© 1996 American Heart Association, Inc.
Articles |
Suppression of Atherosclerotic Changes in Cholesterol-Fed Rabbits Treated With an Oral Inhibitor of Neutral Endopeptidase 24.11 (EC 3.4.24.11)
the Division of Cardiology, Kumamoto University School of Medicine, Kumamoto City, Japan.
Correspondence to Kiyotaka Kugiyama, MD, Division of Cardiology, Kumamoto University School of Medicine, Honjo 1-1-1, Kumamoto City, Japan 860.
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Abstract |
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Neutral endopeptidase 24.11 (NEP), widely distributed in the body, hydrolyzes and inactivates a number of endogenous vasoactive peptides, some of which could alter various functions of cells present in the arterial wall. Recently NEP has been found to exist in the vascular endothelium. The aim of this study was to assess the influence of chronic NEP inhibition by daily administration of UK79300 (candoxatril), an orally active NEP inhibitor (NEPI), on the development of atherosclerotic changes in high-cholesterol-fed rabbits. Male New Zealand White rabbits were fed for 8 weeks as follows: normal rabbit diet (Normal, n=15), 1.5% cholesterol diet (Cholesterol, n=15), or 1.5% cholesterol diet containing NEPI (20 mg·kg-1·d-1) (Cholesterol+NEPI, n=15). At the end of the dietary period, NEPI treatment was found to suppress the surface area of the aorta covered by plaques (% surface area: Cholesterol, 59±6 versus Cholesterol+NEPI, 36±7, P<.01) and decreased contents of cholesterol and cholesterol esters in the aortas. NEPI also reduced plasma total cholesterol by 27% of Cholesterol rabbits (1781±130 mg/dL). The endothelial function, estimated by the endothelium-dependent relaxation of the isolated aortas in response to acetylcholine, was preserved in Cholesterol+NEPI rabbits compared with that in Cholesterol rabbits. NEP enzymatic activities in plasma and the particulate fraction of the homogenates from the aortas in Cholesterol rabbits were both increased, 3.1- and 3.9-fold, respectively, above those in Normal rabbits, but the activities in Cholesterol+NEPI rabbits were significantly lower than those in Cholesterol rabbits. UK73967, an active form of UK79300, or phosphoramidon partly reversed the atherosclerotic impairment of relaxation of the isolated thoracic aortic rings from Cholesterol rabbits in response to exogenous additions of C-type natriuretic peptide (CNP) and substance P, which are NEP substrates known to exist endogenously in the vascular endothelium. The results suggest that the increased NEP activity plays a significant role in atherogenesis, and NEPIs might be therapeutically useful in the prevention of atherosclerosis. Reduction of plasma cholesterol and suppression of degradations in the arteries of endogenously released CNP, substance P, or possibly other kinins known to have anti-atherosclerotic actions may at least partially contribute to the inhibitory effects of NEPIs on atherosclerotic changes.
Key Words: neutral endopeptidase 24.11 • atherosclerosis • endothelium-dependent relaxation • endothelium • C-type natriuretic peptide
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Introduction |
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Neutral endopeptidase 24.11, a membrane ectoenzyme, cleaves a variety of peptides at the amino side of hydrophobic amino acids.1 2 3 4 5 NEP is widely distributed in the body and is especially abundant in the kidney, lung, and intestine.1 2 3 4 Recently, the enzyme was also found to exist in vascular endothelial cells.6 7 NEP could hydrolyze and inactivate a number of endogenous vasoactive peptides circulating in the peripheral blood or produced at vascular walls.8 9 10 11 Therefore, endothelial NEP could metabolize vasoactive peptides and reduce their local concentrations at the arterial walls, resulting in attenuation of the effects of the peptides on vascular functions. In this context, NEP inhibition might potentiate the vascular responsiveness to these endogenous vasoactive peptides. Substance P and other kinins existing in vascular endothelium12 13 are the substrates of NEP, and they can stimulate the endothelial release of vasodilators such as prostaglandins, endothelium-derived relaxing factor(s), and nitric oxide, which are shown to be capable of inhibiting growth of vascular smooth muscle cells and migration of monocytes.14 15 16 17 18 CNP, the third member of the natriuretic peptide family, is known to be abundant in the brain.19 Recently it has been shown to be produced in vascular endothelial cells.20 21 CNP relaxes vascular smooth muscle, suppresses proliferation of smooth muscle cells, and inhibits intimal thickening of rat carotid arteries in vivo.22 23 24 Thus, CNP acts as a paracrine factor and regulates vascular tone and growth. Recently, CNP has also been shown to be hydrolyzed by NEP.25 Therefore, one might expect that an NEPI could potentiate the antiproliferative effects of CNP and substance P or other kinins by inhibition of their degradation at the site of arterial endothelium. This study was aimed to assess the influence of chronic NEP inhibition by daily administration of UK79300 (candoxatril), an orally active NEPI,11 26 27 on the development of atherosclerotic changes in high-cholesterol-fed rabbits.
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Methods |
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Animal Experiments
Fifty-three male New Zealand White rabbits weighing between 2.5 and 2.9 kg were housed individually in wire-bottomed cages in an air-conditioned room at 20°C and 50% humidity with 12-hour light/12-hour dark cycles. After an adaptation period of 1 week, the animals were separated into three equal groups, which were randomly assigned to one of three dietary and therapeutic regimens: (1) standard chow (n=17, Normal rabbits), (2) chow containing 1.5% cholesterol (n=18, Cholesterol rabbits), and (3) chow containing 1.5% cholesterol and UK79300 (candoxatril, an orally active NEPI; Pfizer Central Research) (n=18, Cholesterol+NEPI rabbits). UK79300 was added to the pelleted diet at an oral dose of 20 mg·kg body weight-1·d-1. This dose was selected because its oral administration consistently lowered plasma NEP activity by 75% of that in normal rabbits. Standard and cholesterol chows were purchased from Cler Japan Inc. The amount of daily diet for each animal was restricted to 120 g during the study period. Water was provided ad libitum. At the 7-week diet period, arterial blood pressure and heart rate in the conscious rabbits were measured by the central ear artery technique. The measurements were performed in the early morning at 2 hours after the rabbits were provided access to the diet. At the end of the 8-weeks diet period, blood samples from the central ear artery after a 24-hour fast were collected into tubes containing EDTA-Na2 (1 mg/mL blood) for the lipids assay and into heparinized tubes for the assay of NEP activity. Then the rabbits were killed under pentobarbital anesthesia (30 mg/kg IV). The entire aorta from the aortic valve to the origin of the renal arteries was excised and placed immediately in ice-cold Krebs-Henseleit buffer. The isolated aortas were cleaned of perivascular tissue. The aorta from origin of the aortic valve to the first intercostal arteries was used for the morphometric assessment of atheromatous plaque area. The aorta from the origin of the first intercostal arteries to the origin of the third intercostal arteries was used for the assays of the tissue lipids and NEP activity and for the immunohistological examination. Four 3-mm-wide rings were cut from the aorta just distal to the origin of the third intercostal arteries immediately after the isolation and were used for the organ-chamber experiments.
Atheromatous Plaque Area
The aortas from the aortic valve to the origin of the first intercostal arteries were opened longitudinally to expose the intimal surface and were fixed in 10% buffered formalin overnight. The preparations were then stained by Sudan IV to reveal sudanophilic plaques and subsequently photographed. The photographs were then copied onto graph paper with magnification (x2), and the outlines of the aorta and the sudan-positive area were delineated on the copied paper. The delineated areas of the aorta and the sudan-positive area were scanned and estimated by the computerized planimetry. The atheromatous areas within each aorta were summed and the extent of atheromatous plaques was expressed as a percentage of surface area with the aorta.
Organ-Chamber Experiments
The rings from each thoracic aorta were suspended by stainless steel hooks in organ chambers filled with Krebs' buffer (composition [mmol/L]: Na+ 144.2, K+ 4.0, Ca2+ 1.5, Mg2+ 1.2, Cl- 123.0, SO42- 1.2, H2PO4- 1.2, HCO3- 25.0, and glucose 5.0). The solution was aerated with 15% O2-5% CO2-80% N2 (PO2=100 mm Hg) and maintained at 37°C. During this procedure, care was taken not to injure the luminal surface. The rings were then stretched to an optimum basal tension of 3 g, and the isometric tension was monitored by means of a force transducer (Minebea) and a polygraph machine (Nihon Kohden). After an equilibration period of 90 minutes, the rings were contracted with 0.3 µmol/L phenylephrine and subsequently relaxed with cumulative additions of ACh, ANP, or SNP. To examine the role of NEP in arterial relaxation, some rings obtained from Normal and Cholesterol rabbits were pretreated for 10 minutes with UK73967 (50 µmol/L) (an active form of UK79300, Pfizer Central Research) or phosphoramidon (50 µmol/L), NEPIs, and then precontracted with 0.3 µmol/L phenylephrine. The rings were then relaxed with cumulative additions of CNP, which is an NEP substrate, or SNP. The vasorelaxation response to substance P, another NEP substrate, was also examined after the treatment with UK73967 (50 µmol/L) in the same manner as examination with CNP, but captopril (1 µmol/L), an inhibitor of angiotensin-converting enzyme, was also added in all preparations tested at 10 minutes before the contraction with phenylephrine to prevent degradation of substance P by angiotensin-converting enzyme. Vasorelaxation was expressed as a percent reduction of the phenylephrine-induced contraction. All of the rings used for the muscle-chamber experiments were cut open after the experiment and then fixed, stained by Sudan IV, and the percentage of the plaque areas in sum of the aortic strips was calculated in the same manner as used for the aortas from the origin of the aortic valve to the first intercostal arteries, as described above.
Lipids Analysis of EDTA-Plasma and Aortic Samples
A part of the frozen aortas from the first intercostal arteries to the third intercostal arteries was blotted and weighed, immediately frozen in liquid nitrogen, and stored at -80°C. The frozen aortas were pulverized at liquid nitrogen temperature and homogenized in 10 vol of chloroform/methanol (2:1, vol/vol) containing 0.001% BHT as an antioxidant. Lipids in the homogenate were extracted by the method described by Chan et al.28 The lipid-containing fraction was dried under nitrogen and then resuspended in isopropyl alcohol.
Total cholesterol, triglycerides, and free cholesterol in the aortic tissues and in plasma were measured using the specific enzymatic kits (Cholesterol C-test Wako, Triglycerides G-test Wako, and Free cholesterol E-test Wako, Wako Pure Chemical Ltd). HDL cholesterol in plasma was measured after precipitation of apoB-containing lipoproteins by heparin and manganese (HDL cholesterol-test Wako, Wako Pure Chemical Ltd). Esterified cholesterol was calculated as the difference between total and free cholesterol. Protein concentrations in the aortic tissues were estimated in whole homogenates by the method of Lowry et al,29 using BSA as the standard.
NEP Assays
NEP activities in plasma and particulate fractions of the homogenates from the aortic samples were measured by the two-step spectrofluorometric assay using the synthetic peptide Glu-Ala-Ala-Phe-4MeO-ß-naphthylamide.30 The reaction was performed in the presence or absence of UK73967 (50 µmol/L), a specific NEPI, and only the activity inhibited by UK73967 was attributed to the NEP activity. The particulate fractions from the aortas were prepared as follows. A part of the aortas at the level between the first and third intercostal arteries was cut and opened to measure the endothelial luminal surface area (in square millimeters) and then homogenized into ice-cold 50 mmol/L Tris-HCl (pH 7.4), using a glass homogenizer, and centrifuged at 4°C for 10 minutes at 600g. The resulting supernatant was centrifuged at 4°C for 60 minutes at 100 000g. The pellet was superficially washed three times with the cold Tris buffer and resuspended in the Tris buffer supplemented with 0.1% Triton X-100. After gentle shaking for 2 hours at 4°C, the suspension was centrifuged at 15 000g for 10 minutes. The supernatant, containing the solubilized enzyme, was used for the assay of NEP activity. All procedures were carried out at 4°C. NEP activities in the aortic tissues were expressed as the degrading activity of the synthetic peptide in 1 mm2 of the endothelial luminal surface area of the aortic samples.
NEP Immunoreactivity
A part of the aortas at the level between the first and third intercostal arteries was isolated and immediately fixed with the Zamboni fixative at 4°C for 6 hours, then rinsed and embedded in OCT compound (Miles Inc), quickly frozen, and stored at -80°C. Frozen tissue specimens were cut into 8-µm-thick sections. The sections were stained by using the immunoperoxidase method as previously reported.31 Briefly, the sections were incubated with nonimmune horse and mouse sera at room temperature for 1 hour and then incubated with or without anti-NEP/CD10 monoclonal antibody (J5, Coulter Clone, 20 µg/mL) or anti–von Willebrand factor polyclonal antibody (DAKO Japan) at 4°C overnight. After washing, the sections were incubated with biotinylated anti-mouse and anti-rabbit IgG (host animal, horse) for NEP and von Willebrand factor, respectively, at 4°C overnight. Thereafter, they were incubated with Vectastain avidin-biotin complex (Vectastain ABC kit, Vector Laboratories) reagent at room temperature for 60 minutes. The final reaction was achieved by incubating with freshly prepared 3,3'-diaminobenzidine tetrahydrochloride solution with 0.01% hydrogen peroxide. The nuclei were counterstained with hematoxylin. The presence or absence of immunoreactive products for anti-NEP/CD10 monoclonal antibody was judged by three independent observers.
Materials
-Human ANP and CNP-22 were purchased from Peptide Institute Inc. UK79300 and UK73967 were supplied by Pfizer Central Research. Captopril was a gift from Sankyo Inc. Other chemicals were from Sigma Chemical Co.
Statistical Analysis
All values were expressed as mean±SEM. Statistical evaluation of the data was performed by Student's t test for unpaired observations. When more than two groups were compared, ANOVA was used. A value of P<.05 was considered significant.
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Responses of Body Weight and Hemodynamics to Diet
Total cholesterol levels at the 4-weeks diet period were <200 mg/dL in four rabbits with high-cholesterol diet (two in Cholesterol rabbits and two in Cholesterol+NEPI rabbits) that were considered to be relatively hyporesponsive to high cholesterol intake and were excluded from the study. The total plasma cholesterol in the remaining 49 rabbits was >500 mg/dL (range, 542 to 2174 mg/dL) at the 4-weeks diet period. There were two deaths in the Normal rabbits and one death each in Cholesterol rabbits and Cholesterol+NEPI rabbits during the dietary period. Although autopsy was performed in these rabbits, the cause of death could not be determined. The remaining 45 rabbits (15 Normal rabbits, 15 Cholesterol rabbits, and 15 Cholesterol+NEPI rabbits) were finally examined for the study. At the 7-weeks diet period, arterial blood pressure and heart rate in the conscious rabbits were measured by the central ear artery technique. As shown in Table 1
, body weight, systolic blood pressure, and heart rate at the 7-weeks diet period did not differ among the three groups of rabbits.
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Aortic Plaque Area, NEP Activity, and NEP Immunoreactivity
The area of sudanophilic atheromatous plaque was significantly smaller in Cholesterol+NEPI rabbits than that in Cholesterol rabbits, as shown in Fig 1 (P<.01). As shown in Fig 2, plasma NEP activity was significantly increased in Cholesterol rabbits (P<.01) but remained unchanged in Cholesterol+NEPI rabbits compared with that in Normal rabbits. There was significant difference in the plasma NEP activity between Cholesterol and Cholesterol+NEPI rabbits (P<.01). Tissue NEP activity in aortas was significantly increased in both Cholesterol and Cholesterol+NEPI rabbits compared with that in Normal rabbits (P<.01 and P<.05, respectively), as shown in Fig 3. However, the activity in Cholesterol+NEPI rabbits was significantly lower than that in Cholesterol rabbits (P<.05). The immunohistochemical staining showed that NEP immunoreactivity was confined mainly to the endothelium and was upregulated in the endothelium in some atherosclerotic aortas, as demonstrated in Fig 4. NEPI treatment did not affect the immunoreactive expression of NEP in the atherosclerotic arteries.
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Lipids in Plasma and Aortic Tissues
Plasma cholesterol concentration at the 8-weeks dietary period (on the day rabbits were killed) was significantly increased in both Cholesterol and Cholesterol+NEPI rabbits (P<.01 in both versus that in Normal rabbits), but the concentration in Cholesterol+NEPI rabbits was lower than that in Cholesterol rabbits (P<.05), as shown in Fig 5. Plasma triglyceride level at the 8-weeks dietary period was significantly increased in Cholesterol rabbits (P<.01 versus that in Normal rabbits). The triglyceride level in Cholesterol+NEPI rabbits tended to be higher than that in Normal rabbits, but there was no significant difference in the level between them. There was significant difference in the triglyceride level between Cholesterol and Cholesterol+NEPI rabbits (P<.05). Plasma HDL cholesterol tended to be higher in Cholesterol+NEPI rabbits than that in Normal and Cholesterol rabbits, but it did not significantly differ among the three groups of rabbits. As shown in Fig 6, tissue concentrations (per milligram tissue protein) of cholesterol and cholesterol esters in the aortas were significantly increased in Cholesterol rabbits (P<.01 in both), but they remained unchanged in Cholesterol+NEPI rabbits compared with those in Normal rabbits. There were significant differences in the concentrations of cholesterol and cholesterol esters between Cholesterol and Cholesterol+NEPI rabbits (P<.05). Tissue contents (milligrams per gram wet tissue) of cholesterol and cholesterol esters in the aortas were also significantly increased in Cholesterol and Cholesterol+NEPI rabbits, but the contents in Cholesterol+NEPI rabbits were significantly lower than those in Cholesterol rabbits (cholesterol and cholesterol ester contents: Normal rabbits, 0.7±0.2 and 0.2±0.1 mg/g wet tissue, respectively; Cholesterol rabbits, 14.5±3.1 and 7.4±1.2 [P<.05 versus the respective values in Normal rabbits]; Cholesterol+NEPI rabbits, 3.6±1.3 and 1.2±0.4 [P<.05 versus the respective values in Normal rabbits and Cholesterol rabbits]; n=7 in each group).
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Organ-Chamber Experiments
As shown in Table 2 and Fig 7, relaxation of the isolated aortas in response to ACh was significantly impaired in Cholesterol and Cholesterol+NEPI rabbits, but the impairment was significantly less in Cholesterol+NEPI rabbits than in Cholesterol rabbits. Vasorelaxation response to ANP, which is an NEP substrate, was also impaired in Cholesterol rabbits, but it was preserved in Cholesterol+NEPI rabbits compared with that in Normal rabbits. Vasorelaxation response to SNP was fully preserved in all three groups. The magnitude of the contraction with phenylephrine was not significantly different among the three groups of rabbits (data not shown). The plaque areas in sum of the aortic rings in Cholesterol+NEPI rabbits were significantly smaller than those in Cholesterol rabbits (percent of atheromatous plaque areas: 20±5% versus 36±7%, n=15 in each, P<.05). As shown in Table 3 and Fig 8, the relaxations to CNP and Substance P in the atherosclerotic aortas from Cholesterol rabbits were impaired compared with those in normal aortas from Normal rabbits. However, the incubation with UK73967 of the atherosclerotic aortas from Cholesterol rabbits attenuated the impairment of the vasorelaxation response to CNP and Substance P. The incubation with phosphoramidon also improved the vasorelaxation response to CNP (EC20 [nmol/L], Normal rabbits: control, 10±2; phosphoramidon, 10±3. Cholesterol rabbits: control, 142±11 [P<.05 versus the control value in Normal rabbits]; phosphoramidon, 48±6 [P<.05 versus the control value in Normal rabbits and Cholesterol rabbits]; n=7 to 9). The addition of UK73967 or phosphoramidon did not affect the contraction with phenylephrine (data not shown) or the vasorelaxation response to SNP (Table 3).
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, Cholesterol (rabbits fed high-cholesterol diet); 
, Cholesterol+NEPI (rabbits fed high-cholesterol diet containing UK79300); 
, Normal (rabbits fed standard diet). *P<.05 vs the respective values in Normal and Cholesterol+NEPI; n=10 to 15 in each group of rabbits.
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Discussion |
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The present study demonstrated that NEP inhibition suppresses atheromatous formation and preserves endothelial function in high-cholesterol-fed rabbits, suggesting that NEP may play a significant role in atherogenesis. NEP is widely distributed in the body and has recently been shown to exist in the vascular endothelium,1 2 3 4 6 7 as also demonstrated in the present study. NEP has been reported to hydrolyze and inactivate a number of endogenous vasoactive peptides, such as bradykinin, substance P, angiotensin I and II, natriuretic peptides, and endothelin.1 2 3 4 5 Thus, NEP could affect arterial responses to these endogenous vasoactive peptides circulating in the blood or produced in the peripheral arterial walls by regulating their local concentrations in the arterial walls.8 9 10 11 Systemic administration of NEPI could increase the endogenous vasoactive peptides, which are cleaved by NEP, in the circulation or in the peripheral tissues.8 10 32 33 34 In fact, recent reports showed that oral intake of UK79300 induced elevation of ANP in serum in patients with heart failure.35 Substance P and other kinins existing in vascular endothelium stimulate endothelial release of nitric oxide and prostacyclin, which are shown to inhibit smooth muscle proliferation and monocyte migration.13 14 15 16 17 18 CNP, the third member of the natriuretic peptide family, is produced mainly in the brain but has been recently found to be also synthesized in the peripheral vascular endothelial cells.19 20 21 CNP has been shown to relax arterial smooth muscle and suppress smooth muscle cell proliferation in vitro and to inhibit intimal thickening of carotid artery after intimal denudation in vivo.22 23 24 Thus, substance P or other kinins and CNP, which exist in the vascular endothelium and could be hydrolyzed by endothelial NEP, exert anti-atherosclerotic effects on the arterial walls. However, the breakdown of these peptides by NEP is suggested to be enhanced in atherosclerotic arteries on the basis of the present findings that NEP enzymatic activities are increased in atherosclerotic arterial walls and plasma with hypercholesterolemia compared with those in nonatherosclerotic arteries and plasma with normocholesterolemia. In addition, long-term treatment with NEPI suppresses the increase of NEP activities in high-cholesterol-fed rabbits, and furthermore, NEP inhibition reverses the atherosclerotic impairment of the arterial response to exogenous additions of substance P and CNP. These results suggest that the in vivo arterial concentrations of substance P or other kinins and CNP endogenously existing in the endothelium could presumably be preserved in cholesterol-fed rabbits treated with NEPI by suppression of degradations in the arterial walls of these peptides, which have anti-atherosclerotic effects. This possible scenario might be one of the mechanisms explaining the inhibition of atheromatous formation by the administration of NEPI. The additional experiment with chronic infusion of CNP in combination with NEPI or HS-142-1, a blocker of natriuretic peptide receptor guanylyl cyclase,36 could clarify the role of natriuretic peptides in atherogenesis. It seems to be difficult to demonstrate the in vivo increase of CNP and substance P or other kinins in the circulation and in aortic tissues in rabbits fed cholesterol with NEPI because their expressions are very low and they act in an autocrine or paracrine manner.12 37 38 39 Furthermore, the anti-atherogenic effects of UK79300 were unlikely to be mediated by ANP, which has been reported to increase in human plasma a couple of hours after single administration,26 27 since plasma ANP level was not increased in the present study after chronic treatment with UK79300 (data not shown). Investigations are now under way to determine the cellular composition of and magnitude of accumulation of smooth muscle cell–derived foam cells in atherosclerotic aortic tissues from rabbits with or without NEPI treatment to elucidate the possible inhibitory effect of CNP, substance P, and other kinins on smooth muscle cell proliferation in atherosclerotic arteries. Suga et al40 reported that natriuretic peptide receptor–B, a biologically active receptor of CNP, is upregulated in the synthetic type of cultured smooth muscle cells, which is known to be the predominant type of smooth muscle cells present in the intimal layer of atherosclerotic arteries, a finding that could also support the regulatory role of natriuretic peptides in atherogenesis.
The present study showed that oral administration of UK79300 decreased the plasma concentration of cholesterol, which may partly contribute to the mechanism(s) of the inhibitory effect of UK79300 on atheromatous formation. However, to our knowledge, there is no report showing relation of NEP and regulation of cholesterol metabolism. There may be unknown effects of NEP and its substrates on the regulation of cholesterol absorption and metabolism, probably at the intestine and liver. It also remains unclear whether the reducing effect of NEPI on total plasma cholesterol levels in hypercholesterolemic rabbits fed a high-cholesterol diet, observed in the present study, may be reproducible in humans with hypercholesterolemia and in animal models such as Watanabe heritable hyperlipidemic (WHHL) rabbits, whose plasma cholesterol level is much lower than that in high-cholesterol-diet rabbits in the present study (
800 mg/dL in humans and in WHHL rabbits versus 2000 mg/dL in high-cholesterol-diet rabbits). When plaque areas were compared in rabbits matched for total plasma cholesterol levels (range, 1000 to 2000 mg/dL), the NEPI-treated rabbits (n=9, mean levels of plasma cholesterol=1513±24 mg/dL) still had significantly smaller values for plaque areas than the rabbits receiving cholesterol alone (n=10, mean levels of plasma cholesterol=1503±28 mg/dL) (percent surface plaque area: Cholesterol+NEPI rabbits, 32±4% versus Cholesterol rabbits, 55±5%; P<.05). Hence, the effect of NEP inhibition on plaque areas may be caused not only by its reducing action on total plasma cholesterol, but other mechanism(s) might also be involved. UK79300 and its active form UK73967 and their substrate peptides had no effect on LDL oxidation by endothelial cells or macrophages or on acyl coenzyme A/cholesterol acyltransferase activity in rat macrophages (data not presented). The suppression of atherosclerotic changes by NEP inhibition was not due to its hypotensive effect, as previously reported in DOCA-salt rats,41 42 because blood pressure in the present rabbits was not persistently affected by the treatment with UK79300.
In conclusion, the increased NEP activities in the arterial tissues and in plasma play a significant role in atherogenesis, and NEPIs might be therapeutically useful in the prevention of atherosclerosis. Reduction of plasma cholesterol and suppression of degradations in the arteries of endogenous CNP, substance P, or possibly other kinins, which are known to have anti-atherosclerotic actions, may at least partially contribute to the inhibitory effects of NEPI on atherosclerotic changes. However, the precise mechanism for the inhibition of atheromatous formation by NEPI remains to be determined.
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Acknowledgments |
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This study was supported in part by grant-in-aid for Scientific Research C07670793, from the Ministry of Education, Science and Culture in Japan, and the Smoking Research Foundation grant for Biomedical Research, Tokyo, Japan.
Received May 30, 1995;
revision received February 23, 1996;
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