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*
Cardiovascular Research, Institute of Physiology, University of
Zürich, Zürich,
Switzerland
Clinical Research, Inselspital, University of Bern, Bern,
Switzerland
Cellular and Molecular Pathology, German Cancer Research Center,
Heidelberg, Germany
Cardiovascular Center and Cardiology, University of
Zürich, Zürich,
Switzerland.
Correspondence to Dr. Thomas F. Lüscher, Professor and Head of Cardiology, University Hospital, CH-8091 Zürich, Switzerland. Phone: 0041 1 255 21 21; Fax: 0041 1 255 42 51; E-mail: cardiotfl{at}gmx.de
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Vasopeptidase inhibition represents a new therapeutic principle in hypertension (5,6,7) and heart failure (8), which includes inhibition of neutral endopeptidase (NEP) in addition to ACE inhibition. NEP catalyzes the degradation of a number of endogenous vasodilator peptides, including atrial natriuretic peptide, brain natriuretic peptide, C-type natriuretic peptide, substance P, and bradykinin, as well as vasoconstrictor peptides, including endothelin-1 (ET-1) (9) and angiotensin II (10). Hence the overall effect of NEP inhibition on vascular tone will depend on the effects of a compound on the procession of these different vasoactive substances and is—especially in molecules, which inhibit other systems as well—difficult to predict. Nevertheless, omapatrilat (O), a new vasopeptidase inhibitor, effectively lowers BP in salt-dependent and volume-dependent as well as in renin-dependent forms of hypertension (11). The combination of ACE and NEP inhibition may be particularly useful in the treatment of hypertension (5, 12,13,14) and heart failure (15,16,17,18,19).
Vasopeptidase inhibitors lower BP in a broader range of conditions than inhibition of ACE or NEP alone, and their effectiveness seems to be independent of the activity of the renin-angiotensin system or the degree of salt retention (20). O is a new vasopeptidase inhibitor that induces long-lasting antihypertensive effects in experimental hypertension (12), greater than those elicited by selective inhibition of either enzyme alone (11). Furthermore, O lowers BP and attenuates cardiac hypertrophy in diabetic hypertensive rats (21). Meanwhile, first clinical data are available, demonstrating hemodynamic benefits of treatment with O in patients with hypertension (14, 22,23,24) and heart failure (8, 25,26,27). The first large-scale clinical study on heart failure indicates reduced morbidity and mortality on treatment with O as compared with ACE inhibitor treatment (28).
Despite obvious clinical benefit of vasopeptidase inhibitors in heart failure and hypertension, their mechanism of action is still poorly understood. A positive influence of O on vessel stiffness (19) and vascular remodeling (29, 30) has been shown before. Also, long-term vasopeptidase inhibition exerts beneficial effects in the renal circulation (26). Influences of vasopeptidase inhibitors on endothelial function in the renal artery may substantially improve renal hemodynamics and therefore contribute to their beneficial systemic effects.
Therefore, this study was designed to investigate the effects of long-term treatment with the vasopeptidase inhibitor O on renovascular endothelial function as well as its effects on vascular hypertrophy in a model of salt-induced hypertension.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Tissue Harvesting
Animals were anesthetized with pentobarbital (50 mg/kg intraperitoneally)
after 8 wk treatment, and blood samples were collected through puncture of the
right ventricle. The renal arteries were removed, dissected free from adherent
connective tissue, and placed immediately into cold (4°C) modified
Krebs-Ringer bicarbonate solution: 118.6 mmol/L NaCl, 4.7 mmol/L KCl, 2.5
mmol/L CaCl2, 1.2 mmol/L MgSO4, 1.2 mmol/L
KH2PO4, 25.1 mmol/L NaHCO3, 0.026 mmol/L
ethylenediaminetetraacetic acid, and 10.1 mmol/L glucose. Under a microscope
(Leica Wild M3C, Heerbrugg, Switzerland), vessels were cleaned of adherent
tissue and cut into 3-mm-long rings.
Organ Chamber Experiments
Renal artery rings were suspended to fine tungsten stir-ups (diameter, 50
µm), placed in an organ bath filled with 25 ml Krebs solution, and
connected to force transducers (UTC 2, Gould Statham, CA) for isometric
tension recording as described
(32). After an equilibration
period of 60 min, renal artery rings were progressively stretched to their
optimal passive tension (2.0 ± 0.2 g) as assessed by the response to
100 mmol/L KCl in modified Krebs solution
(33). Rings were precontracted
with norepinephrine (approximately 70% of 100 mmol/L KCl) and relaxations to
acetylcholine (ACH; 10-10 to 10-5 mol/L) or sodium
nitroprusside (SNP; 10-11 to 10-5 mol/L) were obtained.
Relaxations to ACH were assessed with and without preincubation of
indomethacin (30 min, 10-7 mol/L) and in the presence or absence of
the nitric oxide synthase (NOS) inhibitor NG-nitro-L-arginine methyl ester
(L-NAME) (preincubation for 30 min, 3 x 10-4 mol/L). In
additional experiments, cumulative concentration-response curves to ET-1
(10-10 to 10-7 mol/L) and big ET-1 (10-9 to
10-7 mol/L) were obtained in quiescent preparations. To avoid the
development of tachyphylaxis, only single concentrations of angiotensin I and
angiotensin II were used (10-7 mol/L). All drugs used in the organ
bath were obtained from Sigma Chemical Co (Buchs, Switzerland) apart from ET-1
and big ET-1, which were purchased from Novabiochem/Calbiochem AG (La Jolla,
CA).
Vascular and Cardiac Hypertrophy
For assessment of vascular hypertrophy, aortic rings were blotted dry and
weighed, and the arterial surface area of opened rings was measured as
described (33). Aortic surface
area was calculated using formulas for diameter and radius of a cylinder for
each individual ring, and values were averaged. After exsanguination of the
animals, hearts were removed, isolated, and snap-frozen in liquid nitrogen.
Wet weight of hearts was measured, standardized for body weight, and reported
as mg heart weight/kg body weight.
Morphologic Analysis of Glomerular Injury
Renal injury was assessed as described previously
(34). Briefly,
paraffin-embedded sections of whole kidneys (5 to 7 µm) stained with
periodic acid-Schiff reagent were viewed by light microscopy at a
magnification of x40 using a Zeiss microscope (Carl Zeiss GmbH, Jena,
Germany). One hundred glomeruli per slide were evaluated. Morphologic
evaluation of glomerular injury was performed using semi-quantitative scoring
methods. Lesions were graded by glomerulosclerosis (grade 1 to 4: 1 to 25%, 26
to 50%, 51 to 76%, and 76 to 100% sclerosis, respectively). The glomerular
injury score was calculated by summarizing the products of severity grade
times the percentage of glomeruli displaying the same degree of severity.
Calculations and Statistical Analyses
Relaxations to agonists in isolated arteries are reported as percent
precontraction in rings precontracted with norepinephrine to about 70% of
contraction induced by KCl (100 mmol/L). The contractions were expressed as a
percentage of 100 mmol/L KCl—induced contractions, which were obtained
at the beginning of each experiment. Results are presented as mean ±
SEM. Functional endothelin-converting-enzyme (ECE) activity was calculated as
the ratio of the contraction to big ET-1 (10-7 mol/L) divided by
the contraction to ET-1 (10-7 mol/L). In all experiments,
n equals the number of rats per experiment. For statistical analyses,
the sensitivity of the vessels to the drugs was expressed as the negative
logarithm of the concentration that caused half-maximal relaxation or
contraction (pD2). Maximal relaxation (expressed as a percentage of
precontraction) or contraction was determined for each individual
concentration-response curve by nonlinear regression analysis with MatLab
software (Math Works Inc., Natick, MA). For comparison between two values, the
unpaired t test or the nonparametric Mann-Whitney test was used when
appropriate. For multiple comparisons, results were analyzed by ANOVA followed
by Bonferroni's correction
(35). Pearson correlation
coefficients were calculated by linear regression. Significance was defined as
P < 0.05.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Vascular Relaxations
In hypertensive animals, maximal endothelium-dependent relaxations to ACH
and sensitivity (pD2 value) in renal arteries were markedly
impaired when compared with control rats
(Figure 1A; P <
0.05). Both O and C improved endothelium-dependent relaxations, but the
maximal relaxation achieved by O was significantly higher than by C
(Figure 1A; P <
0.05 versus C) and was comparable to the control animals.
Preincubation with the NOS inhibitor L-NAME blunted relaxations to ACH in all
groups completely (Figure
2).
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In contrast to endothelium-dependent relaxations, maximal endothelium-independent relaxations to the NO donor SNP (Figure 1B) in renal arteries were comparable in all groups. Preincubation with indomethacin (10-7 mol/L) did not alter maximal relaxations or sensitivity (pD2 value) to either ACH or SNP.
Vascular Contractions
Contractions of renal arteries to ET-1 were reduced in Dahl rats on a
high-salt diet (Figure 3A;
P < 0.05) and were normalized by long-term administration of O or
C, respectively (P < 0.05 versus placebo-treated,
salt-fed Dahl rats for maximal response;
Figure 3A). In addition, renal
artery contractions to big ET-1 were markedly reduced in salt-sensitive
hypertension (Figure 3B;
P < 0.05). Treatment with O but not with C (P < 0.05
for maximal contractions versus O) normalized contractions to big
ET-1 (Figure 3B). Therefore,
functional ECE activity, expressed as the ratio of the contraction to
10-7 mol/L big ET-1 divided by the contraction to 10-7
mol/L ET-1, was significantly lowered in salt-sensitive hypertension
(Figure 4; P < 0.05
versus controls). ECE activity was normalized by O
(Figure 3; P < 0.05
versus placebo treatment) but was not significantly affected by C. In
addition, ECE activity was blunted by incubation with O (10-7
mol/L) in vitro (data not shown).
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The effectiveness of ACE inhibition, as assessed by determination of functional ACE activity, did not differ between O and C (0.28 ± 0.04 versus 0.33 ± 0.06, respectively; NS). Also, ACE activity, as determined by the ratio of the contraction to angiotensin I (10-7 mol/L) divided by the contraction to angiotensin II (10-7 mol/L), was significantly reduced by either C or O compared with the control group (0.74 ± 0.08; P < 0.01).
Vascular and Cardiac Hypertrophy
After 8 wk of salt feeding, standardized heart weight of salt-sensitive
rats was significantly elevated (4.9 ± 0.4 versus 3.2 ±
0.3 g/kg in control rats; P < 0.05;
Figure 5A), indicating cardiac
hypertrophy. Increase in heart weight was prevented by O (3.7 ± 0.4
g/kg; P < 0.05 versus high-salt diet;
Figure 5A) but not by C (4.3
± 0.3 g/kg, NS). In parallel, aortic weight increased in salt-induced
hypertension (0.31 ± 0.018 versus 0.22 ± 0.02
mg/mm2 in control rats; P < 0.05,
Figure 5B). Vascular
hypertrophy was prevented by O (0.23 ± 0.019 mg/mm2;
P < 0.05 versus high-salt diet;
Figure 5B) but not by C (0.28
± 0.02 mg/mm2, NS). The difference in aortic hypertrophy
between C-treated and O-treated animals reached statistical significance
(P < 0.05).
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Morphologic Analysis of Glomerular Injury
High-salt diet induced marked glomerulosclerosis in salt-sensitive Dahl
rats (glomerulosclerosis index, 22.0 ± 4.6 versus 9.4 ±
3.2 in control rats on standard chow). Both O and C reduced glomerular
alterations (glomerulosclerosis index, 16.6 ± 2.6 and 9.6 ± 2.0,
respectively), but only the reduction of glomerular damage by C reached
statistical significance (P < 0.05 versus salt diet).
Both compounds tended to lower the percentage of sclerotic glomeruli
(Figure 6), but a significant
reduction of affected glomeruli was only achieved by C in grade 1 and grade 4
sclerotic glomeruli (Figure 6; P < 0.05 versus salt diet).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Salt-sensitive hypertension is associated with impaired endothelial function in the aorta (33, 36). Here we extend this observation to renal arteries in which we documented impaired endothelium-dependent relaxations to ACH as well as reduced contractile responses to ET-1 and big ET-1. Long-term treatment with the ACE inhibitor C improved vascular responsiveness but did not restore endothelial function to a degree comparable to O—even though achieved BP and inhibition of functional and biochemical ACE activity in renal arteries was comparable in the two treatment groups (21). In vitro inhibitory constants of O against ACE and NEP are in the nanomolar range (11); therefore, sufficient inhibition of both enzymes can be assumed. Thus, with equipotent ACE inhibition, the different effects of the two drugs on renovascular endothelial function must be related to properties of O other than ACE inhibition.
Vasopeptidase inhibitors simultaneously block ACE and NEP (10), therefore, the metabolism of several vasoactive peptides, such as angiotensin, natriuretic peptides, bradykinin, and ET-1, and their clearance is altered. In accordance with recent findings (33), plasma ET levels were elevated in place-bo-treated animals with salt-induced hypertension (37), although functional ECE activity was decreased. Thus, clearance of ET must be reduced in this model. As pure NEP inhibitors cause vasoconstriction due to decreased breakdown of ET-1 (38), selective blockade of this enzyme may not be appropriate under these conditions. In contrast, the combined ACE and NEP inhibitor O normalized both plasma ET-1 levels (37) as well as functional ECE activity. It has to be remembered, however, that the predictive value of ET-1 plasma levels on local, and in particular renal, ET tissue levels is rather limited (39). However, these findings certainly reflect the complex influence of vasopeptidase inhibition on the endothelin system including inhibition of ET-1 degradation (38) as well as inhibition of ET-1 generation from big ET-1 (40). In any case, lowering of plasma ET-1 (37) and elevation of ECE activity demonstrate normalization of this altered paracrine system by O in this model of hypertension, and this may be one constituent that contributes to renovascular protection of vasopeptidase inhibitors.
In this study, we only investigated large renal arteries. Whether or not these alterations also occur in renal arterioles is uncertain, particularly because the endothelin system exhibits tissue specificity. Distribution of endothelin isoforms and their receptors differs in the renal cortex and medulla (39). Also, alterations in vascular structure and function in salt-induced hypertension are heterogenous, depending on the size and vascular bed involved (41, 42). As in large renal arteries, however, endothelium-dependent relaxations of renal resistance vessels are impaired in salt-sensitive Dahl rats (43). Correspondingly, improvement of vasorelaxation in large renal arteries may contribute to normalization of renal plasma flow and therefore may be beneficial for both renal function and lowering of BP.
As in the aorta of salt-sensitive Dahl rats (33), ACH-induced relaxations in renal arteries were blunted in the presence of the NOS inhibitor L-NAME and therefore are mediated by NO. In renal arteries of hypertensive salt-sensitive Dahl rats, endothelium-dependent relaxations to ACH, but not the response to SNP, were impaired; therefore, reduced bioavailability of NO must be involved, which is in accordance with recent findings of reduced endothelial NOS protein expression in this model (44). Therefore, restoration of NO bioavailability may contribute to the normalization of endothelium-dependent relaxations in both treatment groups, but it cannot account for the greater endothelial protection by O as compared with C. Hence, other properties of O, such as the reduced breakdown of natriuretic peptides, are most likely involved.
Besides functional changes, structural vascular and parenchymal alterations, such as cardiac and vascular hypertrophy (33), occur in salt-induced hypertension. O prevented vascular hypertrophy to a greater extent than C did. This is in line with the effect of O in resistance arteries of stroke-prone spontaneously hypertensive rats (30) and salt-sensitive Dahl rats (29).
Surprisingly, assessment of glomerular morphology revealed only moderate improvement of glomerulosclerosis by O but a significant reduction of the glomerulosclerosis index by C. The lack of renal tissue protection by O contrasts with the marked improvement of vascular remodelling. Although the morphologic assessment may be prone to greater variation than that of larger vessels, this finding requires further investigation of the mechanisms by which vasopeptidase inhibition influences glomerular scarring. Differential, tissue-specific effects of O reflect its complex interaction with local regulatory systems. Possibly the beneficial effect of O on ET plasma levels is not paralleled by similar changes of intrarenal ET levels; if so, elevated renal ET-1 may promote glomerular damage (34). Furthermore, characteristics of the model used in this study have to be taken into account because O has very recently been demonstrated to effectively reduce glomerulosclerosis in Goldblatt hypertension (45).
In conclusion, this study demonstrates marked improvement of renovascular endothelial function by long-term treatment of salt-sensitive hypertension with equipotent dosages of O or C. In contrast to C, O completely restored renovascular NO-mediated relaxation and ECE activity and prevented vascular hypertrophy. Therefore, vasopeptidase inhibition may represent an interesting, new approach in the treatment of hypertension and renovascular disease. In addition to recently published data on vasopeptidase inhibition in hypertension (14, 22,23,24, 46) and heart failure (8, 26,27,28), a number of large clinical studies—in part already under way (25, 47)—will be necessary to further evaluate the future clinical role of vasopeptidase inhibitors in the treatment of cardiovascular and renovascular disease and to better understand their beneficial effects.
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Acknowledgments |
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