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1Division of Pediatric Cardiology, Children's Hospital of Pittsburgh, Pittsburgh, Pennsylvania; 2Cell Biology and Anatomy and the Cardiovascular Developmental Biology Center, Medical University of South Carolina, Charleston, South Carolina; 3Magee-Womens Research Institute, Pittsburgh, Pennsylvania
Submitted 6 October 2005 ; accepted in final form 11 April 2006
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ABSTRACT |
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-actin content was similar between groups, more cell layers expressed smooth muscle -actin, and myocyte cell shape was markedly rounder in VAL embryos. Additionally, aortic and perivascular collagen type I and III content significantly increased in load-exposed VAL vessels. Increased production of these proteins is consistent with the observed increase in aortic PWV and decreased strain in VAL passive aortic segments. Thus the embryonic dorsal aorta is sensitive to increased arterial load and adapts by altering its material properties via changes in collagen content.
arterial mechanics; pulse-wave velocity; chick; collagen
The embryonic dorsal aorta has unique architectural features but has a functional role similar to the mature circulation. In contrast to the mature aorta, the tunica media of the dorsal aorta during midembryogenesis consists of only a few layers of proliferating and differentiating cells expressing vascular smooth muscle cell (VSMC) markers (18, 20, 28), including smooth muscle -actin (SMA). These cells, as well as the surrounding mesenchymal cells, produce ECM proteins (1, 17, 18, 27, 28), although some proteins that influence adult arterial mechanical properties, such as elastin, are not synthesized until later developmental stages (1, 27). Nevertheless, the contours of pressure waveforms recorded in the early embryonic chick dorsal aorta are strikingly similar to adult mammalian aortic waveforms (6, 16).
Similar to the adult system, embryonic cardiovascular tissues remodel in response to changes in loading conditions. Increased cardiac load in the chick embryo triggers an increase in ventricular function (19) and leads to compensatory changes in ventricular structure (7, 30, 31) and material properties (24). When compared with the heart, the impact of altered load on embryonic arterial remodeling has received much less attention, even though it is likely a significant factor in normal and pathological arterial development. Alterations in aortic compliance during key stages of embryogenesis may not only impact arterial maturation but may also play a significant role in the development of cardiac structural and functional abnormalities and anomalies associated with compromised microcirculation.
Lucitti et al. (23) previously showed that within 1 h of increasing arterial load in Hamburger-Hamilton (12) stage (HH) 21 chick embryos, arterial impedance parameters increase and compliance decreases (23). These parameters, including characteristic impedance, remain altered at HH24 and HH27. Characteristic impedance is an index of arterial resistance at the site of measurement, and when measured in the dorsal aorta, it yields information about aortic stiffness. We therefore concluded that sustained increases in arterial load during development trigger an increase in dorsal aortic, as well as global arterial stiffness. However, because of the invasive nature of micropuncture and the limited number of high-frequency moduli available to calculate characteristic impedance using the micropressure servonull system (34, 35), independent measures of aortic properties are needed to corroborate these data.
In the current study, we tested the hypothesis that a chronic increase in arterial load leads to an increase in dorsal aortic stiffness during embryogenesis. We measured pulse-wave velocity (PWV), a noninvasive index of local arterial properties, to determine the impact of increased load on aortic stiffness in vivo and to compare with our previous in vivo index of aortic stiffness, characteristic impedance. To assess changes in material properties after a prolonged exposure to increased arterial load, we tested the properties of isolated aortic segments in vitro. Finally, we examined the impact of increased load on the aortic morphology and the expression of three proteins known to influence vascular material properties: SMA, procollagen I, and collagen III. Understanding how the embryonic dorsal aorta responds to hemodynamic forces is critical to identifying potential origins of cardiovascular disease in children and adults.
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METHODS |
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Fertilized chicken (Gallus gallus) eggs were obtained from Utah State University (Logan, UT) and incubated blunt-side up at 37–38°C and at a relative humidity of 
65%. After 3.5 days (HH21), each egg was removed to a temperature-controlled bench environment. Each embryo underwent either vitelline artery ligation (VAL) to chronically increase proximal dorsal aortic mechanical load or sham treatment (CON). For VAL, 10-0 monofilament was used to permanently occlude the right lateral vitelline artery, and CON embryos were treated as previously described (23).
PWV
PWV was measured after 1 h of postintervention recovery (HH21), at HH24 (14 h), or at HH27 (30 h). We used a dual-channel 20-MHz pulsed Doppler velocimeter (model 202, Triton Technology, San Diego, CA) that is linear up to 100 mm/s for steady (y = 0.95 x +1.4, r2 = 0.990) and pulsatile (y = 0.92 x +8.2, r2 = 0.915 at peak) flow (33). Two 0.5-mm custom probes (Iowa Doppler Products, Iowa City, IA) measured simultaneous arterial blood flow velocity at two locations along the dorsal aorta (Fig. 1). The probes were mounted on micromanipulators, and the probe angle was measured with a protractor for signal correction. The probe angle and the pulse distance were kept constant for all embryos at each stage. The phasic velocity output was digitally sampled at 2,500 samples/s using a customized LabVIEW (National Instruments, Austin, TX) environment. The arterial segment between the probes was imaged using a video camera (model 70, Dage-MTI, Michigan City, IN) mounted on a dissecting microscope.
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t, where d is the distance between measurement sites and t is wave-front transit time. We measured the imaged distance between the two probes using image analysis software (Scion Image, Scion, Frederick, MD) calibrated to an etched glass standard. To determine the actual sampling site on the image, we calibrated the range setting by creating a flow phantom. Polyethylene tubing was connected to a programmable microsyringe pump (Micro4, WPI, Sarasota, FL) and filled with day 17 chick embryo blood diluted in a nonheparinized PBS solution to a 20% hematocrit. Erythrocyte clotting was not evident. At preset range settings, we measured the distance between the probe and the polyethylene tubing at both the onset of an output signal and maximum signal, which coincided with the lumen of the tubing. Using the calibrated range distances, the measured probe angles, and the measured interprobe distance, we calculated the actual distance (d) between the two Doppler sampling sites within the dorsal aorta (Fig. 2).
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Isolated Vessels
At HH27, embryos were removed from the egg and pinned to a silicone-coated Petri dish. The dorsal aorta was carefully dissected from the surrounding tissue using microsurgical tools. The isolated aorta was transferred to an isobaric arteriograph (Living Systems Instrumentation, Burlington, VT), flushed to remove residual blood, and secured at both ends with the dorsal aspect at the right side of the cannulae. The suspended vessels were bathed in a 65:35 HEPES-Roswell Park Memorial Institute Medium 1640 (Invitrogen, Carlsbad, CA) solution maintained at pH 7.4 and 37°C. The arteriograph monitors intraluminal pressure with a solid-state pressure transducer and maintains pressure via a servo-controller and a peristaltic pump. The operator adjusts pressure via a vernier scale dial. The dial was calibrated against a water manometer and was used to set intraluminal pressure. The arteriograph was then placed on an inverted microscope stage, and vessel diameter was determined by using a mounted camera and an integrated electronic dimension analyzing system (Living Systems Instrumentation, Burlington, VT). Vessels were briefly prestressed by inflation to 3 mmHg, and vessels exhibiting signs of wall tears (obtained during dissection) were discarded.
Active and Passive Tone
After a 15-min equilibration period at 1 mmHg, inner and outer vessel diameter and wall thickness were recorded. Pressure was increased to 2 mmHg, and measurements were recorded after a 4-min equilibration period. The protocol was repeated at 3 mmHg. Intraluminal pressure was then reset to 1 mmHg. After we replaced the bath with a calcium-free HEPES solution containing 1 x 10–4 M papaverine and 1 x 10–4 M EGTA and allowed a 10-min equilibration period, passive arterial properties were determined by repeated step pressure increases from 1 to 3 mmHg. Midwall stress was calculated as 8P(ro·ri)2/[(ro2 – ri2)(ro + ri)2], where P is lumen pressure, ro is the outer radius, and ri is the inner radius. Midwall strain was calculated as [(ro + ri)/2] – [(r1o + r1i)/2]/[(r1o + r1i)/2], where r1o is the outer radius at 1 mmHg (unstressed radius) and r1i is the inner radius at 1 mmHg. Data from vessels unable to main intraluminal pressure at any point in the protocol were discarded. Seven vessels did not complete the protocol due to the presence of small tears in the wall or suture area that were not evident during the initial inflation or were accidentally damaged during bath changes.
Frozen Sections and Immunohistochemistry
At HH27, a second-order vitelline artery was incised, and embryos were infused with 10 µl of 4% paraformaldehyde (PFA) in PBS via an opposite vitelline vein. Embryos were harvested with vessels in a noninflated state, immersed in 4% PFA in PBS overnight, then in PBS for 8 h, and then in a 15% glucose-PBS solution overnight. Embryos were submerged in a warmed solution of distilled water, gelatin (15%), and sucrose (7.5%). After being cooled, gelatin blocks were carved to optimize aortic orientation during slicing and snap frozen in liquid nitrogen-cooled methylbutane. Samples were stored at –80°C. Serial 20- to 30-µm cryostat sections were made, and slides containing slices made through the cranial aspects of the lung buds and crop were identified. Selected slides were rehydrated with PBS, incubated in blocking solution (5% goat serum in PBS) for 1 h, and incubated overnight in blocking solution containing antibodies to SMA (1:300, Clone 1A4, Sigma, St. Louis, MO) or collagen III (1:300, Developmental Studies Hybridoma Bank, Iowa City, IA) overnight at 4°C. Rinsed sections were then exposed to the appropriate secondary antibody (AlexaFluor 594, Sigma), phalloidin 488 (1:200, Sigma) and Draq5 (1:1,000, Biostatus, Leicestershire, UK) for 1 h at room temperature, rinsed, and then sealed. Antibody, F-actin (phalloidin) and nuclear (Draq5) marker concentrations, as well as controls for nonspecific staining and autofluorescence, were determined through a series of preliminary experiments (not shown). Sections were imaged with an Olympus FluoView 500 using predetermined and fixed settings to allow for comparison between embryos and groups. Confocal stacks were processed with Imaris (Bitplane, St. Paul, MN) image analysis software packages and rendered with Adobe Photoshop (Adobe Systems, San Jose, CA). All frozen sections were processed at the University of Pittsburgh.
Image Quantification
For fluorescence quantification of confocal images, the lateral dorsal aortic wall was manually identified, and its perimeter was traced using Metamorph image analysis software (Molecular Devices, Sunnyvale, CA). An area of equal width was traced in the perivascular mesenchyme. Images were then separated by laser line. A fixed predetermined signal threshold level was applied to the image, and pixels above this level were considered positive for fluorescence signal. The percentage of positive pixels was calculated for the pixels defined within the wall and perivascular regions. Both the right and left lateral walls were analyzed and averaged to produce one value per embryo. To determine the number of cell layers expressing SMA, three identical positions (with respect to the dorsal-ventral axis) were identified for the right and left lateral walls. The number of cell layers expressing any SMA was counted and averaged to yield one value per embryo. All imaging and processing were performed at the Center for Biologic Imaging, University of Pittsburgh Medical Center.
Paraffin Sections
PFA-fixed, stage-matched embryos were processed for paraffin sectioning at 3 µm. Selected wax sections were cleared in Histoclear II (National Diagnostics, Atlanta, GA) and stained with hematoxylin and eosin, dehydrated through graded ethanol baths, cleared in xylenes, and mounted in Permount (Sigma-Aldrich). Images of each slide were captured, and dorsal aortic area and perimeter were determined using Metamorph. Three values for each parameter were calculated and then averaged to yield one value per embryo. The absence of elastin was confirmed with Verhoeff/Vangieson stain.
Immunolabeling for procollagen type I was performed using antibodies SP1.D8 and M-38 (Developmental Studies Hybridoma Bank). Sections were cleared as above, permeabilized in 0.5% Tween-20 (Roche Applied Science, Indianapolis, IN) in PBS, and blocked in 3% bovine serum albumen in PBS. Sections were labeled with hybridoma supernatant diluted 1:1 in PBS containing 0.05% Tween-20. Primary antibodies were immunolocalized with Cy3-conjugated donkey anti-mouse IgG secondary antibodies (Jackson Immunochemicals, West Grove, PA). Nuclei were labeled with Hoechst 33342 (Molecular Probes, Eugene, OR), and the slides were mounted in Gel-Mount (Biomeda, Foster City, CA). Micrographs were generated on a Leica DMRB HC microscope equipped with a Spot LCD camera supported by a PC workstation. Images were processed using Adobe Photoshop 7.0. All paraffin processing was performed at the Medical University of South Carolina.
Statistics
Data are reported as means ± SE. For PWV and histological experiments, group comparison for each stage was performed by using independent t-tests. For isolated vessel experiments, groups were compared at each pressure using repeated-measures ANOVA. Statistical significance was determined at a level of 5% alpha error between groups for a single measure (P < 0.05).
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RESULTS |
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PWV
Dorsal aortic diameter in vivo did not change immediately or 1 h after ligation or sham procedure (P = 0.50) (data not shown). One hour after ligation or sham procedure (HH21), PWV increased in VAL embryos (P < 0.01) (Fig. 3). Time-averaged velocity (TAV) and both peak proximal and distal blood velocities decreased in VAL embryos (Table 1) (P < 0.01, P < 0.01, and P < 0.01, respectively). Ejection time was shorter in VAL embryos (P < 0.01), yet heart rate was similar between the groups (P = 0.20) (Table 1). At both HH24 and HH27, PWV remained elevated in VAL embryos (P < 0.01 and P = 0.03, respectively). TAV was similar between groups at HH24 (P = 0.77) but was lower in VAL embryos at HH27 (P = 0.04).
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Stress. Arteries from VAL (n = 11) embryos experienced a higher wall stress when inflated at 2 mmHg (12.9 ± 1.5 vs. 8.19 ± 0.9 mmHg) for CON (n = 22) (P < 0.01) and 3 mmHg (28.5 ± 3.0 vs. 15.3 ± 1.20 mmHg for CON) (P < 0.01) in physiological buffer. At 2 mmHg, the outer radius was larger in VAL embryos (258.6 ± 13.0 vs. 210.5 ± 9.1 µm for CON, P < 0.01), and wall thickness was smaller (38.6 ± 2.8 vs. 50.5 ± 3.5 µm for CON, P = 0.04). At 3 mmHg, the outer radius remained larger in VAL vessels (293.6 ± 16.0 vs. 234 ± 10.7 µm for CON, P < 0.01), and wall thickness remained smaller (31.1 ± 2.9 vs. 43.1 ± 2.2 µm for CON). After exposure to the calcium-free, passive buffer, wall stress was similar between groups in vessels at both 2 mmHg (22.8 ± 2.6 vs. 18.0 ± 3.1 mmHg, P = 0.25) and 3 mmHg (49.2 ± 9.2 vs. 33.9 ± 3.0 mmHg, P = 0.12) (Fig. 4). The outer wall radius was similar between groups at 2 mmHg (340.7 ± 10.9 for VAL and 311.6 ± 9.3 µm for CON, P = 0.19) and 3 mmHg (352.7 ± 22.6 for VAL vs. 349.9 ± 6.3 µm for CON, P = 0.90). Wall thickness was also similar between groups at 2 mmHg (32.0 ± 4.8 for VAL and 44.9 ± 7.7 µm for CON, P = 0.19) and 3 mmHg (26.2 ± 3.9 for VAL and 32.4 ± 3.7 µm for CON, P = 0.27).
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Histology
Dorsal aortic area and perimeter values were similar between CON (57.7 ± 15 mm2, n = 6) and VAL (50.0 ± 8.6 mm2, n = 12) (P = 0.66) embryos. Despite this variation, cells comprising the aortic wall and their nuclei were round and wide, relative to the elongated and flattened cells of CON walls. This was evident in both paraffin and frozen sections (Fig. 5).
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The dorsal aortic wall in both CON (n = 8) and VAL (n = 8) embryos expressed SMA, although expression appeared to be more widespread in VAL embryos (Fig. 5). A higher number of cell layers expressed SMA in VAL embryos (2.6 ± 0.1) compared with CON embryos (1.8 ± 0.2) (P < 0.01), although the proportion of SMA-positive area to the total wall area was similar between groups (P = 0.50) (Table 2). Similarly, although collagen III fibers encircled both VAL (n = 7) and CON (n = 10) aortas, expression was higher in both the VAL aortic wall (2.9 ± 0.4% of total area) and adjacent mesenchyme (3.2 ± 0.6%) than the CON wall (0.9 ± 0.4%) or mesenchymal area (1.1 ± 0.2%) (P = 0.02 and P = 0.01, respectively, Table 2). We were unable to obtain consistent staining using collagen III antibodies in paraffin sections for comparison with frozen sections. In paraffin sections, both antibodies to procollagen type I showed that the protein was expressed at greater levels in VAL (n = 6) embryos than in CON (n = 6) embryos (Fig. 5). We were unable to obtain consistent staining for procollagen type I in frozen tissues.
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DISCUSSION |
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Wave velocity is modulated by factors including mean circulatory filling pressure, arterial tone, wall morphology, and matrix characteristics (25) and can be expressed by the Bramwell-Hill equation: PWV = [(dP·V)/(dV·
)], where P is pressure, V is volume, and is fluid density. In the present study, we noted an increase in PWV 1 h after VAL. However, VAL did not increase aortic outer diameter immediately or 1 h after ligation compared with control embryos (data not shown), suggesting that an increase in aortic volume does not play a significant role in the observed increase in PWV. Because VAL removes a portion of total arterial volume without affecting venous return, we suspect that blood was quickly shunted from the large conduit arteries to the relatively compliant extra-embryonic arterioles and the venous system. However, we were not able to visualize internal diameter in vivo, and it is possible that aortic volume and wall compression (influencing wall stress) increased enough to enhance PWV. Alternatively, the acute increase in PWV observed at HH21 may have resulted, in part, from a change in arterial tone caused by a local vasoconstrictor or a myogenic response. The tunica media is reportedly only one-cell thick in HH21 chick embryos (1), and VSMCs at this stage exhibit an immature phenotype, and its ability to respond to acutely increased luminal pressure is not known.
A sizable literature describes how forces associated with chronically altered arterial loads change the structural and mechanical properties of vessels (4, 9–11, 13–15, 21, 22, 26), including increasing collagen production (5, 29) and smooth muscle cell proliferation (2, 3). These adaptations enhance vascular stiffening and increased wave propagation. In the present study, we observed a sustained increase in PWV in VAL embryos through HH27. We suspect that chronic exposure to increased arterial load triggers load-induced remodeling of the dorsal aortic wall in a similar manner to that observed in adult tissues and in vitro preparations. The differential contribution of vascular myocytes (imparting "active" arterial properties) and extracellular matrix proteins (imparting "passive" arterial properties) to aortic mechanics after arterial loading cannot be ascertained by using PWV but can be assessed by investigating the properties of isolated aortic segments.
The mechanics of mature arterial walls depend in a large part on the activity of VSMCs and the ECM proteins elastin and collagen (25). Intact mature arterial segments oppose increases in intraluminal pressure via the myogenic response. Step increases in intraluminal pressure trigger VSMC constriction and result in an increase in vessel stiffness without a significant change in lumen diameter or wall thickness. When calcium is functionally removed from vascular myocytes and a myogenic response cannot occur, arterial mechanics depend on the relative content and spatial orientation of elastin and collagen fibers (32). Elastin imparts elasticity to the arterial wall and is responsible for the linear relationship between stress and strain at low vessel distention levels. Collagen has a very high elastic modulus and is responsible for the increasingly steep slope of the stress-strain curve as distension increases. The relative proportion of these proteins influences the elastic modulus of the vessel over a range of intraluminal pressures.
The mechanics of the embryonic aorta are largely unknown. Cells adjacent to aortic endothelium exhibit the presumptive VSMC marker SMA as early as HH17 (day 3 of a 21-day incubation period) in chick embryos (18, 20, 27). However, markers associated with increased specification to the mature phenotype, such as 1E12, calponin, and smooth muscle myosin (8, 20) are not apparent until later stages. The functional properties of vascular myocytes under various hemodynamic conditions at various stages of maturation are not yet defined. In regard to passive mechanics, elastogenesis does not occur in the dorsal aorta until approximately HH29 (day 6 or 7), and elastin was not observed in HH27 embryos used in this study (data not shown). Therefore, passive mechanics at HH27 may depend primarily on the organization and orientation of collagen.
Our histological findings demonstrate a robust presence of SMA-positive cells in the HH27 dorsal aorta. In CON vessels, cells tended to be flattened and organized into definable layers, whereas in VAL vessels, they appear rounder and more loosely organized, consistent with the attributes of less mature cells (38). Although more wall layers expressed SMA in VAL embryos, the intracellular organization of these fibers and functional maturity of the cells expressing them was not determined. It is unlikely that these immature cells can initiate a robust myogenic response after acutely increased intraluminal pressure. This speculation is evidenced by the observed increase in stress and strain as pressure changed from 2 to 3 mmHg in isolated arterial segments. However, after the removal of available calcium, stress and strain increased from baseline values, indicating that VSMCs at this stage do exhibit significant tone. Unfortunately, the low range of operating arterial pressures and vessel fragility hindered the generation of stress-strain curves, the "gold standard" for evaluating passive arterial mechanics.
In physiological buffer, VAL vessels displayed a higher stress at a given pressure than CON vessels. This was because wall thickness was smaller and vessel outer diameter was larger at both 2 and 3 mmHg, which is in direct opposition to the measurements taken from histological sections. The observed increased amount of collagen type III in the VAL perivascular mesenchyme may provide critical opposition to distention in vivo that is absent when the vessel is removed to an in vitro environment. Furthermore, the rounded VAL VSMCs may compress and flatten without external support, allowing for an increase in wall stress without a concomitant increase in strain. Interestingly, when calcium was removed from the buffer, aortic wall thickness and diameter became similar to CON vessels. It is tempting to propose that the relatively mature phenotype of the CON VSMCs indicates the onset of a myogenic response and that these cells are able to resist changes in wall thickness and diameter in the face of increasing pressure more efficiently than the rounder, less mature VAL smooth muscle cells.
After calcium removal, stress was similar between the groups, although strain was larger in CON vessels, indicating that they were more distensible than VAL vessels. Collagen types I and III are rigid structural proteins. An overabundance of these proteins in the dorsal aortic wall as observed in this study would likely limit distensibility (strain) in the absence of smooth muscle tone and contribute to a vessel with increased stiffness.
It is worth noting that unlike ex vivo studies, manipulation of one parameter in an intact system generally causes compensatory changes in another parameter in order for the system to regulate. We previously showed that chick embryos display a capacity to regulate arterial resistance and flow to maintain pressure. In that study, embryos chronically reduced aortic flow to compensate for the increased load after VAL. In this study, TAV was also reduced in VAL embryos at HH21 and HH27. It has been shown that decreased flow and shear stress causes medial hyperplasia and a general decrease in vessel and lumen diameter (9, 10). This morphological trend was observed in our histological sections. We therefore cannot rule out that our results are due to a combination of increased arterial load and decreased flow or shear stress. Nevertheless, it is evident that the hemodynamic milieu has a significant impact on arterial mechanics and remodeling during embryogenesis. Because increased aortic stiffness can increase ventricular afterload and impede vital blood flow to developing embryonic organs and extra-embryonic nutritional sources, factors that impact the structure and function of the central arteries may play an important role in the development of cardiac and microcirculatory abnormalities.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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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.
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) showed increased stress at 2 and 3 mmHg than CON vessels (
). In a calcium-free "passive" state, stress and strain values were similar at 2 mmHg but VAL vessels were considerably stiffer at 3 mmHg (
). Passive stress and strain values were higher than active values in CON embryos, indicating that vascular smooth muscle cells (VSMCs) exert tone and influence distension at this stage. No difference was observed between active and passive strain in VAL vessels at 2 and 3 mmHg, although passive stress was higher at both pressures. This suggests that the relatively immature VSMCs do not exert the same degree of tone as control vessels and that increased levels of nonelastic matrix proteins contribute to stiffer vessel mechanics in response to increased arterial load. Data are means ± SE.