Reduced Vessel Elasticity Alters Cardiovascular Structure and Function in Newborn Mice

Reduced Elastin Increases LV Pressure and the Absence of Elastin Impairs Cardiovascular Function

Elastic vessels are critical for proper cardiovascular function in a closed, pulsatile circulatory system. To evaluate the effects of reduced vascular elasticity, we measured LV blood pressure and cardiovascular function in P1 mice with 100%, ≈60%, and 0% elastin levels. We used ultrasound visualization to guide a small gauge needle into the ≈1-mm-diameter LV cavity and recorded blood pressure and heart rate with a fluid-filled catheter system (Figure 1). The increased LV pressure in Eln^+/− and Eln^−/− mice is clear, despite the damped the pressure waveform. The calculated systolic pressure is increased 25% in Eln^+/− and 100% in Eln^−/− mice compared to WT and the heart rates are similar in all genotypes. WT pressures are comparable to previous studies in newborn mice.^3,6,14

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Figure 1. Reduced elastin levels correlate with increased LV pressure. Representative LV pressure waveforms (a), average calculated systolic pressure (b), and average heart rates (c) for all genotypes are shown. N=5 (WT and Eln^+/−) and N=3 (Eln^−/−). *P<0.05 between all genotypes.

Cardiovascular function was determined by echocardiography and the measured parameters are shown in Table 1. WT and Eln^+/− values are similar to previous studies of newborn mice.¹⁵ Eln^−/− mice show a trend toward increased LV diameter at diastole (a sign of LV dilatation) and increased LV mass. At systole, the inner diameters of the ascending aorta are almost equal in all 3 genotypes (Figure 2). However, the percentage diameter change between systole and diastole is ≈80% less in Eln^−/− ascending aorta than WT and Eln^+/−, indicating a very stiff, noncompliant artery at physiological pressures. Ejection fraction and cardiac output are ≈35% lower in Eln^−/− than WT and Eln^+/−. Eln^−/− mice also show a trend toward decreased blood flow in the proximal ascending aorta, which is consistent with reduced ejection fraction and cardiac output. The maximum physiological shear stress can be calculated from the cardiac output, the aortic inner diameter at systole and by assuming a constant blood viscosity of 4 cp.³ The calculated shear stresses are 36, 39 and 19 dyn/cm² in WT, Eln^+/−, and Eln^−/− aortae, respectively.

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Figure 2. The reduced compliance of Eln^−/− ascending aorta is illustrated by representative M-mode images from the echocardiographic studies. Note the small change in inner diameter of the Eln^−/− aorta between end-systole (IDs) (mm) and end-diastole (IDd) (mm) and that all genotypes have approximately the same end-systolic diameter.

In the echocardiographic studies, the heart rates of anesthetized mice (388±23 bpm) are significantly lower than unanesthetized mice (441±32 bpm), but the heart rates are not significantly different between genotypes. Eln^−/− mice are easily identifiable by their impaired heart function and tortuous, stiff vessels. Movies of the aorta for each genotype are included in the supplemental data (Movie in the online data supplement). Regions of dilation and stenoses are observed for Eln^−/− mice in the ascending and descending aorta and pulmonary artery, as evidenced by abrupt changes in the Doppler flow profiles (Figure 3).

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Figure 3. Elastin insufficiency alters vascular morphology. The heart (H), ductus arteriosus (D), ascending aorta (AA), right subclavian (RS), right carotid (RC), left carotid (LC), left subclavian (LS), and descending aorta (DA) are labeled in representative morphologic images (a). Eln^+/− arteries have smaller diameters, longer lengths, and a sharp bend at the RS (white arrow). Eln^−/− arteries have stenoses, dilations, and tortuosity (black arrows). The eosin dye did not flow into the Eln^−/− left carotid (LC), but the artery can be identified by blood clots in the stenosed regions (black arrowhead). Scale bars=0.5 mm. Areas 1 and 2 in the Eln^−/− image correspond to regions where the blood flow velocities were measured with Doppler ultrasound and are shown in b. Low flow (1) is present in the dilated region, and high flow (2) is present in the stenosed region (b). Scale bars=20 cm/sec.

The normalized total heart weight in Eln^−/− mice (8.0±1.5 mg/g) is significantly higher than WT (6.9±0.8 mg/g) and Eln^+/− (7.0±1.0 mg/g). LV hypertrophy, as indicated by the trend toward increased LV mass, is likely responsible for the increase in total heart weight. The body weight is not significantly different between genotypes and averages 1.35±0.18 g for all mice.

Low Elastin Levels Correlate With Longer Vessels and Altered Cardiovascular Morphology

Gross morphological analysis of the vascular network in P1 Eln^−/− mice shows longer, tortuous arteries with numerous stenoses and dilations (Figure 3). Variations in the blood flow profile and abrupt changes in arterial diameter are also seen by ultrasound, confirming the presence of stenotic lesions in live animals. In contrast, the basic pattern of Eln^+/− arteries resembles WT with some interesting differences. Eln^+/− arteries have smaller unloaded diameters, longer lengths between branches, and a sharp bend at the right subclavian artery (Figure 3). The average in vivo lengths of WT, Eln^+/−, and Eln^−/− ascending aortae are 1.47±0.08, 1.59±0.08, and 2.38±0.05 mm, respectively, and are significantly different between all genotypes.

Elastin Deficiency Alters the Arrangement of SMCs in the Vessel Wall

The gross morphology shows that elastin deficiency changes the geometry and patterning of developing blood vessels. We used electron microscopy to determine the effects of elastin deficiency on the ultrastructural arrangement of SMCs and extracellular matrix (ECM) in the newborn vessel wall (Figure 4). WT aorta looks similar to previous electron microscopic images of newborn rat and mouse aortae^16,17 and has circumferentially arranged SMCs and elastic laminae that extend from the intima through ≈2/3 of the wall thickness. The laminae are not yet continuous but will progressively thicken and become continuous by P7.² The thickest laminae are the central units, whereas the layers closest to the intima and adventitia are thinner. The adventitia consists of circumferentially oriented cells within an ECM rich in collagen and proteoglycans. The Eln^+/− aorta has thinner but a higher number of elastic laminae (≈11 compared to ≈8 in WT) that occupy a greater percentage of the vessel wall and extend into the adventitia. The Eln^−/− aorta has SMCs that are highly disorganized compared to WT or Eln^+/−. Cells near the adventitial interface are organized circumferentially, but those in the middle of the wall show various orientations, are surrounded by a collagen-rich matrix, and have few cell-cell interactions. SMCs near the intima have less collagen in the ECM and are oriented longitudinally which, together with increased proliferation and enhanced migration, contributes to obliteration of the arterial lumen.¹⁸ The Eln^−/− aorta has significantly higher DNA amounts (58.5±4.5 ng) than Eln^+/+ (48.2±2.4 ng) and Eln^+/− (48.6±3.2 ng) because of the hyperproliferation of SMCs.

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Figure 4. Eln^+/− and Eln^−/− mice have unique vascular wall organization. Representative electron microscopic images taken at ×700 original magnification of the proximal ascending aorta in each genotype are shown. The adventitia is at the top of the images. Note the additional elastic laminae near the adventitial interface in Eln^+/− aorta and disorganized SMCs near the intimal interface in Eln^−/− aorta (black arrows). Scale bars=11 μm.

Mechanical Differences in Vessels With Differing Elastin Levels

The mechanical properties of the vessel wall, combined with the applied in vivo pressures and forces, produce stresses and strains on the wall that may affect the developmental SMC phenotype. Growth during development can also produce residual stresses and strains in the vessel wall. To determine the mechanical properties and applied stresses and strains in elastin insufficient vessels, we measured the unloaded aortic geometry and OA and performed ex vivo mechanical tests. The unloaded outer diameters are not significantly different between genotypes (Table 2). The unloaded inner diameter is 20% larger and the unloaded thickness is 20% smaller in WT aorta compared to Eln^+/− and Eln^−/−. The circumferential residual strain, characterized by the OA, is 35% smaller in Eln^−/− aorta compared to WT and Eln^+/−.

At all pressures >5 mm Hg, the outer diameter of Eln^−/− aorta is significantly smaller than WT and Eln^+/− (Figure 5). Yet at the systolic pressure for each genotype (Figure 1), the diameters are approximately the same (0.51 to 0.55 mm), as confirmed by the echocardiographic studies (Table 1 and Figure 2). The small diameter change with pressure for Eln^−/− aorta is consistent with the echocardiography data and accounts for the low compliance values. At all pressures <25 mm Hg, Eln^−/− compliance is significantly lower than WT and/or Eln^+/−. The slope of the Eln^+/− pressure–diameter curve is slightly different than WT, which provides altered compliance at some pressures. The axial force data are not shown because at the in vivo length the force remains approximately constant with increasing pressure and the forces are not significantly different between genotypes.

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Figure 5. Eln^−/− mice have reduced aortic diameter and compliance in vitro. Average outer diameter (OD) (a) and average compliance (C) (b) vs pressure. N=10 (WT), N=12 (Eln^+/−), and N=6 (Eln^−/−). *P<0.05 for Eln^−/− compared to WT and Eln^+/−; ‡P<0.05 between WT and Eln^−/−; &P<0.05 between Eln^+/− and Eln^−/−; $P<0.05 between WT and Eln^+/−; #P<0.05 for Eln^+/− compared to WT and Eln^−/−.

The circumferential stress is significantly lower in Eln^−/− aorta compared to WT and Eln^+/− at all pressures (Figure 6). The axial stress is not significantly different between genotypes at any pressure. At all pressures >5 mm Hg, the circumferential stretch is greater in Eln^+/− aorta than WT and Eln^−/−. At the systolic pressures for each genotype (Figure 1), the circumferential stresses are almost identical in all 3 genotypes, the axial stresses are approximately double in Eln^−/− aorta compared to WT and Eln^+/−, and the circumferential stretch ratio is ≈10% higher in Eln^+/− aorta compared to WT and Eln^−/−.

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Figure 6. Eln^−/− aorta has altered circumferential stresses, and Eln^+/− aorta has altered circumferential stretch ratios. Average circumferential stress (a), axial stress (b), and circumferential stretch ratio (c) vs pressure are shown. N=10 (WT), N=12 (Eln^+/−), and N=6 (Eln^−/−). @P<0.05 between all genotypes; *P<0.05 for Eln^−/− compared to WT and Eln^+/−; #P<0.05 for Eln^+/− compared to WT and Eln^−/−.

Cardiovascular Properties of Newborn Mice With no Elastin (Eln^−/−)

Because Eln^−/− mice are viable throughout fetal development when many of the critical cardiovascular structural and hemodynamic changes are occurring, they provide unique insight into how a closed circulatory system develops when vertebrate vessels cannot provide the elastic recoil required for normal heart function. Newborn Eln^−/− mice have tortuous, stenotic arteries that show little diameter change between systole and diastole. Without elastic arteries, LV pressure increases in an attempt to maintain cardiac output and perfusion pressure through the smaller, stiffer Eln^−/− aorta. The high blood pressure and increased afterload lead to cardiac hypertrophy and impaired cardiac function.

Although hypertrophic cardiac remodeling is expected with high blood pressure and noncompliant vessels, it is remarkable that cardiac development proceeds to birth with arteries as stiff and tortuous as those in Eln^−/− mice. Based on morphological comparison, however, most of the adverse changes in vessel wall structure occur during the last few days of development. At embryonic day (E)15.5, the aortae in WT and Eln^−/− mice look similar by histology and both have ≈8 circumferential cell layers.¹⁸ There is little elastin or collagen in the wall at this stage, so the stresses are borne mostly by the cells. Because wall structure is similar, hemodynamic forces are most likely similar in the 2 genotypes. There is a dramatic increase in elastin and collagen production beginning around E15,^21,22 which suggests that the wall is strengthening in response to increased hemodynamic stimuli. When elastin cannot be produced at appropriate levels to accommodate increased wall stress, as in the Eln^−/− aorta, SMCs proliferate to lower the wall stress by increasing the wall thickness. As a result, at P1, the physiological circumferential stress in the thicker Eln^−/− aorta is approximately equal to WT, despite the almost 2-fold higher LV pressure in Eln^−/− mice. Eln^−/− SMCs proliferate at the inner wall of the aorta, increasing the thickness as well decreasing the inner diameter, which partially normalizes the reduced shear stress caused by decreased cardiac output. For reasons that are not yet understood, SMC proliferation continues in the Eln^−/− aorta until the lumen becomes occluded and the animal dies.

The similarity in structure between WT and Eln^−/− aorta at E15.5 shows that elastin is not necessary for the recruitment of SMCs to the developing aortic wall nor is it required for their initial circumferential organization. It may, however, play an important role in maintaining the layered architecture by providing a direct signal to SMCs^23,24 or, in its polymerized form, creating a physical barrier and/or landmark that serves to spatially compartmentalize SMCs. By P1, the elastic laminae are almost continuous in WT aorta and each SMC is in contact with the elastin above and below it in the wall.¹⁷ The loss of this physical restraint in vessels without elastin may contribute to the changes in SMC organization and phenotype observed in Eln^−/− mice. In vitro, mechanical strain induces proliferation in SMCs plated on collagen, fibronectin, and vitronectin, but not on elastin or laminin, suggesting that ECM-specific signals and mechanical strain and/or stress work coordinately to alter SMC phenotype.²⁵

Newborn Mice With Reduced Elastin Levels (Eln^+/−)

In contrast to the altered cardiac function in Eln^−/− mice, functional parameters in newborn Eln^+/− mice are comparable to WT values. The heart shows normal ejection fraction and cardiac output, despite longer arteries with smaller unloaded diameters. As adults, Eln^+/− mice have increased stroke volume and cardiac output,⁹ suggesting that the Eln^+/− cardiovascular system continues to remodel after birth. The outer diameters of newborn Eln^+/− and WT aorta are not significantly different at any pressure, but the outer diameter of adult Eln^+/− aorta is significantly smaller than WT at most pressures,^9,11 again suggesting additional changes after birth. Although newborn Eln^+/− mice have higher LV pressures than WT, the pressure increase is lower than what is seen in Eln^−/− mice. The 25% pressure difference between Eln^+/− and WT mice at P1 is similar to the pressure difference in adult mice^9,11; therefore this major hemodynamic parameter is established in Eln^+/− mice before birth and persists into adulthood. The pressure increase at birth, combined with both pre- and postnatal remodeling, allows Eln^+/− mice to develop unique cardiovascular structure and hemodynamic parameters that are “normal” for this genotype. At this new cardiovascular set point, adult Eln^+/− mice are able to maintain cardiac and cardiovascular function despite significantly smaller arteries and a lifetime of increased blood pressure. This is accomplished without the pathological vessel wall remodeling observed in adult onset hypertension. The initial conditions that determine this cardiovascular set point also allow for a modified aging process in Eln^+/− arteries,²⁶ showing that developmental modifications have consequences throughout the lifespan of the animal.

One of the unique features of adult Eln^+/− arteries is an increased number of SMC layers and elastic laminae in the vessel wall.¹⁰ In this study, we show that these layers are present at birth and appear in the adventitia. We have speculated that in elastin insufficiency, SMCs cannot make enough elastin to accommodate circumferential forces, and the increased number of lamellar units is a developmental adaptation to normalize wall tension.⁹ The elevated blood pressure in newborn Eln^+/− mice supports our hypothesis; higher pressure means higher wall tension, but the addition of 3 lamellar units to the wall brings tension per lamellar unit back to within 10% of WT values.

The extra SMC layers in the Eln^+/− aorta are associated with a decrease in adventitial area, suggesting that adventitial cells give rise to these structures. Numerous studies show that the adventitia is important in sensing wall stress and that adventitial cells can be activated in response to hypertension or other forms of vascular injury.²⁷ During development, Eln^+/− vessels experience more circumferential stretch than WT with each cardiac cycle because of their increased pressure and smaller inner diameters. The increased stretch could trigger adventitial cells to differentiate into SMCs that produce extra layers of elastin. Recent findings show that the adventitia contains a progenitor cell population capable of differentiating into SMCs that repopulate the wall.^28,29 These cells appear in the adventitia in late embryonic and early postnatal development,²⁹ at the same time that large hemodynamic and structural changes are occurring in the vessel wall.

Axial Stress, Tortuosity, and Residual Strain

Axial stress in arteries is often neglected, but there is increasing evidence that arteries remodel to normalize axial tension and the resulting stresses.^30,31 Arteries can grow in response to increased axial tension but cannot shorten and will actually grow and become tortuous with decreased axial tension.³² Tension is regulated by the amount of axial stretch applied to each vessel in vivo. The estimated in vivo stretch ratio of all P1 aortae in this study is 1.01. This is lower than the stretch ratio in adult WT and Eln^+/− aortae,¹¹ but the stretch ratio is known to increase with postnatal development in mice.³ Gross morphological analysis found that Eln^−/− arteries have increased length and are tortuous, implying that they grew in response to decreased axial tension at some point during development. Similar, but less extreme, vascular abnormalities occur in Eln^+/− arteries and other mouse models where elastic fiber structure or assembly is perturbed through gene inactivation.^11,33–35 Removing elastin by treatment with elastase eliminates the axial traction force, whereas treatment with collagenase has no effect.³⁶ Therefore, it is possible that arteries lacking elastin cannot sustain the required axial tension necessary for normal, nontortuous development.

Arteries experience circumferential residual strain that may be caused by differential growth at the inner and outer wall and serves to normalize the transmural strains.^37,38 The OA in adult Eln^+/− aorta is larger than WT, but the intramural strain distributions are almost identical at physiological pressures.¹¹ The OA in P1 Eln^+/− aorta is not significantly different from WT, but OA may be another parameter that changes in postnatal remodeling. The P1 Eln^−/− aorta, in contrast, has a significantly smaller OA than WT and Eln^+/− aortae. This is contrary to the increased OA in normal adult vessels with increased pressure and with increased SMC growth at the intimal surface.^12,39,40 The unexpected decrease in Eln^−/− OA shows that ECM is an important component in growth models for residual strain and that developmental adaptations should be treated differently than adult remodeling.

The average OA and geometry were used to calculate the transmural strain distribution for each genotype. WT and Eln^+/− aortae have 5 to 9% higher strains at the outer wall compared to the inner wall, whereas Eln^−/− aorta has 6% higher strains at the inner wall compared to the outer wall. The increased strain and consequently increased stress at the inner wall may contribute to the medial hyperplasia in the Eln^−/− aorta.

Stenotic Lesions and Elastin Levels

The aortic stenoses in Eln^−/− mice arise from medial, not intimal hyperplasia, which is similar to the pathology seen in stenotic lesions in humans with supravalvular aortic stenosis (SVAS) (Online Mendelian Inheritance of Man no. 185500). SVAS is caused by loss of function mutations that lead to functional elastin haploinsufficiency.⁴¹ Humans and mice respond to elastin haploinsufficiency differently: humans develop focal stenotic lesions in the aorta and other great vessels, whereas there is a general narrowing of arterial vessels without focal lesions in mice. Stenoses have been observed, however, in adult hBAC-mNULL mice with elastin levels ≈30% of normal.⁴² Adult hBAC-mNULL mice have mean pressures ≈50 mm Hg higher than WT, whereas adult Eln^+/− mice have mean pressures ≈40 mm Hg higher.¹¹ There may be a threshold combination of decreased elastin and increased blood pressure that determines whether the developmental vessel wall remodeling is advantageous or pathological, and the threshold may be different in humans and mice. Characterizing this delicate balance between elastin levels, mechanical stimuli, SMC phenotype, and aortic wall structure will be important in developing strategies for treating and preventing stenotic lesions in SVAS.

Conclusion

Our results show that vascular elasticity is essential for vessel wall development and cardiovascular function in vertebrates. Without elastic vessels to provide an elastic reservoir, high blood pressure and stiff, stenotic arteries cause impaired LV function in newborn Eln^−/− mice. The high blood pressure and stiff vessels also lead to SMC proliferation and vessel lumen narrowing as the cells attempt to normalize wall stress by increasing wall thickness. In the absence of elastin to provide an axial traction force, the arteries become longer and tortuous, which only increases the afterload on the heart. Thus, without elastin, a closed, pulsatile, high pressure circulatory system cannot operate. When elastin content is reduced to ≈60% in mice, however, the developing cardiovascular system can adapt to produce normal cardiac function despite significant changes in the vessel wall structure, mechanics, and hemodynamics. The major adaptations occur in late fetal development and persist into adulthood, defining a new cardiovascular set point for these mice. This functional adaptation can only occur when the ECM is being formed and appropriate gene sets are being expressed.