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CARDIOVASCULAR
Department of Pharmacology, Faculty of Pharmacy, Comenius University, Bratislava, Slovak Republic (J.K., P.M., Z.B., A.G.); and Laboratoire de Pharmacologie, Faculté de Médecine, Université Catholique de Louvain, Bruxelles, Belgium (T.G.)
Received May 23, 2005; accepted July 12, 2005.
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Abstract |
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). In atherosclerosis, lesion formation depends upon calcium-regulated cellular processes, such as chemotaxis, adhesion, migration, proliferation, lipid uptake, and necrosis. As reported by Henry (1990), interventions acting on cell calcium uptake, including treatment with calcium-chelating agents lanthanum trichloride and calcium antagonists, may retard atherogenesis in fat-fed animals in the absence of hypolipidemic effects. Several randomized control clinical trials, including the International Nifedipine Trial of Antiatherosclerotic Therapy, Verapamil Hypertension Atherosclerosis Study, and Prospective Randomized Evaluation of the Vascular Effects of Norvasc Trial with amlodipine, have substantiated the inhibitory effect of calcium channel blockers on the progression of atherosclerosis (for reference, see Godfraind, 2004). More recently, in the European Lacidipine Study on Atherosclerosis (Zanchetti et al., 2002), lacidipine was compared with atenolol. The authors reported that this CCB had a greater efficacy than the 
-blocker on carotid intima-media thickness progression and number of plaques per patient, despite a smaller ambulatory blood pressure reduction. They concluded that the antiatherosclerotic effect of lacidipine seems independent of the blood pressure-lowering effect. This conclusion was in agreement with Cristofori et al. (2000) who reported that lacidipine reduces the development of atherosclerotic lesions in female ApoE-deficient mice that were fed a normal chow.
ApoE KO mice (also named ApoE-deficient, ApoE-/-) are considered as a model of human atherosclerosis. They develop spontaneous hypercholesterolemia and atherosclerotic lesions that are augmented by a lipid-rich Western-type diet (Nakashima et al., 1994). Moreover, even on a normal chow diet, these mice exhibit several functional alterations of the cardiovascular system, including endothelial dysfunction (Plump et al., 1992; Bonthu et al., 1997; Barton et al., 1998; d'Uscio et al., 2001) that had been proposed to trigger initial molecular and cellular events in atherogenesis (Libby and Galis, 1995). This dysfunction may be evidenced by reduced endothelial nitric oxide-mediated vasorelaxation, which influences vascular tone and hemodynamics (d'Uscio et al., 2001). Pratico et al. (1998) have reported that oxidative stress is increased in the ApoE-deficient mouse on a normal diet and can be suppressed by oral administration of vitamin E, reducing by approximately 60% aortic atherosclerosis area without reduction of elevated cholesterol. However, Thomas et al. (2001) have shown that vitamin E had a moderate antiatherogenic effect located only in the aortic root when ApoE KO mice were on a high-fat diet.
The purpose of the present experiments was to test the hypothesis proposed by Cristofori et al. (2000) that it is unlikely that the antiatherosclerotic properties of lacidipine could be related to the well known antihypertensive effect of the drug, because the dosage they used (3.0 mg/kg/day) did not affect blood pressure values in ApoE-deficient mouse. Indeed, lacidipine has an antioxidant capacity comparable with vitamin E (van Amsterdam et al., 1992), and the dose of 3.0 mg/kg/day seems to be bioequivalent as antioxidant to the amount of vitamin E supplemented by Pratico et al. (1998) in ApoE KO mice that were fed a normal diet. Experiments were designed to evaluate the antiatherosclerotic action of lacidipine in male ApoE-deficient mice, thus avoiding the effect of estrogens on atherosclerosis (Bourassa et al., 1996). Mice were fed high-fat diet to appraise the action of the CCB in a condition that reduced the protective action of antioxidant (Thomas et al., 2001). We compared male ApoE-/- mice exposed for 8 weeks to a lipid-rich Western-type diet (WD) with or without lacidipine at 1 and 3 mg/kg/day (Lac1 and Lac3) to male ApoE-/- mice receiving a standard chow (ND) with regard to blood pressure, cardiac weight, plasma renin activity (PRA), plasma ET-1, renal structure, level in kidney extracts of thiobarbituric acid-reactive substances (TBARS) marker of oxidative stress (Virdis et al., 2002), lipids level, degree of atherosclerotic lesions, and responsiveness to NO-dependent vasorelaxation. Endothelial dysfunction in ApoE-/- vessels is focal, depending on the localization of atherosclerotic lesions (Crauwels et al., 2003). Hence, in vitro vascular studies were performed on aortic arch segments precontracted by norepinephrine.
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Materials and Methods |
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Unless otherwise stated, at the age of 8 weeks, ApoE-/- mice were divided at random into two groups that were exposed to either normal chow (ND, laboratory diet for specific pathogen-free mice containing 3.5% anhydrous fat; Machal, Prague, Czechoslovakia) or to lipid-rich Western-type diet (WD, adjusted calories diet; 42% is from anhydrous milk fat, and 0.15% is from cholesterol; catalog number, TD88137; Harlan Teklad, Madison, WI) without or with lacidipine. Lacidipine (GlaxoSmithKline, Uxbridge, Middlesex, UK) was administered daily by gavage for either 8 or 10 weeks at dose levels of 0 (control), 1.0 (Lac1), and 3.0 (Lac3) mg/kg body weight in 0.5% methyl cellulose (Sigma-Aldrich, St. Louis, MO) at a standard dose volume of 10 ml/kg body weight. Housing facilities and all experimental protocols were following the ethical rules of our institutions.
After 8 weeks (or eventually 20 weeks) of exposure to treatment, animals were anesthetized with thiopental (45 mg/kg i.p.) and approximately 1-ml blood sample was collected by bleeding with a syringe from vena cava caudalis into ice-cold tubes containing 1.6 mg of potassium EDTA that were immediately centrifuged. Both kidneys, as well as the whole aorta, were excised. The ascending aorta was cut in the closest point to the left ventricle for functional studies. The kidney tissue was snap-frozen and stored in liquid nitrogen until further study.
Biochemical Measurements. Blood samples were taken in chilled EDTA tubes, kept at 4°C, centrifuged at 1500 rpm for 15 min, frozen in 100-µl aliquots, and kept at -20°C until further determination. Plasma cholesterol and lipoproteins were determined using the commercial kit Ecoline 25 (Merck, Darmstadt, Germany).
Plasma renin activity [nanogram of angiotensin I (Ang I)/milliliter/hour] was estimated by measuring the rate of Ang I formation with the subsequently generated Ang I quantified by an enzyme immunoassay (Bachem, Bubendorf, Switzerland). ET-1 concentration in plasma was also estimated by an enzyme immunoassay using commercial kits (from Peninsula Laboratories, Belmont, CA).
Measurement of TBARS in Kidney. Lipid peroxidation, as evidenced by the formation of TBARS, was assayed by the method of Ohkawa et al. (1979). The reaction mixture in a total volume of 10 ml containing 0.2 ml of tissue homogenate, 0.2 ml of 8.1% sodium dodecyl sulfate, 1.5 ml of 20% acetic acid, 1.5 ml of distilled water, and 5 ml of n-butanol and pyridine mixture was heated at 95°C for 60 min. The pink-colored chromogen formed by the reaction of 2-thiobarbituric acid with the breakdown products of lipid peroxidation was read at 535 nm.
Assesment of Atherosclerosis. The extent of the atherosclerotic lesions in the aortic en face preparations was quantified by morphometry on photographs. Whole aortas (from heart to iliac bifurcation) were placed into ice-cold (4°C) physiological solution. They were photographed against a black background in a standard fashion, allowing plaques and even minor fatty streaks to seem whitish in contrast to the darker opaquely transparent normal tissue. From digitized gross photographs, intimal plaque area was detected by setting a calibrated gray value threshold with image-analysis software (Pentium IV; Intel, Santa Clara, CA) in Impor 4.0 Professional (Kvant sro, Bratislava, Slovakia) and in Adobe Photoshop 5.0 LE (Adobe Systems, Mountain View, CA). The size of objects (plaques, aorta area) in image was estimated from the amount of pixels corresponding to the inspected objects. The real size was calculated after calibration of pixel image with known scale. Computation was performed by custom-developed software. Data were expressed as a percentage of the total vascular area.
Examination of Kidneys. Kidneys obtained from three mice randomly selected in each group were examined with regard to renal morphology, glomerular abnormalities, and interlobular arteries using standard histological methods. In brief, sections (7 µm) of frozen kidneys from all treatment groups were stained with either hematoxylineosin or with Masson's trichrome solution (for better staining of extracellular matrix proteins). Masson-stained preparations were viewed with light microscopy at 40x. Sections of kidneys were examined on a blinded basis for the level of glomerular and microvascular injury. Forty-five samples (containing at least 20 glomeruli/sample) were studied in each group. The proportion of glomeruli expressing increased extracellular matrix deposition was calculated in each group.
Sections of kidneys stained with hematoxylin-eosin were examined on a randomized basis for the size of glomeruli area and of intrarenal arteries. Intrarenal arteries were examined with regard to the size of the lumen and the vessel wall in the sections used for glomeruli studies. Objects selected were sections perpendicular to the longitudinal axis of the artery without visible atheromatous lesions showing a regular pattern. For microscopic imaging a Meopta objective (10x, numerical aperture 0.30; Meopta, Finchley, UK) was used in a Meopta microscope. The detection system was based on Minitron CCD camera (Minitron Elektronic Gmbh, Ingolstadt, Germany) connected to MiroVideo DC30 frame-grabber (Pinnacle Systems, Mountain View, CA) interfaced to PC (Pentium II). All images were digitized with a 24-bit resolution (768 x 576 pixel, 96 dpi). Captured image sequences were preprocessed with image-analysis software Pentium IV in Impor 4.0 Professional and in Adobe Photoshop 5.0 LE. The size of objects was estimated by processing image pixel as described above. Data were expressed in µm2.
Vascular Reactivity. Aortic arch was placed in a physiological solution (118.6 mM NaCl, 4.7 mM KCl, 2.5 mM CaCl2, 1.2 mM MgSO4, 1.2 mM KH2PO4, 25.1 mM NaHCO3, and 11.1 mM glucose) on a silicon plateau on ice. Adventitial tissue was removed very carefully, avoiding damage of endothelium, and one ring of 2.5-mm long was cut in the ascending part.
Rings were mounted onto small Ni-Co/Fe metal triangles connected to an isometric force transducer in a glass chamber containing 20 ml of physiological buffered solution heated to 37°C and aerated with the a mixture of 94% O2/6% CO2, pH 7.4. Resting tension was set to 10 mN. Indomethacin (10-5 M) was included in all of the solutions to avoid prostaglandin-mediated effects. During a 1-h resting period, the solution was changed every 20 min. The artery contraction was induced by the addition of norepinephrine at concentrations varying from 10-9 to 3 x 10-6 M. A dose-dependent response to ACh was elicited within a concentration range between 10-9 and 3 x 10-6 M when the response to norepinephrine had reached a steady state. This was achieved with norepinephrine 3 x 10-6 M, a concentration that evoked in individual preparations a contraction approaching the maximum value. Then the preparation was washed and stabilized for 1 h with changing of bathing solution containing the NO synthase inhibitor N
-nitro-L-arginine (L-NNA; 3 x 10-4 M) every 20 min. A second dose-effect curve was performed by the addition of norepinephrine. After reaching stable contraction, a dose-dependent response to the NO donor S-nitroso-N-acetylpenicillamine (SNAP) was assessed in preparations preconstricted with norepinephrine within a concentration range as above for ACh. In a separate protocol, relaxation to SNAP (10-8 M) of arteries isolated from WD-exposed ApoE-deficient mice was measured without or with pretreatment with superoxide dismutase (SOD; 74 U/ml for 5 min). Manually assessed data were fed into a computer and analyzed with the Prism software (GraphPad Software Inc., San Diego, CA).
Calculations and Statistical Analysis. Data are given as mean ± S.E.M. (otherwise stated, n = number of animals). Contractile responses are expressed in millinewtons, and relaxations of isolated vascular rings are expressed as percent relaxation of contraction to norepinephrine (3 x 10-6 M). Curve fitting by nonlinear regression and entire curve comparisons were performed with Prism software. This allowed the estimate of values of maximal response, of pD2 values, of their mean ± S.E.M., and the significance of difference between entire curves. For multiple comparisons, results were analyzed with the use of ANOVA followed by Bonferroni's correction. For comparison between two values, the unpaired Student's t test was used. A value of p < 0.05 was considered significant.
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Results |
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In lacidipine-treated WD mice, there was a significant reduction of the SBP when compared with lacidipine-untreated group. This reduction was dose-dependent (difference between Lac1 and Lac3: *** p < 0.001). The relative cardiac mass (mg/g) was reduced down to 4.6 ± 0.1 with lacidipine at 1 mg/kg/day (*** p < 0.001). Reduction of cardiac hypertrophy was similar with the two dosages of lacidipine. At the end of the treatment period, the body weight was not significantly different in the various groups studied (Table 1).
Biochemical Measurements. Plasma levels of total and LDL cholesterol were markedly elevated in ApoE-deficient mice exposed to Western-type diet as compared with ApoE-deficient mice that were fed normal diet (*** p < 0.001; Table 2). In WD ApoE-deficient mice, treatment with lacidipine had no effect on plasma levels of total cholesterol, VLDL+ LDL cholesterol (Table 2), or HDL cholesterol (for ApoE-/- WD and ApoE-/- WD + Lac3, respectively, equal to 1.62 ± 0.15 and 1.59 ± 0.16 mM; n = 6).
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As shown on Table 2, PRA (nanogram of Ang I/milliliter/hour) was augmented by Western-type diet (*** p < 0.001 for ApoE-/- ND versus ApoE-/- WD). This augmentation was significantly prevented by lacidipine (*** p < 0.001). The concentration of ET-1 in plasma (picogram/milliliter) was also increased by Western-type diet (** p < 0.01 for ApoE-/- ND versus ApoE-/- WD). At the two dosages used, lacidipine significantly prevented increase of ET-1 plasma level in WD mice. Estimates of PRA and ET-1 levels in lacidipine-treated mice were not significantly different from their estimates in ApoE-/- ND.
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Vascular Reactivity. Best-fit values of maximal contraction to norepinephrine in Arch segments were equal to 2.35 ± 0.26 mN in ApoE-/- mice fed with ND. In ApoE-/- mice fed with WD without lacidipine, values were equal to 2.04 ± 0.18 mN, with lacidipine at 1 mg/kg/day to 2.29 ± 0.17 mN and to 2.20 ± 0.10 mN with lacidipine at 3 mg/kg/day. pD2 values were equal to 7.17 ± 0.05, 7.10 ± 0.11, 7.06 ± 0.17, and 7.16 ± 0.10, respectively (n = 6 in each group, p = not significant). After exposure of vessels to L-NNA, a NO synthase blocker, pD2 values of norepinephrine were unchanged (data not shown) but maximal contractions to norepinephrine were 3- to 4-fold higher than before L-NNA.
In aortic arch of both ApoE-/- ND and WD mice, endothelium-dependent relaxation of precontracted segments in response to ACh was completely blocked by the NOS inhibitor L-NNA. When compared with ND mice, NO-mediated endothelium-dependent relaxations to ACh were reduced in WD ApoE-/- mice (Fig. 1, *** p < 0.001). Figure 2 illustrates endothelium-independent relaxations to the NO donor SNAP; a significant shift of the dose-effect curve to the right was observed in arch segments from ApoE-/- mice exposed to Western diet (*** p < 0.001). Treatment of WD mice with lacidipine at the two dosages used increased ACh-evoked relaxation significantly and prevented the reduction of sensitivity to SNAP (*** p < 0.001; Figs. 1 and 2). Figure 2, inset, illustrates relaxations to SNAP (10-8 M) of preparations from WD mice treated or not with SOD and of preparations from ND mice. SOD significantly improved endothelium-independent relaxation in WD preparations (*** p < 0.001), which reached the level of relaxation observed in ND vessels.
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Quantification of Atherosclerotic Lesions. Atheromatous lesions were observed in the aortic tract in all ApoE-deficient mice. The distribution of atheromatous lesions was not uniform. Site of predilection for lesion development was the curvature of the aortic arch. In the thoracic tract, focal spotty lesions were seen at the branching of the arteries. The distribution of lesions was not apparently affected by the treatment with lacidipine (Fig. 3). Results of morphometry of en face aortic preparations are reported in Table 3. The extent of atherosclerosis was increased by Western-type diet. This increase was prevented significantly to a similar degree by both dosages of lacidipine. This effect of lacidipine, which was detected after 8 weeks of treatment, was maintained during 20 weeks of treatment despite the large increase of lesions extension noticed in 28-week-old mice (between 8 and 20 weeks; difference in atherosclerosis extension: *** p < 0.001).
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Histological Studies
Kidney Glomeruli. Histological examination of preparations stained with Masson's trichrome showed extracellular matrix deposition in glomeruli of ApoE KO mice; the percentage of glomeruli showing collagen deposits was augmented by Western-type diet and was significantly reduced by lacidipine treatment (Fig. 4, top; see also Supplemental Data for Masson-stained kidney).
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Vascular Morphometry. Observations of vessels morphology in WD ApoE-deficient mice compared with ND ApoE-deficient mice revealed vascular changes consisting of increased thickness of the tunica media of intrarenal arteries accompanied by luminal narrowing. An equal number of vessels with an external diameter between 150 and 55 µm was randomly selected in each group. The median value of their external diameter amounted to 100 µm. Quantitative analysis showed that luminal narrowing, increased thickness of the tunica media, and a consequent significant increase of the wall-to-lumen ratio due to Western-type diet were significantly prevented by lacidipine (Table 4).
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Discussion |
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). We observed that kidney levels of TBARS, markers of lipid peroxidation, were higher in WD ApoE-/- mice than in ND ApoE-/- mice, an indication that WD-dependent augmentation of LDL cholesterol was accompanied by increase of lipids peroxidation. Wanner et al. (1997) have reported that oxidized LDL induces the formation of oxygen radicals not only in arteries but also in glomeruli and juxtaglomerular cells, causing stimulation of renin release. It is likely that this process was operating in ApoE-deficient mice exposed to Western-type diet giving rise to PRA. According to Muller et al. (1998), the uptake of circulating renin by cardiac and vascular tissue allows a local production of functionally active angiotensin II. Direct evidence has been provided for the proatherogenic role of angiotensin II in ApoE-/- mice. It has been shown that, for the same increase in blood pressure, angiotensin II administered via osmotic minipumps for 8 weeks evoked a significantly larger atherosclerosis than did norepinephrine (Weiss et al., 2001). Ang II may play a role in the development of atherosclerosis, probably via its effect on activation of oxidative stress, leading to increased oxidized LDL incorporation into the vascular wall, and its proinflammatory action (Schiffrin, 2002). Furthermore, Ang II increases vascular ET-1 levels (Hsu et al., 2004). This cascade seemed to be present in ApoE-deficient mice exposed to Western diet in which we noted a positive correlation between PRA and ET-1 plasma levels (Fig. 5a). Endothelin antagonists may reduce the development of atherosclerosis, indicating a role for this peptide in the pathogenesis of this process (Barton et al., 1998). Therefore, prevention of Western-type diet-induced atherosclerosis by lacidipine might at least partly be related to reduction of PRA and subsequently to the reduction of ET-1 production, a hypothesis consistent with the positive correlation shown in Fig. 5b between PRA and the extension of atherosclerosis.
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Kidney histology of WD ApoE-deficient mice showed severe alterations characterized by accumulation of extracellular matrix proteins in glomeruli and structural changes in intrarenal arteries, mainly a reduction of the lumen of intrarenal vessels. Atherosclerosis-dependent lowering of renal blood flow has been documented by in vivo hemodynamic studies in ApoE-deficient mice exposed to Western-type diet (Gervais et al., 2003). Renal vessels have a high density in Ca2+channels characterized by an enhanced expression of L-type channels located predominantly at the sites of bifurcations of renal resistance arteries (Goligorsky et al., 1995). ROS can activate L-type calcium channel through oxidation of sulfhydryl residues at an extracellular site of the channel (Sims and Harvey, 2004). Lowering of renal blood flow in WD ApoE-/- mice might be related to ROS-evoked Ca2+channel activation, which amplifies, in vessels preconditioned with ET-1, response to vasoconstrictors in a manner highly sensitive to CCBs (for reference see Godfraind, 2004). Kidney damage leading to abnormal regulation of renin secretion as observed in WD ApoE-deficient mice is not unique, because it has also been found in spontaneous hypertensive rats (Kyselovic et al., 2001; Hu et al., 2004). In those rats, renoprotection by CCBs has been attributed to local hemodynamic changes (Zhou et al., 2002) that may be related to the enlargement of lumen area of vessels (Sabbatini et al., 2002; Skov and Mulvany, 2004). In WD ApoE-/- mice, we observed that lacidipine prevention of kidney injury coexisted with lumen enlargement of intrarenal vessels (Table 4) and that there was a positive correlation between wall-to-lumen ratio considered as an hemodynamic index (Sabbatini et al., 2002) and the proportion of Masson-positive glomeruli in kidney (Fig. 5c). This indicates that lacidipine contributed to renal protection and reduction of PRA by an action on Ca2+ channels improving renal blood flow, a view consistent with the positive correlation between wall-to-lumen ratio and PRA (Fig. 5d).
Information provided by vasorelaxation experiments reveals the complexity of the mechanism of the protective action of lacidipine in atherosclerosis. ACh relaxation of precontracted vessels, which is mediated by endothelium, was incomplete in aortic arch from ApoE-/- mice. This is consistent with observations made in their thoracic aorta (d'Uscio et al., 2001). Impairment of endothelial function in ApoE-/- mice exposed to Western-type diet has been attributed to a reduction of endothelial NO synthase activity and to an increased production of 
causing inactivation of NO (d'Uscio et al., 2001). Experimental studies in isolated mouse vessels have shown that ox-LDL and superoxide impaired endothelial NO function (Jiang et al., 2001; Lu and Kassab, 2004). Antioxidants improve NO-dependent relaxation (d'Uscio et al., 2001; Sato et al., 2002). We have reported that vitamin E and CCBs, including lacidipine, reduce plasma LDL oxidation as well as the formation of oxidation-specific epitopes in arteries of stroke-prone spontaneous hypertensive rats (Napoli et al., 1999). In the present experiments, we observed that the reduction of SNAP-evoked relaxation in WD mice was suppressed after SOD treatment, indicating that loss of responsiveness to SNAP was due to NO destruction by ROS (Fig. 2). It is likely that lacidipine prevented the reduction of endothelium-dependent vasorelaxation and the loss of sensitivity to the NO donor SNAP observed in aortic arch isolated from mice exposed to Western-type diet by reducing oxidative stress either by its direct scavenging effect reported by Van Amsterdam et al. (1992) and confirmed by Ursini (1997) or by an indirect action resulting from a reduced NADPH oxidase activation through reduction of SBP and decrease of renin production. However, even if antioxidants may improve ACh-dependent vasorelaxation in ApoE-deficient mice exposed to high-fat diet as reported with vitamin C by Matsumoto et al. (2003), studies with vitamin E have shown that the antiatherosclerotic action of this nonenzymatic antioxidant is largely operating in ApoE-deficient mice fed a normal diet (Pratico et al., 1998) but not in ApoE-deficient mice fed a high-fat diet (Thomas et al., 2001). Therefore, the protective action of lacidipine on atherosclerosis extension in WD ApoE-/- mice cannot be attributed only to antioxidant capacity of the molecule. Evidence provided in the present study indicates that the calcium-antagonistic effect is also involved in this protective action, mainly through improvement of kidney perfusion resulting in renoprotection and in reduction of PRA.
It is worth noting that the present observations showing that lacidipine at 3 mg/kg/day reduced SBP are in contrast with the report of Cristofori et al. (2000), assuming that the antiatherosclerotic action of this CCB could not be related to blood pressure reduction, because lacidipine at 3 mg/kg/day had been shown not to affect blood pressure values in ApoE-deficient mouse. The dose-dependent reduction of SBP by lacidipine reported in the present study could have also contributed to the antioxidant action, because lowering of SBP diminishes shear stress in vasculature and thus activation of NADPH oxidase, the enzyme responsible for superoxide generation (Hwang et al., 2003), resulting in lesser lipid peroxidation. However, data showed that the protective action was similar for the two dosages of lacidipine so far studied, suggesting that other factors could intervene such as those involved in the tissue selectivity of CCBs (Godfraind, 2004). This hypothesis deserves further investigations.
In summary, we observed that lacidipine was acting as a highly powerful antiatherosclerotic agent in ApoE-deficient mice fed a Western-type diet. This action was associated with preservation of NO-dependent vascular relaxation and renoprotection. We concluded that inhibition of Western-type diet-evoked alterations was related to both antioxidant and vasoactive properties of this calcium channel blocker.
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Footnotes |
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Article, publication date, and citation information can be found at http://jpet.aspetjournals.org.
ABBREVIATIONS: CCBs, calcium channel blockers; NO, nitric oxide; ApoE-/- KO, apolipoprotein E-deficient knockout; SNAP, S-nitroso-N-acetylpenicillamine; PRA, plasma renin activity; ET-1, endothelin-1; TBARS, thiobarbituric acid-reactive substance(s); WD, Western-type diet; ND, normal diet or standard chow; SBP, systolic blood pressure; Ang, angiotensin; L-NNA, N-omega-nitro-L-arginine; SOD, superoxide dismutase; LDL, low-density lipoprotein; HDL, high-density lipoprotein; VLDL, very low-density lipoprotein; ROS, reactive oxygen species; mN, millinewton; Ach, acetylcholine; ANOVA, analysis of variance; Lac1 and Lac3, lacidipine at 1 and 3 mg/kg/day.
The online version of this article (available at http://jpet.aspetjournals.org) contains supplemental material. 
Address correspondence to: Dr. Theophile Godfraind, Laboratoire de Pharmacologie, Faculté de Médecine, Université Catholique de Louvain, 54 Avenue Hippocrate, UCL5410, B1200 Bruxelles, Belgium. E-mail: godfraind{at}farl.ucl.ac.be
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