0.08†† 6% NaCl＋los 331±6 4.0±0.06† 6% NaCl＋HCTZ 319±5 4.0±0.07＊,† 6% NaCl＋(los＋HCTZ) 321±5 3.5±0.07＊,††† Values are given as mean±SEM. oma, omapatrilat; los, losartan; HCTZ, hydrochlorothiazide. ＊ p<0.05 and ＊＊＊ p<0.001 as compared to the 0.5% NaCl controls. † p<0.05, †† p<0.01 and ††† p<0.001 compared to the 6% NaCl controls.

Heart Weight and Body Weight The high sodium diet induced cardiac hypertrophy as assessed by the heart weight to body weight ratio (Table 1). The relative cardiac weight was 4.3 mg/g in the controls on a high salt diet compared to 3.8 mg/g in the low salt diet controls ( p<0.001). All drug treatments significantly lowered the relative cardiac weight in comparison to that of the controls on a high salt diet, but combination treatment was the most efficient of the drugs studied. There was no difference among the groups in the body weight gain. Fig. 1. The development of systolic blood pressure (SBP) in the control groups on a 0.5% or 6% NaCl diet during the 10- week study period (A). The high salt content of the diet in- Cardiac Morphology creased SBP more rapidly and to a higher level than the SBP Cardiac samples from the high salt control group had signifi- ＊＊＊＊ in the controls on a low salt diet. p<0.05, p<0.001 vs. cantly more pathological changes in comparison with all the the 0.5% NaCl controls. B: The effect of medication on SBP other groups (Fig. 2). The large epicardial arteries showed in SHR on a 6% NaCl diet during the 10-week study period. marked adventitial fibrosis and thickening of the medial lay- The 6% NaCl diet began at week 0 and the medications were started at week 2 (arrow). There were no significant differ- er. There were also some inflammatory cells in the perivas- ences between the groups at week 0 or at week 2. All drug cular areas. The small intramuscular arteries showed variable treatments significantly decreased the SBP during the med- amounts of concentric hypertrophy narrowing the luminal ication period compared with that in the controls, except for space. In the cardiac muscle there were numerous scars indi- HCTZ at week 4. At the end of the experiment, the SBP in the cating previous infarcts. Omapatrilat and losartan decreased omapatrilat-treated group was equal to the SBP in the combi- the myocardial damage score to the same level found in the nation-treated group and significantly lower than those in the low salt SHR controls (Fig. 3). In contrast, HCTZ alone or in losartan- (p<0.001) and HCTZ-treated groups (p<0.001). combination with losartan conferred only partial protection against salt-induced myocardial damage. pared to the controls during the high salt diet (Fig. 1B). At CVF the end of the experiment, the antihypertensive effect of omapatrilat was equal to that of the combination of The high salt diet increased the amount of interstitial colla- losartan＋HCTZ ( p＝non significant) and more pronounced gen deposition and fibrosis in SHR ( p<0.01) compared to compared to monotherapy with losartan or HCTZ ( p<0.001, the low salt controls (Fig. 4). All drug treatments prevented respectively). the development of cardiac fibrosis. However, treatment with omapatrilat was most effective in decreasing the CVF. Grönholm et al: Cardioprotection in SHR on a High Salt Diet 613

Fig. 2. Sections of ventricular tissue were stained with the Masson’s trichrome technique. Each sample was scored from 0 to 3 according to morphological changes (as described in the text) to determine the cardiac morphological score. The low salt control group and the losartan- and omapatrilat-treated groups (A, C, D, respectively) showed normal cardiac histology, whereas the high salt controls had marked perivascular ﬁbrosis extending also into the interstitial areas (B). Magniﬁcation:×200.

Fig. 3. Cardiac morphology score after 8 weeks of medication compared to that in the non-treated control rats. The Fig. 4. The amount of cardiac collagen after the 8-week high dietary salt was associated with significantly more treatment period as shown by collagen volume fraction pathological changes in cardiac structure compared to the (CVF) measurement. Treatment with omapatrilat significant- low salt diet controls. Omapatrilat and losartan almost com- ly decreased the CVF in comparison to the other drugs pletely prevented the development of pathological lesions, studied. ＊＊ p<0.01 vs. the 0.5% NaCl controls. † p<0.05, while the effect of HCTZ or the combination of losartan and ††† p<0.001 vs. the 6% NaCl controls. HCTZ was less significant. ＊ p<0.05, ＊＊ p<0.01, ＊＊＊ p<0.001 vs. the 0.5% NaCl controls. † p<0.05, ††† p<0.001 vs. the 6% NaCl controls. 614 Hypertens Res Vol. 27, No. 8 (2004)

CTGF Expression PRA and Plasma Aldosterone The high salt diet increased the myocardial CTGF expres- The high salt diet tended to decrease PRA, but this difference sion 2.25-fold (Fig. 5). The salt-induced increase in CTGF did not reach statistical significance (Table 2). Omapatrilat, expression was prevented by all drug treatments. However, losartan, and the drug combination increased PRA. The com- losartan decreased CTGF more effectively than omapatrilat bination of losartan and HCTZ increased PRA more than or HCTZ ( p<0.05 for both groups). omapatrilat. The high salt diet decreased the plasma aldosterone concentration by 50% in comparison to the low salt diet controls ( p<0.05) (Table 2). Omapatrilat and losartan Apoptosis tended to decrease the plasma aldosterone level compared to The number of apoptotic cardiomyocytes increased by 125% the high salt control group, although this difference was not in the high salt diet control group compare to the low salt di- statistically significant. In contrast, HCTZ increased plasma et controls ( p<0.01) (Fig. 6). Omapatrilat, losartan, and the aldosterone. drug combination prevented the salt-induced increase in cardiomyocyte apoptosis, whereas HCTZ did not significantly influence the number of apoptotic cells.

Fig. 6. The effect of different medications on cardiomyocyte apoptosis in comparison to controls on a low or high salt Fig. 5. Connective tissue growth factor (CTGF) score after diet. The amount of apoptosis in the control group on a high the 8-week treatment period. The high dietary salt upregulat- salt diet was increased compared with that in the low salt ed the expression of cardiac CTGF in comparison to that of diet controls. All the drugs except for HCTZ decreased the the control group on a low salt diet. All drug treatments pre- cardiomyocyte apoptosis compared to the 6% NaCl control vented CTGF upregulation compared to the 6% NaCl con- group. Omapatrilat and losartan reduced the amount of trol group. The CTGF expression was signiﬁcantly sup- apoptosis most effectively, while HCTZ did not affect the pressed in the losartan-treated group compared to the omap- number of apoptotic cells. There was no signiﬁcant differ- ＊＊ atrilat- or the HCTZ-treated group. ＊＊ p<0.01 vs. the 0.5% ence between omapatrilat and losartan treatment. p<0.01 † ††† NaCl controls. † p<0.05, ††† p<0.001 vs. the 6% NaCl con- vs. the 0.5% NaCl controls. p<0.05, p<0.001 vs. the trols. 6% NaCl controls.

Table 2. Plasma Renin Activity (PRA), Cardiac Angiotensin Converting Enzyme (ACE) Inhibition and Plasma (P-) Aldosterone at the End of the Study

Group PRA (ng/ml) Autoradiography of cardiac ACE (%) P-aldosterone (µg/l) 0.5% NaCl control 3.0±0.4 100.0±8.5 160.0±31.7 6% NaCl control 1.0±0.2 108.2±13.6 79.1±19.9＊ 6% NaCl＋oma 18.6±2.0＊＊＊,††† 12.1±1.0＊＊＊,††† 68.1±4.7＊ 6% NaCl＋los 20.8±1.5＊＊＊,††† 109.4±11.9 64.1±6.7＊＊ 6% NaCl＋HCTZ 5.4±1.0 96.5±14.7 146.5±44.3 6% NaCl＋(los＋HCTZ) 24.5±1.7＊＊＊,††† 65.7±6.7＊＊,††† 101.2±16.3 Values are given as mean±SEM. oma, omapatrilat; los, losartan; HCTZ, hydrochlorothiazide. ＊ p<0.05, ＊＊ p<0.01 and ＊＊＊ p<0.001 as compared to the 0.5% NaCl controls. ††† p<0.001 compared to the 6% NaCl controls. Grönholm et al: Cardioprotection in SHR on a High Salt Diet 615

treatment was used in this study. The high salt intake markedly induced cardiac hypertro- Autoradiography of ACE phy, as assessed by the heart weight to body weight ratio. Autoradiography was used to determine the expression and The antihypertrophic effects of all drug treatments were sig- activity of ACE in the heart. The high sodium diet did not in- nificantly greater than those in the high salt controls. The fluence cardiac ACE expression (Table 2). Omapatrilat in- combination treatment, which together with omapatrilat was hibited cardiac ACE by 88%. In contrast, neither losartan nor the most efficient as a blood pressure-lowering agent, was al- HCTZ influenced cardiac ACE expression. so the most effective in decreasing the relative cardiac weight. Discussion Although omapatrilat reduced the blood pressure level more effectively than losartan, the beneficial effects on car- The main findings of this study are: 1) the antihypertensive diac morphology did not differ between these drugs. Both effect of the VPI omapatrilat was superior to that of the ARB provided almost complete protection against the develop- losartan in SHR on a high salt diet; 2) both omapatrilat and ment of pathological lesions. Thus, the AT1 blockade by losartan were equally effective in preventing the detrimental losartan appears to have additional, blood pressure-indepen- effects of dietary salt on myocardial morphology as well as dent, cardioprotective effects. Furthermore, losartan was in preventing a salt-induced increase in cardiomyocyte apop- more effective than omapatrilat in decreasing cardiac CTGF tosis; 3) as compared to losartan, omapatrilat was more ef- expression. CTGF has been reported to mediate the stimula- fective in preventing extracellular matrix accumulation when tory actions of TGF-β on extracellular matrix synthesis (22) assessed by myocardial collagen volume fraction; and 4) the and to enhance the proliferation of fibroblasts and endothe- drug combination of losartan and HCTZ decreased the blood lial cells, as well as to induce apoptosis in vascular smooth pressure effectively, but its cardioprotective effect was less muscle cells (51). On the other hand, omapatrilat was superi- pronounced. or to losartan in decreasing the cardiac interstitial collagen Previous studies have shown that a high salt intake ele- content. The high dietary salt intake weakens the efficacy of vates blood pressure and promotes cardiac fibrosis in SHR most antihypertensives, including ARBs, while the VPIs re- and also induces LVH and cardiac fibrosis in normotensive tain their antihypertensive efficacy independently of the Wistar-Kyoto rats (WKY) (46, 47). The proposed mecha- renin- and salt-status. The higher SBP level in the losartan- nisms of this pressure-independent LVH are the extracellular treated group may have resulted in the higher CVF compared volume expansion and the increased activity of the sympa- to the omapatrilat-treated group. thetic nervous system associated with high sodium chloride Experimental and clinical findings indicate that loss of intake (46). In addition, the high salt diet has been shown to cardiomyocytes by apoptosis contributes to the myocardial induce overexpression of cardiac TGF-β mRNA in both remodeling seen in hypertensive heart disease (30, 52, 53). SHR and WKY (47). In the present study the high dietary Ang II promotes both cardiac hypertrophy and apoptosis via salt concentration was clearly associated with local patholog- the AT1-receptors in cardiomyocytes (30, 54). Long-term ical changes in cardiac structure. As expected, the high salt pharmacological intervention with an ACE inhibitor or an intake increased the blood pressure level. The high content ARB prevents cardiomyocyte apoptosis in SHR (28, 29). In of salt in the diet also increased the cardiac interstitial colla- this study the rats on a high salt concentration diet had sig- gen deposition and fibrosis, significantly induced the expres- nificantly more apoptotic cardiomyocytes than rats on a low sion of the fibrogenic and pro-apoptotic CTGF, and acceler- salt diet. Both omapatrilat and losartan effectively reduced ated the rate of myocardial apoptosis. the degree of cardiomyocyte apoptosis to the level seen in The blood pressure-lowering efficacy of the VPI omapatri- the control group on the low salt concentration diet. Combi- lat was greater than that by monotherapy with losartan or nation treatment also decreased cardiomyocyte apoptosis HCTZ, and was as effective as combination treatment with compared to that of the high salt control group, but the effect losartan and HCTZ. This is in agreement with previous stud- was not as obvious as with losartan alone. Even though com- ies showing that the antihypertensive efficacy of VPIs was bination treatment was as effective as omapatrilat in reduc- superior to that of conventional treatments (48, 49). The si- ing blood pressure, there was a significant difference in favor multaneous inhibition of NEP and ACE results both in in- of omapatrilat in the ability to prevent apoptosis. Thus, the creased availability of natriuretic peptides and bradykinin, prevention of cardiomyocyte apoptosis by different types of and in reduced formation of Ang II, thereby decreasing vas- antihypertensive regimen is not directly connected to the cular tone and lowering blood pressure by dual mechanisms blood pressure-lowering efficacy of the drugs, but rather to (31, 49, 50). Under conditions of salt loading, most antihy- their ability to regulate apoptosis at the tissue level. pertensives, and particularly drugs inhibiting the RAS, lose Surprisingly, the significant cardioprotective effect of some of their efficacy due to the suppressed renin activity. If losartan, as assessed by the cardiac morphological score, was a diuretic is then added to the medication, the antihyperten- impaired when using losartan in combination with HCTZ in sive effect is usually regained, which is why the combination spite of the improvement in blood pressure control. More- 616 Hypertens Res Vol. 27, No. 8 (2004) over, losartan used as a monotherapy was more effective than combination treatment in reducing the expression of Acknowledgements CTGF. There was no significant difference between losartan and the combination treatment in decreasing the interstitial We wish to acknowledge Ms. Terhi Suvanto, Ms. Tuula collagen content. Earlier studies have given conflicting re- Riihimäki, Ms. Riina Hatakka and Ms. Saara Merasto for their sults in regard to the effect of HCTZ on myocardial fibrosis excellent technical assistance. in SHR; the results range from prevention of cardiac hypertrophy and fibrosis (55) to increasing myocardial collagen References concentration in HCTZ-treated SHR (56). In general, the conventional antihypertensive drugs, such as diuretics, seem 1. Olivetti G, Melissari M, Balbi T, et al: Myocyte cellular to have minor antihypertrophic and antifibrotic effects on the hypertrophy is responsible for ventricular remodelling in heart in comparison to ACE inhibitors and ARBs (57, 58). the hypertrophied heart of middle aged individuals in the The reason for the less beneficial cardiac structural effects absence of cardiac failure. Cardiovasc Res 1994; 28: of the combination treatment with losartan and HCTZ com- 1199–1208. pared to losartan monotherapy is not clear. Plasma renin ac- 2. Lopez B, Gonzales A, Varo N, Laviades C, Querejeta R, tivity was highest in rats receiving combination therapy, sug- Diez J: Biochemical assessment of myocardial fibrosis in hypertensive heart disease. Hypertension 2001; 38: gesting that HCTZ activated the RAS, which could counter- 1222–1226. act the effects of AT1-receptor blockade. On the other hand, 3. Dostal DE: Regulation of cardiac collagen: angiotensin and cardiac ACE activity was down-regulated in rats receiving cross-talk with local growth factors. Hypertension 2001; the combination of losartan and HCTZ. The aldosterone lev- 37: 841–844. el in the HCTZ- and the combination-treated groups tended 4. Hefti MA, Harder BA, Eppenberger HM, Schaub MC: Sig- to be higher than in the groups receiving omapatrilat or naling pathways in cardiac myocyte hypertrophy. J Mol losartan. It has recently been demonstrated both in animal Cell Cardiol 1997; 29: 2873–2892. and human studies that aldosterone is locally produced in the 5. Ruiz-Ortega M, Lorenzo O, Ruperez M, et al: Role of the heart (59–61) and acts via mineralocorticoid receptors in the renin-angiotensin system in vascular diseases. Hyperten- – myocardium to enhance extracellular matrix and collagen sion 2001; 38: 1382 1389. deposition (62, 63). The differences in renin activity and al- 6. Higaki J, Aoki M, Morishita R, et al: In vivo evidence of the importance of cardiac angiotensin-converting enzyme dosterone levels may at least partly explain the impairment in the pathogenesis of cardiac hypertrophy. Arterioscler of cardiac structure in diuretic-treated rats. Thromb Vasc Biol 2000; 20: 428–434. In conclusion, both omapatrilat and losartan exerted car- 7. Varo N, Etayo JC, Zalba G, et al: Losartan inhibits the dioprotective properties beyond the blood pressure-lowering post-transcriptional synthesis of collagen type I and re- effect in SHR with high salt intake. During the medication verses left ventricular fibrosis in spontaneously hyperten- period there were no statistically significant differences be- sive rats. J Hypertens 1999; 17: 107–114. tween the group treated with omapatrilat and the group re- 8. Brooks WW, Bing OHL, Robinson KG, Slawsky MT, ceiving the combination of losartan＋HCTZ, and these Chaletsky DM, Conrad CH: Effect of angiotensin-convert- groups attained the same blood pressure level at the end of ing enzyme inhibition on myocardial fibrosis and function the experiment. Thus, the difference in cardiac structural in hypertrophied and failing myocardium from the sponta- – changes between the omapatrilat- and combination-treatment neously hypertensive rat. Circulation 1997; 96: 4002 4010. 9. Brilla CG, Janicki JS, Weber KT: Cardioreparative effects groups was attributed to effects of omapatrilat other than the of lisinopril in rats with genetic hypertension and left ven- blood pressure-lowering effect. When compared to tricular hypertrophy. Circulation 1991; 83: 1771–1779. monotherapy with losartan, omapatrilat was more effective 10. Baba HA, Iwai T, Bauer M, Irlbeck M, Schmid KW, as an antihypertensive and in decreasing collagen deposition, Zimmer HG: Differential effects of angiotensin II receptor whereas losartan was superior to omapatrilat in preventing blockade on pressure-induced left ventricular hypertrophy CTGF upregulation. Both drugs, however, beneficially pre- and fibrosis in rats. J Mol Cell Cardiol 1999; 31: 445–455. vented the development of cardiac lesions and cardiomyo- 11. Varo N, Iraburu MJ, Varela M, Lopez B, Etayo JC, Diez J: cyte apoptosis. Chronic AT1 blockade stimulates extracellular collagen The present study underscores the importance of blockade type 1 degradation and reverses myocardial fibrosis in of the RAS in the treatment of hypertensive heart disease. spontaneously hypertensive rats. Hypertension 2000; 35: – Theoretically, the VPIs offer an interesting approach to the 1197 1202. 12. Varagic J, Susic D, Frohlich ED: Coronary hemodynamic therapeutic strategy for cardiovascular diseases. Still, as a and ventricular responses to angiotensin type 1 receptor in- consequence of the complex pharmacology of these drugs, hibition in SHR. Hypertension 2001; 37: 1399–1403. more studies are needed to clarify the exact mechanisms and 13. Pathak M, Sarkar S, Vellaichamy E, Sen S: Role of myo- interactions of the dual ACE/NEP inhibition at the tissue lev- cytes in myocardial collagen production. Hypertension el. 2001; 37, 833–840. 14. Tsutsumi Y, Matsubara H, Ohkubo N, et al: Angiotensin II Grönholm et al: Cardioprotection in SHR on a High Salt Diet 617

type 2 receptor is upregulated in human heart with intersti- S37–S43. tial fibrosis, and cardiac fibroblasts are the major cell type 32. Trippodo NC, Robl JA, Asaad MM, Fox M, Panchal BC, for its expression. Circ Res 1998; 83: 1035–1046. Schaeffer TR: Effects of omapatrilat in low, normal, and 15. Lijnen PJ, Petrov VV, Fagard RH: Induction of cardiac fi- high renin experimental hypertension. Am J Hypertens brosis by transforming growth factor-beta1. Mol Genet 1998; 11: 363–372. Metab 2000; 71: 418–435. 33. Tikkanen T, Tikkanen I, Rockell MD, et al: Dual inhibition 16. Mezzano SA, Ruiz-Ortega M, Egido J: Angiotensin II and of neutral endopeptidase and angiotensin-converting en- renal fibrosis. Hypertension 2001; 38: 635–638. zyme in rats with hypertension and diabetes mellitus. Hy- 17. Finckenberg P, Inkinen K, Ahonen J, et al: Angiotensin II pertension 1998; 32: 778–785. induces connective tissue growth factor gene expression via 34. Burrell LM, Droogh J, Man in’t Veld O, Rockell MD, calcineurin-dependent pathways. Am J Pathol 2003; 163: Farina NK, Johnston CI: Antihypertensive and antihyper- 355–366. trophic effects of omapatrilat in SHR. Am J Hypertens 18. Gupta S, Clarkson MR, Duggan J, Brady HR: Connective 2000; 13: 1110–1116. tissue growth factor: potential role in glomerulosclerosis 35. Bäcklund T, Palojoki E, Grönholm T, et al: Dual inhibition and tubulointerstitial fibrosis. Kidney Int 2000; 58: of angiotensin converting enzyme and neutral endopepti- 1389–1399. dase by omapatrilat in rat in vivo. Pharmacol Res 2001; 44: 19. Moussad EE, Brigstock DR: Connective tissue growth fac- 411–418. tor: what’s in a name ? Mol Genet Metab 2000; 71: 36. Makino N, Sugano M, Otsuka S, Hata T: Molecular mecha- 276–292. nism of angiotensin II type I and type II receptors in car- 20. Duncan MR, Frazier KS, Abramson S, et al: Connective diac hypertrophy of spontaneously hypertensive rats. Hy- tissue growth factor mediates transforming growth factor pertension 1997; 30: 796–802. beta-induced collagen synthesis: down-regulation by 37. Nunez E, Hosoya K, Susic D, Frohlich ED: Enalapril and cAMP. FASEB J 1999; 13: 1774–1786. losartan reduced cardiac mass and improved coronary he- 21. Chen MM, Lam A, Abraham JA, Schreiner GF, Joly AH: modynamics in SHR. Hypertension 1997; 29: 519–524. CTGF expression is induced by TGF-beta in cardiac fibro- 38. Wienen W, Schierok HJ: Effects of telmisartan, hy- blasts and cardiac myocytes: a potential role in heart fibrosis. drochlorothiazide and their combination on blood pressure J Mol Cell Cardiol 2000; 32: 1805–1819. and renal excretory parameters in spontaneously hyperten- 22. Ihn H: Pathogenesis of fibrosis: role of TGF-beta and CT- sive rats. JRAAS 2001; 2: 123–128. GF. Curr Opin Rheumatol 2002; 14: 681–685. 39. Finckenberg P, Lassila M, Inkinen K, et al: Cyclosporine 23. Oemar BS, Werner A, Garnier J-M, et al: Human connec- induces myocardial connective tissue growth factor in tive tissue growth factor is expressed in advanced athero- spontaneously hypertensive rats on high-sodium diet. sclerotic lesions. Circulation 1997; 95: 831–839. Transplantation 2001; 71: 951–958. 24. Hamet P, Richard L, Dam T-V, et al: Apoptosis in target 40. Saraste A, Pulkki K, Kallajoki M, Henriksen K, Parvinen organs of hypertension. Hypertension 1995; 26: 642–648. M, Voipio-Pulkki L-M: Apoptosis in human acute myocar- 25. Liu Y-H, Yang X-P, Sharov VG, et al: Effects of an- dial infarction. Circulation 1997; 95: 320–323. giotensin-converting enzyme inhibitors and angiotensin II 41. Palojoki E, Saraste A, Eriksson A, Pulkki K, Kallajoki M, type 1 receptor antagonists in rats with heart failure: role of Voipio-Pulkki L-M: Cardiomyocyte apoptosis and ventric- kinins and angiotensin II type 2 receptors. J Clin Invest ular remodeling after myocardial infarction in rats. Am J 1997; 99: 1926–1935. Physiol Heart Circ Physiol 2001; 280: H2726–H2731. 26. Yamamoto S, Sawada K, Shimomura H, et al: On the na- 42. Fyhrquist F, Soveri P, Putula L, Stenman U: Radioim- ture of cell death during remodeling of the hypertrophied munoassay of plasma renin activity. Clin Chem 1976; 22: human myocardium. J Mol Cell Cardiol 2000; 32: 250–256. 161–175. 43. Mervaala EMA, Pere A-K, Lindgren L, et al: Effects of di- 27. Gonzalez A, Lopez B, Ravassa S, et al: Stimulation of car- etary sodium and magnesium on cyclosporine A-induced diac apoptosis in essential hypertension: potential role of hypertension and nephrotoxicity in spontaneously hyper- angiotensin II. Hypertension 2002; 39: 75–80. tensive rats. Hypertension 1997; 29: 822–827. 28. Diez J, Panizo A, Hernandez M, et al: Cardiomyocyte 44. Burrell LM, Farina N, Risvanis J, Woollard D, Casely D, apoptosis and cardiac angiotensin-converting enzyme in Johnston CI: Inhibition of NEP, the degradative enzyme for spontaneously hypertensive rats. Hypertension 1997; 30: natriuretic peptides, in rat kidney after oral SCH 42495. 1029–1034. Clin Sci 1997; 93: 43–50. 29. Fortuno MA, Ravassa S, Etayo JC, Diez J: Overexpression 45. Kohzuki M, Johnston CI, Chai SY, et al: Measurement of of Bax protein and enhanced apoptosis in the left ventricle angiotensin converting enzyme induction and inhibition us- of spontaneously hypertensive rats: effects of AT1 blocking quantitative in vitro autoradiography: tissue selective ade with losartan. Hypertension 1998; 32: 280–286. induction after chronic lisinopril treatment. J Hypertens 30. Fortuno MA, Ravassa S, Fortuno A, Zalba G, Diez J: Car- 1991; 9: 579–587. diomyocyte apoptotic cell death in arterial hypertension: 46. Mervaala EM, Himberg JJ, Laakso J, Tuomainen P, mechanisms and potential management. Hypertension Karppanen H: Beneficial effects of a potassium- and mag- 2001; 38: 1406–1412. nesium-enriched salt alternative. Hypertension 1992; 19: 31. Burnett JC Jr: Vasopeptidase inhibition: a new concept in 535–540. blood pressure management. J Hypertens 1999; 17: 47. Yu HCM, Burrell LM, Black MJ, et al: Salt induces myo- 618 Hypertens Res Vol. 27, No. 8 (2004)

cardial and renal fibrosis in normotensive and hypertensive 56. Su JZ, Chen SC, Wu KG, et al: Effects of perindopril, pro- rats. Circulation 1998; 98: 2621–2628. pranolol, and dihydrochlorothiazide on cardiovascular re- 48. Campese VM, Lasseter KC, Ferrario CM, et al: Omapatri- modelling in spontaneously hypertensive rats. Pharmacol lat versus lisinopril; efficacy and neurohormonal profile in Sin 1999; 20: 923–928. salt-sensitive hypertensive patients. Hypertension 2001; 38: 57. Frohlich ED: Local hemodynamic changes in hypertension: 1342–1348. insights for therapeutic preservation of target organs. Hy- 49. Weber MA: Vasopeptidase inhibitors. Lancet 2001; 358: pertension 2001; 38: 1388–1394. 1525–1532. 58. Motz W, Vogt M, Scheler S, Strauer BE: Pharmacothera- 50. Stewen P, Mervaala E, Karppanen H, et al: Sodium load in- peutic effects of antihypertensive agents on myocardium creases renal angiotensin type 1 receptors and decreases and coronary arteries in hypertension. Eur Heart J 1992; bradykinin type 2 receptors. Hypertens Res 2003; 26: 13: 100–106. 583–589. 59. Silvestre J-S, Robert V, Heymes C, et al: Myocardial pro- 51. Hishikawa K, Nakaki T, Fujii T: Connective tissue growth duction of aldosterone and corticosterone in the rat. J Biol factor induces apoptosis via caspase 3 in cultured human Chem 1998; 273: 4883–4891. aortic smooth muscle cells. Eur J Pharmacol 2000; 392: 60. Yamamoto N, Yasue H, Mizuno Y, et al: Aldosterone is 19–22. produced from ventricles in patients with essential hyper- 52. Narula J, Haider N, Virmani R, et al: Apoptosis in myo- tension. Hypertension 2002; 39: 958–962. cytes in end-stage heart failure. N Engl J Med 1996; 335: 61. Ito T, Yoshimura M, Nakamura S, et al: Inhibitory effect of 1182–1189. natriuretic peptides on aldosterone synthase gene expres- 53. Olivetti G, Abbi R, Quaini F, et al: Apoptosis in the failing sion in cultured neonatal rat cardiocytes. Circulation 2003; human heart. N Engl J Med 1997; 336: 1131–1141. 107: 807–810. 54. Kajstura J, Cigola E, Malhotra A, et al: Angiotensin II in- 62. Zannad F, Dousset B, Alla F: Treatment of congestive heart duces apoptosis of adult ventricular myocytes in vitro. J failure: interfering the aldosterone-cardiac extracellular ma- Mol Cell Cardiol 1997; 29: 859–870. trix relationship. Hypertension 2001; 38: 1227–1232. 55. Contard F, Glukhova M, Marotte F, et al: Diuretic effects 63. Grandi AM, Imperiale D, Santillo R, et al: Aldosterone an- on cardiac hypertrophy in the stroke prone spontaneously tagonist improves diastolic function in essential hyperten- hypertensive rat. Cardiovasc Res 1993; 27: 429–434. sion. Hypertension 2002; 40: 647–652.