Published online before print August 8, 2005, doi: 10.1161/01.HYP.0000182154.61862.52
(Hypertension. 2005;46:932.)
© 2005 American Heart Association, Inc.
Part 2 Original Articles |
Evaluation of Vascular Function in Apolipoprotein E Knockout Mice With Angiotensin-Dependent Renovascular Hypertension
From the Laboratory of Transgenes and Cardiovascular Control (R.M.P.A., V.A.P. S.S.M., E.C.V.), Physiological Sciences Graduate Program, Biomedical Center, Federal University of Espirito Santo, Brazil; and College of Health Sciences of Vitoria (EMESCAM; E.C.V.), Vitoria, ES, Brazil.
Correspondence to Elisardo C. Vasquez, PhD, Physiological Sciences Graduate Program, CBM, UFES, Av. Marechal Campos 1468, Vitoria, Brazil 29042-755. E-mail evasquez{at}terra.com.br
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Abstract |
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It is known that the endothelial function is compromised in atherosclerosis and arterial hypertension and that angiotensin is an important factor contributing to both pathophysiologies. The aim of this study was to evaluate the vascular function in a hypercholesterolemia/atherosclerosis model, in the angiotensin II–dependent 2-kidney 1-clip (2K1C) hypertension model and when both conditions coexist. Eight-week-old apolipoprotein E knockout (apoE; n=20) and C57BL/6 (C57; n=20) mice underwent a 2K1C or sham operation and were studied 28 days later. Mean arterial pressure was higher in apoE—2K1C and C57–2K1C (126±3 and 128±3 mm Hg) when compared with the apoE–Sham and C57–Sham (103±2 and 104±2 mm Hg, respectively; P<0.05). The vascular reactivity to norepinephrine (NE; 10–9 to 2x10–3 mol/L), acetylcholine (ACh), and sodium nitroprusside (SNP; 10–10 to 10–3 mol/L) was evaluated in the mesenteric arteriolar bed through concentration–effect curves. NE caused vascular hyper-reactivity in apoE–Sham, apoE–2K1C, and C57–2K1C (maximal response 146±5, 144±5, and 159±4 mm Hg, respectively) compared with C57–Sham (122±7 mm Hg; P<0.05). The ACh-induced relaxation was smaller (P<0.05) in apoE–2K1C and C57–2K1C (maximal response 53±3% and 46±3%) than in apoE–Sham and C57–Sham mice (78±5% and 73±4%). SNP-induced vascular relaxation showed similar concentration–effect curves in all groups. We conclude that in C57–2K1C mice, the increased reactivity to NE and the decreased endothelium-dependent relaxation contribute to the maintenance of hypertension. The apoE mouse, at early stages of atherosclerosis, shows hyper-reactivity to NE but does not have endothelium dysfunction yet. However, the concurrence of both pathophysiologies does not result in additive effects on the vascular function.
Key Words: atherosclerosis • mice • hypertension, renovascular • apolipoproteins • mesenteric arteries • mice
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Introduction |
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Atherosclerosis and arterial hypertension are multifactorial diseases recognized as the main causes of acute cardiovascular events. These pathophysiologies are characterized by endothelial dysfunction,1,2 and angiotensin II seems to be involved in both diseases.3
Over the past decades, the availability of new investigative tools, including the homozygous apolipoprotein E knockout (apoE) mouse, has contributed to the understanding of the atherosclerotic process. ApoE is a constituent of VLDL synthesized by the liver and mediates high-affinity binding of apoE-containing lipoprotein particles to LDL receptors, and thus is responsible for the cellular uptake of these particles.4 Therefore, apoE mice develop marked hypercholesterolemia and spontaneous atherosclerosis.4,5 In this experimental model, an endothelial dysfunction has been reported under a Western-type cholesterol-rich diet6–8 but not under a normal chow diet.9
Arterial hypertension has also been associated with changes in endothelial function and in vascular smooth muscle cell reactivity to contractile agents.10 In addition, arterial hypertension induced by endogenous or administered angiotensin II aggravates the atherosclerotic process in apoE mice.11–13 Therefore, the findings that the renin-angiotensin system is implicated in the pathogenesis of atherosclerosis14–16 and that it also plays a pivotal role in the development and maintenance of 2-kidney, 1-clip (2K1C) renovascular hypertension in rats17 and mice18 justify the importance of evaluating the vascular function in an animal model of atherosclerosis with concurrent development of renovascular hypertension.
The present experiments were designed to test the vascular function of the mesenteric arteriolar bed in the apoE mouse at the early stage of spontaneous atherosclerosis, in the isogenic C57BL/6 (C57) mouse with angiotensin II–dependent 2K1C renovascular hypertension, and in the mouse with these 2 pathophysiologies.
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Methods |
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Animals
Experiments were conducted on young (8 weeks of age; averaging 23 g) male C57 and apoE mice obtained from the animal facilities of the biomedical center at the Federal University of Espirito Santo. They were fed a normal chow diet and water ad libitum. All procedures were used in accordance with the institutional guidelines for animal research, which are equivalent to the National Institutes of Health guide for the care and use of laboratory animals.
Renovascular Hypertension
The 2K1C renovascular hypertension was produced in C57 (n=9) and apoE (n=10) using U-shaped stainless steel clips (0.12 mm opening width; Exidel SA). The clip was placed around the left renal artery according to the procedure described by others,18,19 and animals were studied 28 days later. Sham-operated C57 (n=9) and apoE (n=10) were used as control animals.
Hemodynamic Measurements
Mice were anesthetized with ketamine/xylazine (91.0/9.1 mg/kg IP), and a catheter (0.040 mm OD x 0.025 mm ID; Micro-Renathane; Braintree Science) was inserted into the right carotid artery for direct arterial pressure measurements. Experiments were performed on conscious, freely moving mice 48 hours after placing the catheter. For arterial pressure recordings, the catheter was plugged into a disposable blood pressure transducer (Cobe Laboratories) connected to a pressure processor amplifier and data acquisition system (Biopac Systems).
Measurement of Plasma Cholesterol Levels
A blood sample was taken from the carotid artery of all the animal groups, and the plasma total cholesterol was measured using a commercial colorimetric kit (Bioclin).
Preparation of the Mesenteric Arteriolar Bed
The superior mesenteric artery was cannulated using a catheter (0.040 mm OD x 0.025 ID; Micro-Renathane; Braintree Science) under thiopental (40 mg/kg IP) anesthesia. The mesenteric arteriolar bed was then taken into a 37°C water container and perfused at a constant rate (2 mL/min) with oxygenated (95% O2–5% CO2 mixture) physiological salt solution using a roller pump (Harvard Apparatus). The solution was composed of the following (in mmol/L): 130 NaCl, 4.7 KCl, 1.6 CaCl2.2H2O, 1.8 KH2PO4, 4.7 MgSO, 1.17 H2O, 14.9 NaHCO3, 0.026 EDTA, and 11.1 glucose. Perfusion pressure was monitored via a T-tube inserted between the pump and the inflow cannula and connected to a pressure transducer (Cobe Laboratories) and a data acquisition system (BioPac Systems). First, we studied the concentration-dependent contraction to 10–9 to 2x10–3 mol/L norepinephrine (NE; Sigma). To study the endothelial function, the vasodilator responses to 10–10 to 10–3 mol/L acetylcholine (ACh; Sigma) and 10–10 to 10–3 mol/L sodium nitroprusside (SNP; Sigma) were calculated as percentages of reductions in the precontractions induced by 5x10–6 mol/L NE (concentration that induces 60% to 80% of the maximal effect).
Statistical Analysis
Data are expressed as means±SEM. Statistical analysis was performed with a 2-way ANOVA followed by the Fisher test for multiple comparisons. Comparisons of mean values between 2 groups were performed by unpaired Student’s t test whenever needed. The significance level was set at P<0.05.
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Results |
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Mean arterial blood pressure (MAP) and total plasma cholesterol levels are summarized in the Table. MAP was significantly higher in C57–2K1C and apoE–2K1C mice when compared with their respective controls C57–Sham and apoE–Sham (P<0.05). As expected, plasma cholesterol levels were significantly higher (4.8-fold; P<0.05) in apoE than in C57 mice. Hypercholesterolemia levels were significantly reduced (24%; P<0.05) in apoE–2K1C compared with the apoE–Sham group.
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Vascular Reactivity
Figure 1 summarizes the concentration–effect curve to NE in the 4 groups. A clear vascular hyper-responsiveness was observed to NE in apoE–Sham and C57–2K1C (maximal response 147±5 and 159±4 mm Hg) groups compared with the C57–Sham group (122±7 mm Hg; P<0.05; Figure 1A and 1C). Renovascular hypertension in apoE did not cause a further increase in vascular reactivity to NE (144±5 mm Hg) compared with apoE–Sham (Figure 1B; P>0.05). Also, no differences were observed between apoE–2K1C and C57–2K1C (Figure 1D; P>0.05).
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The endothelium-dependent relaxation in response to ACh for all groups is shown in Figure 2. The vascular responsiveness to ACh was significantly impaired in the C57–2K1C (maximal response 46±3%, P<0.05) but not in the apoE–Sham group (78±5%; P>0.05) compared with the C57–Sham group (73±4%; Figure 2A and 2C). Renovascular hypertension caused a significant impairment of the vascular reactivity to ACh in apoE mice compared with apoE–Sham (maximal response 53±4% versus 78±5%; P<0.05; Figure 2B). No significant differences in the response to ACh were observed between hypertensive groups (Figure 2D).
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The endothelium-independent vascular smooth muscle relaxation to SNP was similar in all groups (P>0.05), as shown by the concentration–effect curves in Figure 3. No significant differences among groups were observed in the EC50.
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Discussion |
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This is the first study to show in the mouse that spontaneous atherosclerosis and renovascular hypertension caused a hyper-responsiveness to NE in the mesenteric arteriolar bed without additional effect when both pathophysiologies coexist in the same animal. On the other hand, the concentration–effect curves showed that 2K1C hypertension but not atherosclerosis impaired endothelium-dependent relaxations to ACh in the mesenteric arteriolar bed.
One of the hallmarks of atherosclerosis is the impairment of endothelial function, which is present even before the existence of vascular structural changes.1 This feature has been based on studies made in human beings20–22 and in experimental models of atherosclerosis accelerated by high-fat diet.6–8,23 The apoE mouse is considered one of the most relevant models for atherosclerosis because it is hypercholesterolemic and develops spontaneous arterial lesions.4,5 Although endothelial dysfunction has been considered one of the early steps in atherosclerosis,1 a normal endothelial function of the aorta was reported recently in apoE mice at 35 weeks of age.9 In the present study, we evaluated the endothelial function of resistance vessels in young (10 to 12 weeks old) apoE mice and also observed a normal endothelial function. The main candidates that could explain the normal endothelial function in apoE fed with a normal chow diet would be the overexpression of NO synthase or an increased production of superoxide dismutase and catalase enzymes. Nevertheless, none of these mechanisms were altered in the apoE mice, even at the age of 35 weeks.9 The novelty in this study is that we observed an increased vasoconstrictor response to NE in apoE mice. This increased vasoconstrictivity to the 
-adrenoceptor agonist in apoE mice at the early stages of hypercholesterolemia without accompanying endothelial dysfunction was also observed in the rabbit model of atherosclerosis.24 On the basis of the morphological analysis of blood vessels of apoE mice in our laboratory (our unpublished data, 2005) and on data from others25 in young atherosclerotic rabbits, we speculate that this hyper-responsiveness to NE could be related to intima-media thickness.
Another goal of this study was to evaluate the vascular function in young 2K1C hypertensive mice. This model is characterized by vascular hypertrophy and increased plasma renin levels,18 and it has been shown that the development of hypertension can be prevented by targeted disruption of the AT1A receptor gene.26 This is the first study to evaluate the resistance vessels function in 2K1C Goldblatt hypertensive mice, and we observed an increased vasoconstriction to NE associated with impaired endothelium-dependent vasorelaxation. These findings were expected because the renin-angiotensin system plays an essential role in 2K1C Goldblatt hypertensive mice, and it is well known that this peptide is a potent vasoconstrictor in addition to its contribution to endothelial dysfunction.1 On the basis of other studies,16 we cannot rule out the involvement of an excessive oxidative stress in the impairment of the endothelial-dependent vasodilatation in the mesenteric arteriolar bed of 2K1C mice. The finding of a greater capacity for contraction and a lesser capacity for relaxation of resistance vessels observed by us in 2K1C hypertensive mice and by others in 2K1C hypertensive rats27 may contribute to the maintenance of renovascular hypertension.
Interestingly, the vascular responsiveness to NE and ACh in the apoE animal with concurrent development of 2K1C hypertension was similar to that observed in C57–2K1C hypertensive animal (ie, an increased vasoconstriction and a decreased endothelium-dependent vasodilatation). The observation that the coexistence of angiotensin II–evoked hypertension in apoE pathology did not exacerbate the increased vascular responsiveness to NE could be attributable to the vasoconstrictor reserve of apoE mice being at its maximum already. On the other hand, the preserved endothelial function of apoE mice was affected by the induction of 2K1C hypertension in the same magnitude observed in C57–2K1C mice, thus, probably by the same mechanisms as were speculated above for the hypertensive animals.
Consistent with previous reports,4,18,28 plasma cholesterol levels were markedly elevated in apoE mice. It is also well known that at 10 weeks of age, these animals already develop atherosclerotic vascular lesions,4 as also confirmed in our laboratory studies (our unpublished data, 2005). Interestingly, this is the first study to show lower levels of hypercholesterolemia in apoE mice with concurrent development of 2K1C hypertension. This result could be explained, at least in part, by the actions of angiotensin II increasing macrophage uptake of oxidized LDL from circulation to the vessel wall via scavenger receptors.11 However, reduced severity of hypercholesterolemia observed in apoE–2K1C mice did not affect the vascular responsiveness compared with apoE–Sham mice.
Perspectives
One of the limitations of our study is that hypercholesterolemia and renovascular hypertension were investigated in young animals, and we cannot rule out the possibility of finding different results in older mice. Further studies are necessary to fully elucidate the mechanisms involved in the vascular dysfunction in atherosclerotic and renovascular hypertensive mice.
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Acknowledgments |
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This work was supported by the National Council for the Development of Science and Technology (CNPq) and funds for science and technology of the city of Vitoria.
Received April 24, 2005; first decision May 16, 2005; accepted May 24, 2005.
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