INTRODUCTION

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LARGE ARTERIES ARE CHARACTERIZED by their elastic properties and ability to synthesize many vasoactive substances. These properties enable the arterial wall to function as a modulator of blood pressure and more generally of cardiovascular hemodynamics (14). It is well recognized that the mechanical properties of large arteries are primarily determined by the composition of the arterial wall. The "passive" biomechanical properties of the arterial wall are influenced predominantly by the extracellular matrix proteins, collagen, and elastin, whereas the "active" properties depend on the activation of vascular smooth muscle cells. Aging, environmental and genetic factors are responsible for the functional (decreased release of vasodilators and increased synthesis of vasoconstrictors) and structural (smooth muscle cell hypertrophy, extracellular matrix accumulation, and degradation of elastin) (19) modifications of the arterial wall and the arterial endothelium. These modifications could lead to a diminution of elasticity and increased vascular stiffness.

Atherosclerosis produces many pathological changes in the arterial wall, one of the most important being a progressive increase in arterial stiffening. It has also been demonstrated that hypertension can increase aortic stiffness (3, 4, 23, 24, 26, 28, 37). In addition, previous studies showed that abdominal aortic aneurysms are characterized by severe stiffening and dilatation of the large arteries distant from the aneurysm location (7, 22). Apolipoprotein E-deficient (apoE-KO) mice are widely used as a model of atherogenesis (30, 36). Chronic treatment of ANG II in apoE-KO mice has been shown to induce aneurysm formation accompanied by hypertension and exacerbated arterial inflammation and atherosclerosis (13, 45, 47). In contrast to norepinephrine-induced hypertension, which only modestly affected the development of atherosclerosis, it has been demonstrated that ANG II-induced hypertension specifically increased the progression of atherosclerosis in apoE-KO mice (47). We therefore hypothesized that ANG II will injure the arterial wall resulting in increased arterial stiffening in this mouse model. Thus, the objective of the present study was to evaluate the elastic properties in relation to the morphological, biochemical, and reactive properties of the aortas from ANG II-treated apoE-KO mice. Aortic stiffness was determined by in vivo measurement of pulse wave velocity (PWV) using a noninvasive Doppler method and by in vitro measurement of vascular elasticity using a modified vessel elastigraph. Our data demonstrated that ANG II increased arterial stiffening using both in vivo and in vitro methods. The accompanying morphological, biochemical, and reactive changes in the arterial wall induced by ANG II, as demonstrated by a significant increase in collagen content, decrease in elastin content, degradation of the internal elastic lamina, and impaired endothelium-independent vasorelaxation may contribute to the increase in arterial stiffening.

	METHODS

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Animals. All animal protocols were approved by the Institutional Animal Care and Use Committee at the University of California at Davis and Berlex Biosciences. Osmotic minipumps (Alzet, model 2004; ALZA, Palo Alto, CA) containing ANG II (1.44 mg · kg¹ · day¹; CalBiochem, La Jolla, CA) were implanted subcutaneously in 6-mo-old male apoE-KO mice for 30 days to promote increased severity of aortic atherosclerotic lesions as previously described (13, 45). Previous research showed that this dose of ANG II induces increased atherosclerosis and suprarenal aneurysms (13). Vehicle controls received osmotic minipumps containing PBS. After 30 days, systolic blood pressure was measured in conscious mice using a tail-cuff system (Kent Scientific, Litchfield, CT). Mice were trained to lie quietly in a restrainer placed on a warm pad for a period of at least 30 min for 1-4 days before the study. On the day of the study, the mice were placed in the temperature-controlled restrainer for 15 min. Blood pressure was then measured and recorded on a data-acquisition system (PowerLab, 16/s, ADInstruments) (n = 4-7/group). Systolic blood pressure was averaged from five consecutive measurements. Mice were euthanized by CO₂ asphyxiation.

Quantification of atherosclerosis and aneurysms. The left and right carotid arteries were dissected, cut open longitudinally, and pinned down individually on silicon-coated petri dishes (n = 4-10/group). Atherosclerotic plaques were visible without staining. The images of the open luminal surface of both carotid arteries were recorded by a digital camera (Sony) mounted on a dissecting microscope. The plaque area was quantified using C-Imaging Systems (Compix, Cranberry Township, PA) and expressed as a percentage of the total luminal surface area of the carotid arteries as previously described (42, 45, 46). The diameter of the suprarenal aorta was measured postmortem by direct measurement of cross sections (n = 7-12/group).

Measurement of vascular stiffening in vivo using a noninvasive measurement of aortic PWV. Noninvasive Doppler measurement of PWV was developed recently to estimate aortic stiffness in mice (18). Anesthesia was induced by placing mice in a closed chamber ventilated with 1.5% isoflurane for 3-5 min (IMPAC 6, VetEquip, Pleasanton, CA). After induction, the mouse was taped supine to ECG electrodes incorporated into a temperature-controlled printed circuit board. The temperature of the mouse was monitored with a rectal probe (Physitemp, Clifton, NJ), and body temperature was maintained at 35 ± 2°C throughout the study by adjusting the temperature of the board. The ECG electrodes were connected to a high-fidelity ECG amplifier with a 0.1- to 2-kHz bandwidth set to record lead II. Anesthesia was maintained during measurements by placing a coaxial tubing set from the anesthesia machine loosely over the face of the mouse. A 20-MHz Doppler probe with a 4-mm focal distance was placed just right of the sternum and angled to record velocity in the aortic arch moving away from the probe at a depth of 2-4 mm. A mark was made on the chest at the aortic arch measurement site and a second mark was made 40 mm distal on the abdomen. A measurement was then taken at the second mark for the abdominal aortic waveform. Aortic PWV was calculated by dividing the separation distance (40 mm) by the difference in arrival times of the velocity pulse timed with respect to the ECG (n = 12-17/group).

Measurement of vascular stiffening in vitro using the elastigraph method. The biomechanical properties of the vessels were also analyzed in vitro with a vessel elastigraph modified to measure arterial elasticity (9, 41). This method provided a sensitive means of assessing vascular stiffness compared with traditional compliance/distensibility methods (8, 32, 39). Two stainless steel rods were inserted through the lumen of an aorta in a parallel fashion while the vessel was immersed in a Krebs-Henseleit buffer. One rod was attached to a motorized controller. This apparatus enabled us to radially stretch a segment of vessel at a constant rate while vessel tension was recorded via a force transducer. In preparation for each stretch, the aortic segments were preconditioned three times at ~10% of their maximal load (maximal tension at the breaking point). The vessel was stretched until breakage. "Stress" (vessel tension developed per vessel surface area; F/A) vs. strain (fractional change in vessel width; w  w₀w₀) curves were generated on 3-mm-length samples of mouse thoracic and suprarenal aorta. From these curves, the following parameters were calculated 1) maximum stress: stress at vessel "breakage," 2) maximum strain: strain at vessel "breakage" (ultimate extensibility), 3) physiological stiffness: the slope of the linear region between 30 and 40% strain of a stress vs. strain curve (low strain Young's modulus), and 4) maximum stiffness: the slope of the linear region at high load values of a stress vs. strain curve (high strain Young's modulus) (Fig. 1). Experiments were performed with three 3-mm segments of each aorta, and the results were averaged (n = 4-6/group).

View larger version (20K): [in this window] [in a new window]   Fig. 1.   Stress vs. strain curve of a thoracic aorta from apolipoprotein E-deficient (apoE-KO) mice treated with ANG II or vehicle. From this curve, the following parameters were calculated 1) maximum stress: stress at vessel "breakage," 2) maximum strain: strain at vessel "breakage" (ultimate extensibility), 3) physiological stiffness: the slope of the linear region between 30 and 40% strain of a stress vs. strain curve (low-strain Young's modulus), and 4) maximum stiffness: the slope of the linear region at high load values of a stress vs. strain curve (high-strain Young's modulus). Note that vessel breakage occurs at a higher stress and lower strain in the ANG II-treated aortas compared with vehicle.

Histology. At the end of each experiment, the aorta was perfused at a constant pressure of 100 mmHg through the heart with PBS followed by warm (37°C) agarose (Sea-Plaque GTG Agarose, low melt; FRMC BioProducts, Rockland, ME) diluted in saline (3% wt/vol) and colored with a green tissue dye. After the agarose had solidified, the abdominal aorta was dissected free from the surrounding connective tissue and pinned onto a wax block before fixation in 10% formalin. Cross sections of aorta (2.5-mm thickness) were made between the superior mesenteric and right renal arteries. A small portion of the right renal artery was left attached to the samples to facilitate orientation of the specimen. These tissues were dehydrated through a graded ethanol series, cleared with xylene, infiltrated with warm paraffin, embedded in paraffin blocks, and cut at 5-µm-thick sections onto gelatin-coated glass slides. Sections were stained with hematoxylin and eosin. Elastin van Gieson- and trichrome-stained cross sections from the aorta were prepared and quantified for extent of elastolysis/aneurysm formation and collagen content, respectively.

Determination of elastin and collagen. Elastin content was measured using the Fastin Elastin Assay (Accurate Antibodies, New York). The elastin concentration was normalized to dry aortic weight according to the manufacturer's suggestion. Collagen content was determined by measuring hydroxyproline using a mini-Woessner microtiter plate method according to Stegemann and Stalder (40). The concentration of hydroxyproline was normalized to vessel length (n = 5/ group) (9).

In vitro nitric oxide-mediated vascular relaxation. Rings (4-mm long) were cut from the distal segment of the thoracic aorta. Each ring was placed inside a 15-ml tissue bath (Schuler, Hugo Sachs Elektronics) filled with Krebs solution (composition in mM: 118 NaCl, 4.7 KCl, 2.5 CaCl₂, 1.2 MgSO₄, 1.17 KH₂PO₄, 20 NaHCO₃, 0.026 EDTA, and 11 glucose) kept at 37°C and bubbled with 95% O₂-5% CO₂. Rings were mounted on stainless steel wires attached to a force transducer (model F30, Hugo Sachs Elektronics) and coupled to a data-acquisition system (MP100WSW, BIOPAC system) for measurement of isometric tension. Rings were incubated for a 30-min period in the organ chamber, then contracted with KCl (40 mM). Fifteen minutes after KCl had been washed out, the preparation was submaximally contracted with U-46619 (1-6 nM). When the contraction to U-46619 reached the plateau, cumulative concentration-response curves to acetylcholine and sodium nitroprusside were consecutively generated. Responses are expressed in percent relaxation. The log concentration of the drug required to produce 50% of the maximum response (pD₂) was determined by computer-assisted interactive nonlinear regression analysis (GraphPad Prism, San Diego, CA) (n = 15-20/group).

Statistical analysis. Data are expressed as means ± SE. Statistical analysis was performed between two groups using two-tailed Student's t-test for unpaired values, one-way ANOVA (with a Bonferroni's post hoc test) for normally distributed populations, and Kruskal-Wallis ANOVA (with a Dunn's post hoc test) for nonnormally distributed populations when comparing groups of three or more. P < 0.05 was considered statistically significant.

	RESULTS

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Chronic infusion (30 days) of ANG II in apoE-KO mice resulted in an increase in systolic blood pressure by 36 mmHg (160 ± 12 vs. 124 ± 4 mmHg, n = 4-7/group, P < 0.05) and carotid plaque area by sixfold (34.6 ± 3.6 vs. 5.0 ± 1.9%, n = 4-10/group, P < 0.001; Fig. 2) compared with vehicle-treated apoE-KO mice. All mice treated with ANG II developed an aneurysm that was consistently localized to the suprarenal region of the abdominal aorta. None of the vehicle-treated mice developed aneurysms. The average outer diameter of the suprarenal aorta from the ANG II-treated group was about twofold greater than those from the vehicle group (1.9 ± 0.2 vs. 0.89 ± 0.02 mm, n = 7-12/group, P < 0.001). Compared with vehicle, the ANG II-treated mice showed a 28% increase in heart weight (178 ± 3 vs. 139 ± 3 mg, n = 16-18/group, P < 0.001) and a 6% decrease in body weight (27.5 ± 0.3 vs. 29.3 ± 0.6 g, n = 16-20/group, P < 0.01). Thus, the ratio of heart/body wt was 35% greater in the ANG II group than in the vehicle group (6.5 ± 0.1 vs. 4.8 ± 0.1, P < 0.001). There were no significant changes in the serum levels of total cholesterol, low-density lipoprotein, glucose, or insulin between the ANG II and vehicle groups (data not shown).

View larger version (81K): [in this window] [in a new window]   Fig. 2.   Representative pictures of the open luminal surface of the left carotid artery from apoE-KO mice treated with ANG II (B) or vehicle (A).

ANG II increased aortic stiffness as measured in vivo by PWV and decreased aortic elasticity as measured in vitro by vessel elastigraph. Noninvasive Doppler measurements were used to determine PWV, which has been established as an in vivo index for vascular stiffness (18). PWV was significantly greater in the ANG II-treated mice (n = 12) compared with the vehicle group (n = 17, P < 0.01; Fig. 3).

View larger version (9K): [in this window] [in a new window]   Fig. 3.   Pulse wave velocity (PWV) measured in apoE-KO mice treated with ANG II or vehicle. Values are expressed as means ± SE. * P < 0.01, significantly different from vehicle.

View larger version (18K): [in this window] [in a new window]   Fig. 4.   Biomechanical parameters of the thoracic and suprarenal aorta from apoE-KO mice treated with ANG II or vehicle. Values are expressed as means ± SE. * P < 0.05, ** P < 0.01, significantly different from vehicle.

ANG II-induced morphological and biochemical changes in the aortic wall. Histochemical staining of the suprarenal aorta with elastin van Gieson and trichrome revealed that the intima of vehicle aortas was lined by a contiguous internal elastic lamina evident with elastin staining, with relatively linear and concentric layers of elastin investing the smooth muscle of the media (Fig. 5A). ANG II-treated mice had defects in the internal elastic lamina, particularly evident at sites of intimal plaque formation (Fig. 5B). In some arteries, larger discontinuities were associated with replacement of the media by fibrous connective tissue. The media of ANG II-treated mice had apparent reduplication of elastin fibers that acutely branched about smooth muscle cells. The aortas from the vehicle group had minimal collagen staining in the media (Fig. 5C). The medial thickening of the aortic wall in ANG II-treated animals was at least partially due to increased extracellular matrix that surrounded smooth muscle cells and stained as collagen with a trichrome stain (Fig. 5D). Much of the adventitial thickening was also attributable to deposition of extracellular fibers that stained as collagen. Biochemical quantification of the elastin and collagen content in the aortas demonstrated that elastin content was significantly decreased, whereas collagen content significantly increased in the ANG II-treated group compared with the vehicle group (Fig. 6).

View larger version (31K): [in this window] [in a new window]   Fig. 5.   Elastin van Gieson and trichrome staining of the suprarenal aorta from apoE-KO mice treated with ANG II or vehicle. A: vehicle elastin staining has continuous internal elastic lamina and concentric elastin fibers between smooth muscle bundles. B: elastin staining in ANG II-treated mice reveals that the internal elastic lamina is discontinuous in the region of intimal plaque. Medial elastin fibers are irregularly separated by smooth muscle cells embedded in amorphous nonstaining extracellular matrix. Some reduplication of elastin fibers is evident. C: trichrome staining of vehicle aortas displayed limited collagen staining in the media. D: trichrome staining of the aortas from ANG II-treated mice demonstrates adventitial matrix staining as collagen. Medial smooth muscle cells are markedly separated by stainable collagen. Original magnifications: ×600 (A, C), ×1,050 (B, D).

View larger version (15K): [in this window] [in a new window]   Fig. 6.   Elastin (A) and collagen contents (B) of the aorta from apoE-KO mice treated with ANG II or vehicle. Values are expressed as means ± SE. * P < 0.05, significantly different from vehicle.

ANG II reduced aortic relaxation. Figure 7 shows concentration-response curves to acetylcholine and sodium nitroprusside in the thoracic aortas in an in vitro organ chamber assay. No differences in the maximal relaxation (E_max: 74 ± 5.3 vs. 78 ± 4.1%) and sensitivity (pD₂: 6.7 ± 0.1 vs. 6.9 ± 0.1) in response to acetylcholine were observed between the ANG II and vehicle groups, respectively. However, the maximal relaxation (E_max: 75 ± 5.6 vs. 87 ± 3.1%, P < 0.05) and sensitivity (pD₂: 7.6 ± 0.1 vs. 8.0 ± 0.1, P < 0.01) in response to sodium nitroprusside were impaired in the aortas from the ANG II-treated mice compared with the vessels from the vehicle-treated animals, respectively.

View larger version (18K): [in this window] [in a new window]   Fig. 7.   Concentration-response curves to acetylcholine (A) and sodium nitroprusside (B) in isolated aortas from apoE-KO mice treated with ANG II or vehicle.

	DISCUSSION

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

The aim of the present study was to examine the effects of chronic treatment of ANG II on the arterial elastic properties in apoE-KO mice. We hypothesized that ANG II will injure the arterial wall resulting in increased arterial stiffening. The results of this study demonstrate that modifications of the biomechanical elastic properties develop during the combined progression of atherosclerosis, hypertension, and aneurysm formation in apoE-KO mice treated with ANG II. ANG II treatment significantly increased arterial stiffening, as measured for the first time using both in vivo and in vitro methods of analysis. In vivo PWV has been used to estimate elasticity and compliance of large vessels in mice (17, 44) while the modified vessel elastigraph is a static in vitro method suitable for examining the passive properties of collagen and elastin in aortic samples (33, 41). The powerful combination of these two techniques allows one to confidently verify the elastic properties of the same aorta under both in vivo and in vitro conditions.

The increase in aortic stiffness in the ANG II-treated aortas is consistent with morphological observations. In contrast to recent findings reported in the literature describing lack of atherosclerosis in the carotid artery of 4- to 5-wk-old apoE-KO mice fed a lipid-rich Western-type diet for 26-30 wk (1, 15), we measured a significant increase in carotid plaque area with ANG II treatment. Although both high-fat feeding (30) and ANG II infusion (13, 45, 47) accelerate the progression of atherosclerosis in apoE-KO mice, the differences in the age of apoE-KO mice, length of treatment, and genetic factors triggering structural vascular change (31) may account for differences in atherosclerosis observed in the carotid artery. Histochemical staining revealed that ANG II induced vascular remodeling with alterations in the distribution of elastin and collagen content in the suprarenal aorta. Nearly all the ANG II-treated animals had breaks in the internal elastic membrane with evident reduplication and reorientation of elastin fibers. In addition, there was a marked increase in collagen in the media. The increased separation of medial cells by collagen implies increased collagen synthesis by smooth muscle cells, as demonstrated in other examples of hypertension (3). The ability of the aorta to counter the force exerted by blood flow is dependent on the specific structure of its wall. In the normal, nonaneurysmal vehicle-treated aorta, the media is arranged in a series of well-defined concentric elastic lamellae. Each layer, bounded by relatively thick elastin bands, contains circumferentially oriented collagen fibers, along with a network of elastin fibers and the layer of smooth muscle cells compacted between adjacent elastic lamellae. The close association of elastin, collagen, and smooth muscle cells in the aortic media is responsible for the viscoelastic properties of the aorta and the reinforcement of the wall against rupture at higher levels of pressure (48). Collagen, together with the elastic laminae, allows the aorta to regain its shape by recoiling from pressure-volume deformation. The marked increase in collagen and increased fragmentation of the elastic laminae, demonstrated by ANG II treatment, lead to diminution of elasticity and tensile strength of the aortic wall. All mechanisms that weaken the aortic wall, the aortic lamina media in particular, lead to higher wall stress (as demonstrated in this study with the in vitro vessel elastigraph), which can induce aortic dilatation and aneurysm formation. The artery wall in the ANG II-treated mice was more susceptible to breakage/wall stress despite increased artery wall thickening. These changes are likely the causes of decreased aortic compliance that may have ultimately contributed to aneurysm formation induced by ANG II. The biochemical analysis revealed that elastin content was decreased and collagen content increased in the aortas from ANG II-treated animals, which is consistent with the above morphological and biomechanical observations. The presence of residual adventitial tissue observed with ANG II treatment, however, may account for some of the changes measured in elastin and collagen content.

Reduction of large artery elasticity has deleterious effects on the cardiac afterload increasing myocardial work. The elevation in systolic pressure causes a disproportionate increase in end-systolic stress, which is an important hemodynamic factor that promotes the development of cardiac hypertrophy (3, 5, 6, 16, 20, 27, 38). Thus, aortic stiffening induced by ANG II may also contribute to the increase in cardiac afterload, causing compensatory cardiac hypertrophy as demonstrated in this study.

In the present study, we hypothesized that in addition to changes in the elastic properties of the artery wall, ANG II treatment also led to an impairment of endothelium-derived nitric oxide (NO) activity. A variety of studies have documented that induction of vascular NAD(P)H oxidase-derived superoxide anion following ANG II infusion accounts for the impaired endothelial function in rats and rabbits (11, 25, 29, 35, 43). Although the induction and localization of NAD(P)H oxidase and superoxide anion have been described in the aortas of ANG II-treated mice, no evaluation of endothelium-derived NO activity has been reported in this species. Surprisingly, in this study, NO-mediated endothelium-independent relaxation but not endothelium-dependent relaxation was impaired in the aortas of ANG II-treated mice. Although we do not have a definite explanation for such results, a few possibilities should be considered. First, because superoxide anion and NAD(P)H oxidase have been reported mainly in the adventitia of ANG II-treated aorta (11, 43), endothelium-derived NO might be relatively protected from this prooxidative system, which could explain the preserved acetylcholine response. However, the adventitia is within the diffusable field of NO and one cannot rule out the possibility of an impaired basal NO activity in these conditions. Second, it has been shown that other endothelium-derived relaxing factors, such as the endothelium-derived hyperpolarizing factors, might act as a back-up mechanism when the endothelial production of NO is impaired or absent (10, 12). Thus, it is possible that the acetylcholine-induced relaxation in ANG II-treated mice aortas is not dependent exclusively on the release of NO, which might be impaired. Third, the significant reduction in the endothelium-independent relaxation to sodium nitroprusside might be secondary to an altered tissue redox potential, which would impair the sodium nitroprusside metabolism. Indeed, the NO release from sodium nitroprusside depends on a reductive enzymatic/chemical catalysis rather than spontaneous liberation (2, 21). Although additional studies will be needed to address these hypotheses, the results of this study show an altered vascular response to NO donors, suggesting an altered tissue redox potential.

The combination of atherosclerosis, hypertension, and aneurysms on the biomechanical elastic properties of the aortic wall in the apoE-KO mice has been explored in this study. We hypothesized that ANG II will injure the arterial wall resulting in increased arterial stiffening. The increase in arterial stiffening may be attributed to the increase in collagen content, decrease in elastin content, degradation of the internal elastic lamina, and an impairment in endothelium-independent vasorelaxation. These changes caused by ANG II treatment may hamper the recoiling function of the aortic wall. Therefore, ANG II-treated vessels may not be protected from pressure-induced damage. In addition to its etiologic role in cardiovascular disease, increased arterial stiffening may be considered as a significant marker and/or an independent cardiovascular risk factor.