Elastin-insufficient mice show normal cardiovascular remodeling in 2K1C hypertension despite higher baseline pressure and unique cardiovascular architecture

Jessica E. Wagenseil, Russell H. Knutsen, Dean Y. Li, Robert P. Mecham

Abstract

Mice heterozygous for the elastin gene (ELN+/−) show unique cardiovascular properties, including increased blood pressure and smaller, thinner arteries with an increased number of lamellar units. Some of these properties are also observed in humans with supravalvular aortic stenosis, a disease caused by functional heterozygosity of the elastin gene. The arterial geometry in ELN+/− mice is contrary to the increased thickness that would be expected in an animal demonstrating hypertensive remodeling. To determine whether this is due to a decreased capability for cardiovascular remodeling or to a novel adaptation of the ELN+/− cardiovascular system, we increased blood pressure in adult ELN+/+ and ELN+/− mice using the two-kidney, one-clip Goldblatt model of hypertension. Successfully clipped mice have a systolic pressure increase of at least 15 mmHg over sham-operated animals. ELN+/+ and ELN+/−-clipped mice show significant increases over sham-operated mice in cardiac weight, arterial thickness, and arterial cross-sectional area with no changes in lamellar number. There are no significant differences in most mechanical properties with clipping in either genotype. These results indicate that ELN+/+ and ELN+/− hearts and arteries remodel similarly in response to adult induced hypertension. Therefore, the cardiovascular properties of ELN+/− mice are likely due to developmental remodeling in response to altered hemodynamics and reduced elastin levels.

  • arterial mechanics
  • extracellular matrix
  • high blood pressure
  • cardiac hypertrophy
  • two-kidney, one-clip hypertension

hypertension induces complex remodeling in both the heart and arteries. In response to pressure overload, the thickness of the heart is increased to normalize systolic wall stress in the left ventricle (11). This concentric cardiac hypertrophy can produce normal function for long periods but eventually may lead to congestive heart failure when the heart can no longer keep up with increased loads (24). In response to the same pressure stimulus, arterial thickness is also increased to normalize wall stress (21). This vascular remodeling decreases arterial wall stress but also decreases arterial compliance, causing the heart to work harder to pump blood through the system, exacerbating cardiac hypertrophy and accelerating cardiac failure. Remodeling of both the heart and arteries can occur through hyperplasia or hypertrophy of cardiomyocytes and smooth muscle cells (SMCs), as well as through the addition of extracellular matrix proteins (13, 31).

In the large arteries, the major extracellular structural proteins are collagen and elastin. Collagen provides strength at high blood pressures (10), whereas elastin provides reversible elasticity and stores energy throughout the cardiac cycle (23). Mice that are missing the elastin gene (ELN−/−) die within a few days of birth due to uncontrolled growth of SMCs that eventually occludes the arterial lumen (17). Mice that are heterozygous for the elastin gene (ELN+/−) live a normal life span but have reduced levels of vascular elastin, significant hypertension, and an increased number of lamellar units (concentric layers of SMCs and the adjacent elastic lamina) in the arterial wall. Humans with supravalvular aortic stenosis, a disease caused by functional heterozygosity of the elastin gene, also show significant hypertension and increased lamellar units in the arterial wall (7, 18). The number of lamellar units is established at birth and does not change in adulthood, even in response to increased hemodynamic stress (34). We previously investigated the geometry and mechanics of ELN+/− arteries and found that, when compared with ELN+/+, the arteries are smaller and thinner, circumferential and longitudinal residual strains are different, and the stretch-stress behavior is similar (30).

The geometry and mechanics of ELN+/− arteries show that the they do not respond to hypertension in the expected manner; the arteries are not increased in the cross-sectional area and thickness to normalize wall stress. ELN+/− mice have slight cardiac hypertrophy, but this does not lead to cardiac failure and premature death (7), showing that the heart also does not respond to hypertension in the expected manner. The lack of remodeling in both the heart and arteries may be due to the adaptation of the ELN+/− cardiovascular system during development, so that increased blood pressure in ELN+/− mice is actually the normotensive value for this genotype. It is also possible that the ELN+/− cardiovascular system is incapable of normal hypertensive remodeling due to reduced elastin gene expression and protein synthesis. To investigate this second hypothesis, we increased blood pressure in adult ELN+/+ and ELN+/− mice using the two-kidney, one-clip (2K1C) Goldblatt model of hypertension. Reduced blood flow to one kidney activates the renin-angiotensin system, increasing blood pressure and stimulating cardiovascular remodeling (32). Four weeks after clipping one renal artery, we measured blood pressure, heart and kidney weights, arterial geometry, mechanics, and histology to determine whether ELN+/+ and ELN+/− arteries remodel in a similar manner when hypertension is induced in adult animals.

MATERIALS AND METHODS

Animals

Wild-type C57BL/6J mice (ELN+/+) and mice bearing a heterozygous deletion of exon 1 in the elastin gene (ELN+/−) (17) were used for all studies. Mice were of matching age [70 days (SD 8)] and weight [26.4 g (SD 2)], and littermates were used whenever possible. All housing, surgical procedures, and experimental protocols were approved by the Institutional Animal Care and Use Committee.

Surgery

Small silver clips were placed on the right renal artery following published procedures (14, 32). Mice were anesthetized with 1.5% isoflurane inhaled through a nose cone and kept warm by radiant heat. All instruments were sterilized by autoclaving, and a sterile field was maintained. A midline incision was made in the abdominal cavity, and the abdominal viscera were carefully removed and covered with moistened sterile gauze. The right renal artery was isolated, and a silver clip (0.102 mm gap size, 1 mm width, and 2 mm length) was placed around the artery to constrict blood flow. The viscera were carefully replaced, and the incision was closed with interrupted 5-0 prolene sutures and a layer of Vetbond tissue adhesive. Sham-operated animals underwent the entire procedure except for the placement of the renal clip. The clips were left in place for 4 wk at which time the mice were euthanized for blood pressure, organ weights, arterial mechanics, and histology. In preliminary studies, clip size and body weight were varied to determine the optimal parameters.

Blood Pressure, Heart Rate, and ANG II Response

Mice were anesthetized with 1.5% isoflurane inhaled through a nose cone and kept warm by radiant heat. A catheter (Millar Instruments, Houston, TX) was inserted into the right common carotid artery, and the blood pressure was monitored for 20 min. The isoflurane concentration was reduced to 0.5% during this period for at least 5 min, and the average heart rate and systolic, diastolic, and mean pressures were recorded. In some animals, the isoflurane was then increased to 1% for ANG II injections. ANG II was injected through the jugular vein on the contralateral side at doses of 100, 500, 1,000, and 3,000 ng/kg.

Tissue Collection and Weights

The heart and both kidneys were removed for weighing. The total heart and left ventricle plus septum were weighed and normalized to body weight for each mouse. The right (clipped) and left (unclipped) kidney were weighed and normalized to mouse body weight. The ascending aorta and left common carotid artery were removed for mechanical measurements. The descending aorta was removed for histology.

Histology and Unloaded Dimensions of the Descending Aorta

The unloaded descending aorta was fixed in 10% formalin for 24 h and then transferred to 70% ethanol. The samples were dehydrated in a graded series of ethanol, mounted in paraffin blocks, and cut into 5-μm sections starting just inferior to the branch of the left subclavian artery. Two sections for each artery were stained with Hart's elastic stain to visualize the boundaries of the intima, media, adventitia, and individual elastic lamellae. The circumferences of the inner and outer elastic lamina and the adventitia were measured from photomicrographs of each section using ImageJ software. The number of elastic lamellae was counted at four equally spaced locations around the circumference.

Unloaded Dimensions of the Ascending Aorta and Carotid Artery

Three narrow rings (1–2 mm long) were cut from the proximal ascending aorta and from the center of the left common carotid artery. For the carotid artery, these rings were cut after the mechanical testing described in Compliance of the carotid artery. The rings were placed in room-temperature physiological saline solution containing (in mM) 130 NaCl, 15 NaHCO3, 5.5 dextrose, 4.7 KCl, 1.2 MgSo4-7H2O, 1.2 KH2PO4, 0.026 EDTA, and 1.6 CaCl2 (pH 7.2), and images were recorded with a stereomicroscope coupled to a video camera. The artery boundaries were determined manually, and the unloaded diameter and thickness were measured with ImageJ software. The dimensions were averaged for the three rings from each artery.

Mechanical Measurements

Opening angle of the ascending aorta.

Each ascending aortic ring was cut radially at the ventral surface. After 30 min to equilibrate, the opened ring was imaged and the opening angle was measured using custom-written scripts in Matlab software. The opening angle was defined as the angle subtended by the lines connecting the midpoint of the inner circumference with the ends of the ring (4, 10) and averaged for the three rings from each artery.

In vivo longitudinal stretch ratio of the carotid artery.

The in vivo longitudinal stretch ratio of the left common carotid artery was measured as done previously (30). Small particles (30–90 μm diameter) of activated charcoal (Sigma, St. Louis, MO) were placed along the length of the carotid artery on the ventral surface. In vivo images were recorded, the artery was then removed and placed in physiological saline solution, and ex vivo images were recorded. ImageJ software was used to determine the centroid of each carbon marker and to calculate the length between successive markers. The in vivo longitudinal stretch ratio (λziv) was determined by dividing the in vivo distance (liv) by the ex vivo (unloaded) distance (L) for each pair of markers and averaged for the length of the artery. Only markers visible in both in vivo and ex vivo images were measured. The artery was then used for compliance testing as described in the following section.

Compliance of the carotid artery.

Compliance measurements of the left common carotid artery were performed using a pressure and force arteriograph (Danish Myotechnology, Copenhagen, Denmark) (6, 7, 30). The artery was secured to metal cannulae with 10-0 silk surgical suture in a bath of physiological saline solution at 37°C. The artery was transilluminated under an inverted microscope connected to a CCD camera and a computer. The arteriograph software continuously tracks the artery outer diameter by locating the change in pixel intensity between the illuminated artery and the dark background. The arteriograph increases or decreases the intravascular (transmural) pressure of the artery segment by changing the fluid flow through the cannulae. The artery was preconditioned for three to five cycles from 0–175 mmHg. The artery length was adjusted during preconditioning to the in vivo length by stretching the artery until it did not buckle in the longitudinal direction, and the force decreased by 0–3 mN as the pressure increased from 0–175 mmHg (30). At the in vivo length, the intravascular pressure was increased from 0–175 mmHg for three cycles in steps of 25 mmHg (12 s/step). The pressure, longitudinal force, and outer diameter were recorded at 1 Hz. After the testing, the artery was removed and three rings were cut to determine the unloaded dimensions.

Data analysis.

Pressure, longitudinal force, and outer diameter from individual test protocols were converted to stress and stretch ratios. Although all three loading cycles were similar, the third cycle was used for further analysis. The deformed inner diameter and thickness were calculated while assuming constant wall volume (6): Math(1) where di, do, l, and t are the deformed inner and outer diameters, length, and thickness and Di, Do, L, and T are the unloaded inner and outer diameters, length, and thickness.

Cylindrical coordinates are used in the stretch and stress notation with θ, z, and r referring to the circumferential, longitudinal, and radial axes, respectively. Shear was neglected because the markers along the length of the artery did not significantly rotate out of the image plane during loading cycles. The mean stretch ratios in each direction (λθ, λz, and λr) were calculated by the following (19): Math(2) The longitudinal stretch ratio (λz) was equal to the in vivo stretch ratio (λziv). Assuming constant wall volume and a thin-walled tube, we defined the mean stresses in the circumferential (σθ) and longitudinal (σz) directions by the following: Math(3) Math(4) where pi is the inner pressure and f is the longitudinal force (22).

Statistics

Unpaired, two-tailed t-tests, assuming unequal variance, were used to determine statistical differences between groups. Linear regression analysis was used to determine statistical correlations between variables. P < 0.05 was considered significant.

RESULTS

Pressures, Heart Weight, and Kidney Weight in Sham-Operated Mice

Sham-operated ELN+/− mice have a systolic blood pressure ∼50% higher than sham-operated ELN+/+ mice but have only mild cardiac hypertrophy (∼20% increase in normalized heart weight) (Table 1) and do not show signs of early cardiac failure or premature death (7). The normalized kidney weights are similar in sham-operated ELN+/+ and ELN+/− mice.

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Table 1.

Pressures, heart rates, and normalized organ weights for sham-operated and hypertensive-clipped mice

Pressures, Heart Weight, and Kidney Weight in Successful 2K1C Mice

Successfully induced hypertension is defined as a mean systolic pressure of at least 15 mmHg above the mean systolic pressure of sham-operated mice. We successfully induced hypertension in 45% of the clipped mice for both genotypes. All pressures are significantly higher in successfully clipped mice than in sham-operated mice, although the percent increases for ELN+/− mice are about half those of ELN+/+ mice (Table 1). Normalized cardiac weights increase 6–12% with clipping in both genotypes. ELN+/+ mice show a significant decrease in the normalized clipped kidney weight versus that in sham-operated mice, whereas ELN+/− mice show a significant increase in the normalized unclipped kidney weight versus that in sham-operated mice.

Because the increases in cardiac weights and pressures are less in ELN+/− than in ELN+/+ mice, the relationships between these variables were investigated for all mice (successful and unsuccessful clip and sham). For each genotype and for both genotypes combined as a group, normalized total heart and left ventricular weights are significantly and positively correlated with systolic, diastolic, and mean pressures. Figure 1 shows that the normalized total heart weight increases linearly with systolic pressure in sham-operated and clipped mice and follows a similar relationship for both genotypes.

Fig. 1.

Normalized total heart weight (HW) is significantly and positively correlated with systolic pressure (Sys P) for all mice. BW, body weight; ELN, elastin gene.

Several mice were excluded from the study due to cardiac abnormalities that may or may not have been caused by the experimental interventions. These abnormalities included 1) very high heart weights (mean heart weight/body weight = 7.9) with normal or low blood pressures in three ELN+/−-clipped mice, one ELN+/+-clipped mouse, and two ELN+/− sham-operated mice; 2) normal heart weight and very low blood pressure in one ELN+/−-clipped mouse; and 3) an ascending aortic aneurysm in one ELN+/+-clipped mouse.

ANG II Response

ELN+/− sham-operated and ELN+/+-clipped mice show attenuated blood pressure responses to infused ANG II compared with ELN+/+ sham-operated mice (Fig. 2).

Fig. 2.

The increase in mean blood pressure (Mean Press) for specified doses of ANG II is significantly reduced with clipping in ELN+/+ mice. The ANG II response is unchanged with clipping in ELN+/− mice. For sham-operated mice, the ANG II response is significantly reduced in ELN+/− compared with ELN+/+ mice. #Significant difference from sham-operated mice for either genotype; *significant difference from ELN+/+ mice for either experimental group; n = 3 ELN+/+-clip, 7 ELN+/+ sham-operated, 4 ELN+/−-clip, and 4 ELN+/− sham-operated mice.

Histology of the Unloaded Descending Aorta

The internal and external elastic laminae have significantly smaller unloaded diameters in ELN+/− than in ELN+/+ descending aorta (Table 2). The unloaded medial thickness is not significantly different between genotypes for sham-operated mice and increases significantly with clipping in both genotypes. Sham-operated ELN+/− mice have ∼1.5 more lamellar units than sham-operated ELN+/+ mice in the descending aorta, and the lamellar number is unchanged with clipping. There are no significant differences in body weights between the mice in each group.

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Table 2.

Histology measurements of the unloaded descending aorta for sham-operated and hypertensive-clipped mice

Dimensions of the Unloaded Ascending Aorta and Carotid Artery

Consistent with previous results (30), the unloaded outer diameter, thickness, and cross-sectional area of the ascending aorta and left common carotid artery are significantly smaller in ELN+/− than in ELN+/+ mice (Table 3). Cross-sectional area and thickness are significantly increased in clipped ELN+/+ and ELN+/− ascending aorta compared with sham-operated ascending aorta. Area, thickness, and outer diameter are significantly increased in clipped ELN+/+ but not in ELN+/− left common carotid arteries compared with sham-operated arteries. When the unloaded cross-sectional area of each artery for all mice in the study (successful and unsuccessful clip and sham) is plotted versus systolic pressure, there is a significant, linear correlation between these parameters for both ELN+/+ and ELN+/− mice (Fig. 3). The thickness is also significantly and positively correlated with systolic pressure for the carotid artery in all ELN+/+ mice and for the ascending aorta in both genotypes. The outer diameter of the ascending aorta is significantly and positively correlated with systolic pressure in all ELN+/− mice.

Fig. 3.

Unloaded cross-sectional area is significantly and positively correlated with systolic pressure for each genotype in the left common carotid artery (A) and ascending aorta (B).

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Table 3.

Unloaded dimensions and in vivo longitudinal stretch ratio of the carotid artery and unloaded dimensions and opening angle of the ascending aorta for sham-operated and hypertensive-clipped mice

Opening Angle of the Ascending Aorta

The opening angle of the ascending aorta increases ∼25% in both genotypes with clipping (Table 3). The opening angle is significantly and positively correlated with systolic pressure in ELN+/+ and ELN+/− mice.

In Vivo Longitudinal Stretch Ratio of the Carotid Artery

For sham-operated mice, ELN+/− left common carotid arteries have smaller in vivo stretch ratios than ELN+/+ arteries (Table 3) (30). The in vivo stretch ratio is not changed with clipping in either genotype.

Mechanical Testing of the Carotid Artery

As shown previously (30), the outer diameter of the carotid artery is smaller at each pressure and the pressure-longitudinal force curve is more horizontal in ELN+/− sham-operated mice than in ELN+/+ mice (Fig. 4). The pressure/diameter and pressure/force relationships are not significantly different between clipped and sham-operated mice for either genotype. The circumferential stretch ratio, circumferential stress, and longitudinal stress versus pressure for the left common carotid artery are shown in Fig. 5. There are no significant differences between ELN+/+ and ELN+/− arteries for the circumferential stretch ratio and longitudinal stress at each pressure. At pressures 100 mmHg and higher, the circumferential stress is smaller in sham-operated ELN+/− arteries than in ELN+/+ arteries, but at the physiological pressure for each genotype (Table 1), the circumferential stresses are similar. In Fig. 5, there are no significant differences between clipped and sham-operated mice for either genotype. The circumferential stretch ratio-circumferential stress relationships are also similar between sham-operated and clipped mice for both genotypes (not shown).

Fig. 4.

Outer diameter (A) and longitudinal force (B) vs. pressure relationships for the left common carotid artery are significantly different between genotypes but do not change with clipping. *Significant difference from ELN+/+ for either experimental group; n = 9 ELN+/+-clip, 7 ELN+/+ sham-operated, 7 ELN+/−-clip, and 7 ELN+/− sham-operated mice.

Fig. 5.

Circumferential (Circ) stretch ratio (A), circumferential stress (B), and longitudinal (Long) stress (C) vs. pressure relationships for the left common carotid artery do not change with clipping. Circumferential stress is significantly reduced in ELN+/− carotid compared with ELN+/+ carotid at the higher pressures. *Significant difference from ELN+/+ for either experimental group; n = 9 ELN+/+-clip, 7 ELN+/+ sham-operated, 7 ELN+/−-clip, and 7 ELN+/− sham-operated mice.

DISCUSSION

Pressures, Heart Weight, and Kidney Weight in Sham-Operated Mice

Humans with diastolic and systolic pressures similar to ELN+/− mice have a 2× and 24×, respectively, increase in risk of death from cardiovascular disease (20). High blood pressure in ELN+/− mice is signaled through the ANG II type 1 receptor and maintained by increased plasma renin levels (7). Mice that express human chymase also have increased blood pressure (systolic pressure of about 30% greater than that in wild-type) mediated through the ANG II type 1 receptor but have a greater degree of cardiac hypertrophy (about a 40% increase in normalized heart weight over that in wild-type) than ELN+/− mice (15). Mice that express human renin in the liver have a systolic blood pressure of about 20% higher than that in wild-type mice and similar absolute values to ELN+/− mice but have normalized heart weights of ∼30% higher than wild-type and 15% higher than ELN+/− mice (28). These examples show that other genetic mouse models with a lifetime of renin-dependent hypertension have increased cardiac hypertrophy compared with our model of elastin haploinsufficiency. ELN+/− mice may develop resistance to cardiac hypertrophy and related diseases due to developmental remodeling of the cardiovascular system in response to altered wall mechanics of arteries with reduced elastin amounts.

Pressures, Heart Weight, and Kidney Weights in Successful 2K1C Mice

Hypertension was induced in adult ELN+/+ and ELN+/− mice using the 2K1C Goldblatt model. A small clip on one renal artery reduces blood flow to the kidney, stimulating renin production and increasing blood pressure. In 2K1C rats, the degree of hypertension can be modulated by clip size, with mean aortic pressures increasing from 135 mmHg in control rats to 145–185 mmHg (depending on the clip size) in clipped rats after 4 wk (25). In mice, the clip size dictates whether hypertension is induced or not but does not modulate the degree of hypertension (14, 32). The clip must be small enough to decrease renal blood flow but not so small as to produce renal ischemia. In preliminary studies, we tried clips with gap sizes ranging from 0.061–0.12 mm and found that a gap size of 0.102 mm for mice weighing between 24–26 g provided the most consistent results. Unloaded ELN+/− arteries have smaller outer diameters than ELN+/+ arteries, but they are approximately equal in diameter at physiological pressure (7), and we found that the same clip size and body weights were optimal for both groups. With these optimal parameters, we successfully increased the systolic pressure by at least 15 mmHg in about half of the clipped mice for both genotypes. The pressure increase and success rate are similar to previous studies (3, 14, 32).

The pressure increases with clipping are less in ELN+/− than in ELN+/+ mice (Table 1). 2K1C hypertension may be more difficult to induce in ELN+/− mice because they are already operating near maximal renin secretion and pressure output (7). Additional hypertension models that do not rely on increased renin activity, such as one-kidney, one-clip (32) or deoxycortocosterone salt pellets (14), may provide greater increases in blood pressure in ELN+/− mice. However, the significant pressure increases in successfully clipped mice for the current study are sufficient to investigate hypertensive cardiac and cardiovascular remodeling.

The relationship between systolic pressure and total heart weight is similar between genotypes (Fig. 1). Hence, after only 4 wk of hypertension, ELN+/+ mice have similar heart weights to ELN+/− mice that have experienced a lifetime of hypertension. Rats with pressure overload caused by aortic constriction exhibit a gradual transition from compensatory cardiac hypertrophy to heart failure that includes augmented hypertrophy and reduced contractile function (1). About 30% of rats die of cardiac failure 1 mo after aortic constriction, and 60% die by 5 mo (29). One would expect successful 2K1C ELN+/+ and ELN+/− mice to follow a similar disease progression. In future studies, it would be interesting to clip the mice for longer periods and observe the rate and extent of cardiac hypertrophy and eventual cardiac failure in each genotype.

Eight and sixteen percent of the clipped ELN+/+ and ELN+/− mice, respectively, were excluded from the study due to cardiac abnormalities that may indicate the early stages of cardiac failure. At physiological blood pressures, ELN+/− mice may be resistant to cardiac hypertrophy, as well as renin-dependent pressure increases, but if the pressure is successfully increased, they may be more prone to cardiac failure. The high blood pressure, increased heart weight, and smaller blood vessels in ELN+/− mice may be the optimized parameters for the elastin-insufficient cardiovascular system, but the heart may have a limited capacity for additional remodeling to accommodate increased hemodynamic stress.

The average weight for the right (clipped) kidney in ELN+/+ mice is reduced 27% compared with that in sham-operated mice, whereas in ELN+/− mice it is reduced 16% (Table 1). If the ELN+/−-clipped kidney weight was reduced more than this, successful hypertension was not achieved. This indicates either that ELN+/− mice cannot produce as much renin from a small, sickened kidney as ELN+/+ mice or that ELN+/− mice need to increase renin production significantly more than ELN+/+ mice to increase the blood pressure. The second hypothesis is supported by the attenuated blood pressure response to infused ANG II of ELN+/− sham-operated mice compared with ELN+/+ mice (Fig. 2). ELN+/+-clipped mice show a 1% increase in the weight of the left (unclipped) kidney, whereas ELN+/−-clipped mice have a 14% increase. The increased mass of the unclipped kidney may be a remodeling response to the increased work required by the kidney to maintain high renin production and increased blood pressure.

ANG II Response

Previous studies have shown that plasma renin activity is increased approximately twofold in ELN+/− compared with ELN+/+ mice (7) and threefold in clipped wild-type versus sham-operated mice (32). The increased renin activity may lead to increased ANG II concentrations and a saturation of the ANG II receptors. The attenuated pressure response in clipped ELN+/+ and sham-operated ELN+/− mice to infused ANG II supports the essential role of ANG II receptors in maintaining hypertension in both 2K1C (2, 12) and ELN+/− mice (7). The dose-response curves in Fig. 2 show that significantly larger amounts of infused ANG II are required in ELN+/− than in ELN+/+ sham-operated mice to produce equivalent pressure changes and may explain why the in vivo pressure increase with clipping is not as high in ELN+/− as in ELN+/+ mice.

Histology of the Unloaded Descending Aorta

The unloaded internal and external elastic laminae diameters are smaller in sham-operated ELN+/− than in ELN+/+ mice, but there are no significant differences between the adventitial diameters (Table 2). It is difficult to define the adventitial diameter in histology slides, and we observed higher variability in the dimensions measured from histology slides than those measured from arterial rings (as done for the ascending aorta and carotid artery). This may have been due to fixation artifacts or slight changes in the plane of section, so that true cross sections of the arteries were not always captured.

The unloaded medial thickness of the descending aorta increases significantly, and the lamellar number does not change with clipping in both genotypes. The increase in thickness helps to normalize the medial wall stress and may be caused by an addition of extracellular matrix proteins, SMC hypertrophy, or SMC hyperplasia in response to increased blood pressure and increased stress. Previous investigators have observed increases in medial thickness with induced hypertension in rats and noted that individual lamellar units increase in thickness with no change in lamellar number (9, 21, 32). Matsumoto and Hayashi (22) showed larger increases in thickness for the innermost lamellar units in the rat thoracic aorta, whereas Fridez et al. (9) showed larger increases in thickness for the outermost lamellar units in the rat carotid artery after 8 wk of induced hypertension. Nonuniform growth at the innermost or intimal surface of the artery would be consistent with the observed increases in the opening angle in this and in previous studies (8, 22).

Unloaded Dimensions of the Ascending Aorta and Carotid Artery

The unloaded thickness and cross-sectional area of the ascending aorta in both genotypes and the left common carotid artery in ELN+/+ mice significantly increase with clipping (Table 3). The cross-sectional area of the ascending aorta and carotid artery in both genotypes increases linearly with pressure (Fig. 3). The slope of the pressure-area line is similar for each genotype but different between the two arteries. This indicates that ELN+/+ and ELN+/− arteries remodel similarly when pressure is increased in adult animals but that each artery has a different remodeling response. The slope of the pressure-area line for the ascending aorta is about three times that of the carotid artery; therefore, the ascending aorta may be more sensitive to changes in pressure, and the pressure increases may not have been large enough in successfully clipped ELN+/− mice to induce significant remodeling in the carotid artery.

The increases in arterial thickness and cross-sectional area in this study are similar to those measured in rats with induced hypertension (8, 16, 33, 35) and in humans with hypertension (26). In response to increased pressure, both ELN+/+ and ELN+/− ascending aortas are remodeled to increase the thickness and cross-sectional area and, consequently, to normalize the circumferential wall stress. Therefore, the lack of hypertensive remodeling in sham-operated ELN+/− arteries, which experience significantly higher pressure than in sham-operated ELN+/+ arteries, is not due to defects in the remodeling response but may be due to developmental adaptations that redefine the normotensive blood pressure for these mice.

Opening Angle of the Ascending Aorta

The opening angle of the ascending aorta increases with clipping in both genotypes (Table 3). The residual strain characterized by the opening angle serves to normalize the transmural circumferential strain (4) and is likely caused by differential growth in the artery wall (27). An increased opening angle would result from more growth at the intimal than adventitial surface, as observed by Matsumoto and Hayashi (22), in rats with induced hypertension. Increased growth at the intimal surface is consistent with the increase in thickness, but not outer diameter, of most arteries in this study after clipping. Fridez et al. (8) also measured increased opening angles in hypertensive rats.

In Vivo Longitudinal Stretch Ratio of the Carotid Artery

Sham-operated ELN+/− left common carotid arteries have smaller in vivo stretch ratios than sham-operated ELN+/+ arteries (Table 3) (30), supporting the assertion that elastin is the arterial component responsible for the in vivo longitudinal retraction force (5). The in vivo stretch ratio does not change with clipping in either genotype. Fridez et al. (8) also found no significant change in the in vivo length for induced hypertension in rat carotid arteries. Increased pressure increases longitudinal stress (Eq. 4), but changes in the artery diameter and thickness may be enough to normalize the stress without inducing remodeling in the longitudinal direction.

Mechanical Testing of the Carotid Artery

There are no significant differences in the relationships between pressure and outer diameter, longitudinal force, circumferential stretch, circumferential stress, or longitudinal stress with clipping in either genotype (Figs. 4 and 5). Fridez et al. (8, 9) observed a downward shift in the pressure/outer diameter relationship of rat carotid arteries after 8 days of induced hypertension, indicating that the hypertensive arteries were less compliant. Zanchi et al. (35) did not observe any changes in the pressure/inner diameter relationship for rat carotid arteries after 1–24 wk of induced hypertension, but the circumferential stresses were equal at mean physiological blood pressure due to increases in the tissue mass. The increases in unloaded outer diameter and thickness in clipped versus sham-operated arteries should decrease the circumferential and longitudinal stresses at identical pressures by about 10%, but this difference may be masked by interanimal variability and errors in the stress calculations induced by uncertainties in the measured parameters (Eqs. 3 and 4). Hypertension may have to be induced for longer than 4 wk to observe measurable changes in the mechanical behavior of mouse carotid arteries.

The changes in unloaded diameter and thickness help to decrease the physiological circumferential stresses in each clipped group. Pressures, in vivo stretch ratios, and unloaded and loaded dimensions can be used to estimate the effects of the geometric changes on the circumferential stresses. Pressures and unloaded dimensions for the ascending aorta and the left common carotid artery were measured in this study. The in vivo stretch ratio and pressure/diameter relationship for the carotid artery were also measured in this study and did not change with clipping. Assuming that the in vivo stretch ratio and pressure/diameter relationship also do not change with clipping in the ascending aorta, we can use data from Wagenseil et al. (30) to estimate these parameters. We calculated that the circumferential stresses at mean physiological pressure are 40–110 kPa higher in clipped than in sham-operated arteries for both genotypes. For the ascending aorta and the ELN+/+ carotid artery, if the clipped arteries had maintained the sham-operated dimensions, the physiological circumferential stresses would have been 80–150 kPa higher than the sham-operated stresses.

In conclusion, ELN+/+ and ELN+/− mice show similar cardiac and cardiovascular remodeling when blood pressure is increased in adult animals. Therefore, the unique cardiovascular properties (i.e., smaller, thinner arteries with increased lamellar units) observed in ELN+/− sham-operated mice with high blood pressure are not due to defects in the normal remodeling response but are likely due to adaptations signaled by altered hemodynamics and reduced elastin levels during development. These adaptations highlight the remarkable plasticity of the developing SMCs and suggest that hypertension in developmental diseases (such as supravalvular aortic stenosis) may require different treatment approaches than adult diseases (such as essential hypertension).

GRANTS

This study was supported by American Heart Association Fellowship 0525800Z (to J. E. Wagenseil) and National Heart, Lung, and Blood Institute Grants HL-53325 and HL-074138 (to R. P. Mecham).

Acknowledgments

We thank Chris Ciliberto for mouse care and genotyping; Conrado Johns and Ludek Cervenka for helpful suggestions and supplies for the renal clip procedures; Ruth Okamoto for some of the Matlab scripts; and Tom Broekelmann, Adrian Shifren, Hideki Sugitani, and Justin Weinberg for helpful discussions.

Footnotes

  • The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

REFERENCES

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