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. Author manuscript; available in PMC: 2020 Jan 1.
Published in final edited form as: Anat Rec (Hoboken). 2018 Nov 9;302(1):153–162. doi: 10.1002/ar.23942

Systematic Analysis of the Smooth Muscle Wall Phenotype of the Pharyngeal Arch Arteries during their Reorganization into the Great Vessels and its association with Hemodynamics

Jessica Ryvlin 1,#, Stephanie E Lindsey 1,#, Jonathan T Butcher 1
PMCID: PMC6312499  NIHMSID: NIHMS995119  PMID: 30312026

Abstract

Early outflow morphogenesis is a critical event in cardiac development. Understanding the mechanical and molecular based morphogenetic relationships at early stages of cardiogenesis is essential for the advancement of cardiovascular technology related to congenital heart defects. In this study, we pair molecular changes in pharyngeal arch artery (PAA) vascular smooth muscle cells (VSMCs) and hemodynamic changes throughout development. We focus on Hamburger Hamilton stage 24 to 36 chick embryos, using both Doppler ultrasound and histological sections to phenotype PAA VSMCs, and establish a relationship between hemodynamics and PAA composition. Our findings show that PAA VSMCs transition through a synthetic, intermediate, and contractile phenotype over time. Wall shear stress magnitude per arch varies throughout development. Despite these distinct hemodynamic and fractional expression trends, no strong correlation exists between the two, indicating that WSS magnitude is not the main driver of PAA wall remodeling and maturation. While WSS magnitude was not found to be a major driver, this work provides a basic framework for investigating relationships between hemodynamic forces and tunica media during a critical period of development.

Introduction

Congenital heart defects (CHDs) are the most severe form of birth defects, affecting 1–2% of live births and estimated 10–30% of deaths from congenital malformations (Mozaffarian et al., 2015). The majority of these heart defects are associated with malformations of the outflow tract (OFT) and pharyngeal arch arteries (PAAs) (Go et al., 2013). Development of the PAAs is a complex system of events that involves the emergence of six major pairs of arch arteries, which sequentially remodel and disappear before forming the mature great vessels, including the aorta and pulmonary artery (Figure 1). Final arch pairs (PAA III, IV, VI) are present by Hamburger Hamilton (HH) stage 24. By HH34, PAA IV has disappeared from the non-flow dominant side (left in chick) (Figure 2). At the cellular and molecular level, the continual remodeling of the PAAs involves cardiac neural crest cell migration (CNCC) and endoderm signaling (Graham, 2003; Macatee et al., 2003; K. L. Waldo & Kirby, 1993; K. L. Waldo, Kumiski, & Kirby, 1996). Local hemodynamic forces likely play a role in whole vessel as well as local cellular remodeling, affecting early differentiation of vascular smooth muscle cells (VSMCs) through changes in endothelium characteristics (Bergwerff, DeRuiter, Poelmann, & Gittenberger-de Groot, 1996). At early stages of cardiac development, before septation of the heart into its four chambers, the PAAs are largely comprised of endothelium surrounded by an externally supportive mesenchyme which separates the primitive vessels from the ectodermal and endodermal epithelium (Bockman, Redmond, & Kirby, 1989; Le Lièvre & Le Douarin, 1975). As the embryo grows, the PAAs are characterized by a gradual increase in medial cell layers and maturation of the tunica media (Bergwerff et al., 1996).

Figure 1. Early and Mature Cardiac Configurations Including OFT and PAAs.

Figure 1.

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A) Single ventricle stage and single atria heart configuration. At HH24 arches present are III,IV,VI B) Mature heart configuration with outflow tract network region denoted by dashed rectangle and detailed image shown to the side. OFT network dotted lines denote areas where cross-sections are shown. dOFT- distal OFT, pOFT-proximal OFT., Ao-Aorta, Pa-pulmonary artery, APS –aortic-pulmonary septum, RA-right atrium, LA-left atrium, RV-right ventricle, LV-left ventricle, A-Artium,V-ventricle.

Figure 2. PAA configurations and Max Velocities.

Figure 2.

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A) PAA configurations for HH24-HH34 geometries with left arches color coded B) Max velocity magnitude as measured by Doppler Ultrasound, standard error bars are shown. N > 7 for all stages C) Doppler velocity evolution of PAA IIIR. Bmode image of HH29 embryo is shown (right) with arches color coded. From right to left, arches IIIL, IVL, and VIL are shown. Arches and dorsal aorta are outlined in dashed lines. The full video of the B-mode is included as video 1.

Formation and remodeling of the PAAs have been shown to involve multiple signaling molecules, that ensure coordinated communication between cells of mesoderm, endoderm, surface ectoderm, and neural crest cells. Cardiac neural crest cells are recruited from surrounding mesenchyme and condense into VSMCs around the endothelium. This spreading of mesenchymal condensations around an endothelial sheath is the first indication of media formation (Hughes, 1943). Endothelial cells can participate in the formation of intimal thickening (Deruiter, Poelmann, Vanmunsteren, Mironov, & Markwald, 1997). While many of the signaling players remain unknown, G-protein-coupled receptor adhesion groups, expressed in endothelial cells, work together to ensure proper PAA formation (Lu et al., 2017). Transforming growth factor –beta, TGFβ, plays a crucial role in neural crest and anterior heart field signaling that aids in the formation of the spiral septum that separates the aortic sac into the pulmonary and aortic arches (Mjaatvedt et al., 2001). TGF-βs have been shown to be shear stress responsive and enhance the expression of SMC-specific proteins including smooth muscle-α-actin, smooth muscle-myosin heavy chain, and smooth muscle calponin) and matrix proteins (fibronectin, collagen, elastin) (Molin et al., 2004; G K Owens, 1998). As the media thickens, premature VSMCs are distinguishable from neighboring non-VSMCs through the expression of muscle specific actins. VSMC maturation is marked by the expression of both contractile and cytoskeletal proteins. Expression of regulatory proteins associated with the myosin-actin complex complete the VSMC phenotype. (Gary K Owens, 1996; Slomp et al., 1997).

Proper PAA development is required for normal cardiac function. Little is known about the biological mechanisms of CHD formation. While underlying genetic mutations are associated with CHDs, genes cannot be mutated to produce malformations as targetedly as flow can be perturbed to induce diseased phenotypes. Hemodynamics guides cardiac morphogenesis. The growing heart is accompanied by changing hemodynamic function ( E B Clark et al., 1986; Edward B Clark et al., 1989). Blood flow exerts forces on the walls of the heart in the form of pressure, circumferential stress, and shear stress. Cells that populate the heart and affect its formation are constantly exposed to these forces during development. Wall shear stress (WSS) represents the shearing force exerted by blood as it drags along the walls (Gjorevski and Nelson, 2010a) and has been shown to play a key role in vascular reactivity (Bartman & Hove, 2005; Gnasso et al., 1996). Endothelial cells, which line the vascular system, act as force sensors and transducers of WSS to adjacent smooth muscle layers (Kamiya & Togawa, 1980). In addition to WSS, VSMCs are also sensitive to changes in pressure (deBlois, Tea, Than, Tremblay, & Hamet, 1997), which may modulate VSMC proliferation and differentiation. Hemodynamics is therefore associated with changing VSMC function, yet few studies quantify a relationship between the two. By HH24 the pulmonary arteries acquire actin expression at their junction with the sixth arch arteries, a time point that coincides with the initiation of flow through these vessels (Bergwerff et al., 1996). The role of hemodynamics in maintaining these functional relationships and the timing of VSMC differentiation in response to changing levels of flow and WSS levels remains to be determined.

VSMCs exhibit a range of phenotype characteristics along a continuum that begins with a synthetic phenotype and ends with a contractile phenotype (Rensen, Doevendans, & van Eys, 2007). VSMC contractile phenotypes, are characterized by contractile filaments and low protein synthesis, while VSMC synthetic phenotypes are characterized by protein synthesis organelles, high growth and migration. Here, we focus on characterizing natural cell expression and blood velocity variations during the HH24 to HH36 remodeling processes. We detail molecular changes in PAA VSMCs phenotype, through the use of contractile markers alpha smooth muscle actin (α-SMA), smooth muscle myosin heavy chain 11 (SM-MHC), and smooth muscle calponin (SM- calponin), and quantify its association with hemodynamic changes. α-SMA localizes contractile filaments in the cell, and it is typically expressed at E9/HH11. SM-calponin is associated with actin in the cytoplasm, and is expressed at E13.5/HH30. SM-MHC also localizes contractile filaments in the cell, and it is usually expressed at E10.5/HH19. A higher expression of the contractile markers indicates a more contractile phenotype in the VSMCs. A lower or loss of expression of contractile markers signifies a synthetic phenotype in VSMCs. Ultimately, establishing quantitative relationships between blood flow and its surrounding tunica media will lead to a better understanding of how cardiac malformations arise and eventually how these malformations can be remedied.

Materials and Methods

Embryo Culture and Preparation

Fertile white Leghorn chicken eggs were incubated blunt-side up in a continuous rocking incubator at 37.5˚C. After 72 hours, embryos were open cultured as described in (Yalcin, Shekhar, Rane, & Butcher, 2010). Embryos were subsequently transferred to a portable incubator and monitored until they reached the desired stages.

Doppler Ultrasound Imaging

Doppler ultrasound imaging system (Vevo 2100, Visualsonics, Inc.) was used for the assessment of cardiac functionality (Figure 2, Video 1). Warm Earle’s balanced salt solution was used as an aqueous conduit between the embryo and the ultrasound transducer. Embryos were kept in a heated water bath at 37.5˚C during imaging. For each embryo, the right PAAs were imaged as the embryo naturally lies on its right side up on the yolk sack. After obtaining Doppler flow curves, embryos were removed from the water bath and flipped in order to obtain access to their left arteries. Embryos were then transferred back to the water bath and the left PAAs imaged. After imaging, embryos were returned to the incubator and allowed to recover. For each embryo, a minimum of 7 Doppler recordings were averaged to obtain a velocity curve.

Histology and Immunofluorescence

Cultured embryos were dissected away from their yolk sac, external membranes removed, and placed into petri dishes. Embryos were flushed with phosphate buffer solution and perfusion fixed with 4% paraformaldehyde (PFA). After 48 hours, embryos were transferred to 70% ethanol and embedded in paraffin wax. Embryos staged HH24 through HH26 were sectioned at 4µm thickness, while embryos post HH26 were sectioned at 5µm thickness using a rotary microtome. Sections were stained with Hematoxylin and Eosin (H&E), and imaged with Zeiss Stereo Discovery V20 to identify vascular structure and vessel wall boundaries for future immunohistochemistry measurements. These images were then processed in ImageJ.

For immunohistochemistry, sections antigen retrieval was done using 10mM sodium citrate buffer. Samples were blocked with 10% goat serum before applying primary antibodies α-SMA (1:500, rabbit polyclonal, Abcam), SM-MHC11 (1:500, rabbit polyclonal, Abcam), and SM-calponin (1:500, goat polyclonal, Abcam). MF20 (1:500, mouse monoclonal, Developmental Studies Hybridoma Bank) primary antibody, a myocardium label is used as a negative control for PAAs. Secondary fluorescence-conjugated antibodies Alexa Fluor 568 (1:500, goat anti-rabbit, Life Technologies), Alexa Fluor 488 (1:500, goat anti-mouse, Life Technologies), fluorescein isothiocyanate (FITC) (1:500, donkey anti-goat, Santa Cruz Biotechnology), and DRAQ5 (1:1000, nuclear counter-stain, Cell Signaling) were applied according to species specificity. Sample signals were detected and imaged with the Zeiss 710 laser-scanning confocal microscope using a 25x water immersion objective. Each channel was imaged using the same Gain across samples to normalize fluorescence across embryos. A total of 3 adjacent sections were analyzed per PAA per embryo. These sections were taken along the length of the vessel at the distal end, midpoint, and proximal end of the vessel. As a minimum of 3 images were taken per section, this amounted to an average of 9 images per PAA per HH stage. Images were then processed in ImageJ by first converting the blue channel (DRAQ5) into a binary image. Nuclei were counted by increasing contrast to a preset value constant across all images, by applying a fill holes function, and then by applying a classic watershed to separate overlapping nuclei, where a minimum cluster of 20 positive pixels were considered one nucleus. Red (α-SMA and SM-MHC) and green (SM-calponin) channels were adjusted in brightness/contrast to a constant value across all images to remove background fluorescence and visualize co-localized nuclei staining. Nuclei were individually counted positive based on a threshold of >50% perimeter co-localization with a given channel, and counts. Positive nuclei were presented as a percentage of total cells positive for expression. Location and circumference specific variation was not found. SMC markers were not analyzed along the radius axis outside of the percentage expression reported. The percentage of cells with SMC phenotype markers reached a maximum closest to the lumen, with expression reducing further away from the lumen.

Wall Shear Stress Calculation

Two-dimensional images of PAAs from Hematoxylin and Eosin (H&E) staining were imported in ImageJ to calculate the inner vessel area. The inner vessel area was calculated by tracing the inner vessel perimeter to obtain a value for area. Diameters were then calculated assuming a circular cross-sectional area. For simplification, blood was assumed to be Newtonian and the vessels were assumed to be incompressible. Poiseuille Flow was used to approximate the wall shear stress, the equation of which is given by,

τ =4×µ×Vmaxd (1)

where µ is the blood viscosity and is given by 3.71 × 10−3 Pa.s, Vmax is the maximum flow velocity from Doppler ultrasound B-mode recordings, and d is the apparent diameter of the vessel as measured by histology multiplied by a constant scaling factor in order to account for dehydration and processing of the embryos for histology and immunofluorescence. Diameter measurements were cross-checked against in-vivo measurements as well as measurements obtained from 3–4 voxel nano-CT scans. A scaling factor of 2.58 was applied across stages. A total of 5 to 21 embryos per stage were averaged to obtain HH24 to HH36 measurements (HH24 N=21, HH26 N=13, HH29 N=23, HH31 N=10, HH34 N=10, HH35 N=16, HH36 N=5).

Vessel Thickness to Diameter Ratio

Individual PAA wall thicknesses and diameter measurements for all Hematoxylin and Eosin (H&E) stained samples were imaged using a Zeiss Stereo Discovery system, and processed with ImageJ. Vessel thicknesses were calculated by taking four wall thickness measurements on opposite sides of the vessel starting at the lumen (Lu) and ending at vessel wall borders (Figure 3). These thickness values were normalized to vessel diameter measurements. Only images of sections depicting transverse plane sections were selected from a collection of samples for vessel wall calculations to ensure accuracy.

Figure 3. Apparent Thickness Diameter Histology Measurements.

Figure 3.

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Thickness normalized to diameter shown across stages for HH24 to HH36 embryos. Lu-Lumen. Arrows indicate where vessel thickness was measured. HH24 N=21, HH26 N=13, HH29 N=23, HH31 N=10, HH34 N=10, HH35 N=16, HH36 N=5. Stars indicate a statistically significant change between stages for combined left and right thickness/diameter dynamics based off a one-tailed unequal variance t-test. (P = 0.004 HH24 to HH26, P = 0.037 HH34 to HH35, P = 0.003 HH35 to HH36).

Statistical Analysis

Fractional expression and hemodynamic changes were compared qualitatively and quantified when possible. Results were summarized in the form of mean and standard error values. Paired T-tests were used where appropriate with P < 0.05 denoting significance. Non-linear regressions in the form of plateau followed by one-phase decay were performed. Regressions were made through the use of GraphPad Prism (GraphPad Software, Inc San Diego, CA) statistical software.

Results

Wall thickness to diameter ratio (Figure 3) varies across stages. There is a significant increase between HH24 to HH26, at which point the vessels themselves are lengthening and rotating as the heart enters the late phase of cardiac looping (Manner, 2000; K. Waldo, Miyagawa-Tomita, Kumiski, & Kirby, 1998). The thickness to diameter ratio also significantly increases from HH34 to HH35 and decreases from HH35 to HH36. HH35 marks a period of period of apotosis for the great vessels, with heightened staining occurring at the branching point (Schaefer, Doughman, Fisher, & Watanabe, 2004). The variation of WSS (HH24-HH34) for each PAA is shown in Figure 4. These calculations are based off max velocity measurements (Figure 2). Results show that WSS decreases over time.

Figure 4. Arch by Arch WSS magnitude variation across stages.

Figure 4.

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WSS arch magnitudes were highly variable. No significant differences exist between stages (one-tailed t-test, unequal variance)

Immunofluorescence staining of α-SMA and cell nuclei in PAA vessel walls (Figure 5, Figure 7) indicates that α-SMA expression is concentrated at the lumen in early stage embryos. As the embryos ages, α-SMA becomes evenly dispersed across the vessel wall. Increased striations in α-SMA staining highlight VSMCs organization in PAA walls, which increases over time. These qualitative trends are quantified in Figure 8A. Results indicate that when quantified, the variation in the fraction of PAA VSMCs that are positive for α-SMA expression is minimal across stages.

Figure 5. α-SMA expression level across stages.

Figure 5.

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Immunofluorescence staining of α-SMA (red) and cell nuclei (blue) in PAA vessel walls of different embryos. (A) A HH24 embryo, show α-SMA localization in the vessel wall (W) near the lumen (Lu). HH26 (B) and HH29 (C) embryos, show larger spreading of α-SMA throughout the vessel wall but sustained localization near the lumen. At HH31 (D), α-SMA can be seen expressed throughout the vessel wall, with an increasingly striated appearance beginning at HH31 and continuing through HH34 (E), HH35 (F), and HH36 (G). Localization of α-SMA near the lumen only begins to disappear after HH35. W - Vessel wall, Lu - Vessel lumen, Scale bar indicates 20 µm.

Figure 7. Full lumen view of PAA immunofluorescence staining.

Figure 7.

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Immunofluorescence staining of α-SMA (red) and cell nuclei (blue) in PAA vessel walls of HH24 (A), HH26 (B), HH29 (C) and HH31 (D) embryos in which the entire vessel wall (w) and lumen (Lu) can be seen. Full lumen views were used for marker quantification.

Figure 8. Quantification of contractile markers’ fractional expression levels.

Figure 8.

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Total number of cells in each PAA wall were counted based on observed nuclei. For A) α-SMA B) SM-calponin and C) SM-MHC. No statistically significant difference were observed between stages for α-SMA, suggesting expression remains constant from HH24 to HH36. SM-calponin results show little to no expression in HH24-HH26 PAAs, whereas expression of SM-calponin significantly increases HH26 to HH29. From HH29 through HH34, SM-calponin stays constant (no significance detected), after which a significant decrease is seen from HH34 to HH35. From HH35 to HH36, SM-calponin stays constant, though the left PAAs are higher in SM-calponin expression than the right PAAs when compared to other embryo ages. For SM-MHC expression, all PAAs stay constant at very low levels from HH24 to HH26. From HH26 to HH29, and from HH29 to HH31, all PAAs significantly increase in expression of SM-MHC. While no change is seen in expression from HH31 to HH34, significant increases in expression of all PAAs are seen from HH34 to HH35 and from HH35 to HH36. Additionally, right PAAs show higher expression of SM-MHC than left PAAs in HH36 embryos. *p<0.05 and **p<0.001. HH24 N=21, HH26 N=13, HH29 N=23, HH31 N=10, HH34 N=10, HH35 N=16, HH36 N=5.

SM-calponin and SM-MHC immunofluorescence staining (Figure 6) help identify intermediate VSMC contractile phenotypes. SM-calponin and SM-MHC expression are concentrated at the lumen in early stage embryos but are evenly dispersed across the vessel wall in older embryos just like α-SMA. SM-calponin appears only after HH26, whereas SM-MHC appears only after HH34. SM-calponin and SM-MHC expression data from Figure 6 are quantified and shown in Figure 8B & C. Quantitative results confirm minimal SM-MHC and SM-calponin expression before HH26 followed by a significant increase. After HH34 SM-MHC expression increases and SM-calponin expression decreases.

Figure 6. SM-Calponin and SM-MHC expression levels across stages.

Figure 6.

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Immunofluorescence staining of cell nuclei (blue), SM-calponin (green), and SM-MHC (red) in PAA vessel walls. HH24 (A) and HH26 (B) show minimal staining of both SM-calponin and SM-MHC in the vessel wall (W). HH29 (C), HH31 (D), and HH34 (E) show significant staining of SM-calponin when compared to previous days. SM-calponin over this period also decreases in localization to the lumen (Lu) over time, with HH34 showing almost no SM-calponin concentration near the lumen. HH35 (F) and HH36 (G) show significant SM-MHC expression when compared to previous stages with a comparable decrease in SM-calponin. From HH24 to HH36, vessel wall striation becomes increasingly organized. Scale bar- 20µm.

In order to study the effect of WSS on PAA VSMC contractile phenotype expression, we combine WSS data and immunofluorescence data (Figure 9). A slight correlation between fractional expression and WSS magnitude exists for α-SMA and SM-calponin (R2 0.10, 0.28 respectively) based off a plateau followed by one phase decay regression. No correlation exists for SM-MHC.

Figure 9. Contractile marker fractional expression WSS correlation.

Figure 9.

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WSS and PAA VSMC phenotype expression relationship independent of stage. Fitted lines represent a plateau followed by one-phase decay regression. SM-MHC failed to converge in a plateau followed by one-phase decay regression.

Discussion

The effects of hemodynamics and WSS on outflow morphogenesis have been an area of intense study and uncertainty. In order to understand how malformations form, we must first understand what drives normal development. Recent studies have focused on characterizing hemodynamic forces in the developing heart (Bharadwaj, Spitz, Shekhar, Yalcin, & Butcher, 2012; Kowalski, Teslovich, Dur, Keller, & Pekkan, 2012; Lindsey & Butcher, 2011; Lindsey et al., 2015; Wang et al., 2009; Yalcin, Shekhar, McQuinn, & Butcher, 2011), though few reports of individual PAA WSS measurements exist (Lindsey et al., 2015; Wang et al., 2009). HH24 Peak flow WSS values of the current study, calculated via Doppler Ultrasound and histology measurements, were in keeping with HH24 peak flow WSS obtained by computational fluid dynamics, as reported by Wang et al. (2009). The role of cellular media such as CNCCs in OFT and PAA reorganization has also been the topic of extensive study (Azhar et al., 2003; Bajolle et al., 2006; Kirby & Hutson, 2010; Liu et al., 2004; Poelmann, Mikawa, & Gittenberger-de Groot, 1998; Turner, Badu-Nkansah, Crowley, van der Flier, & Hynes, 2015). While many of the tunica media studies have been qualitative, rather than quantitative, their studies are nevertheless consistent with current results, namely, the gradual increase in medial cell layers from HH24 onward, gradual increase in organization of the tunica media into continuous fibers (Bergwerff et al., 1996), the distinguishable “compact dense zone” that marks the vessel wall (Hughes, 1943), as well as the “marked increase” in vessel wall thickness between day 5 and 7. The relationship between these two factors, hemodynamic forces and cellular media composition was recently explored by Espinosa et al. (2018). They found that increase in tunica-media content was more broadly affected by vitelline vein ligation than increase in wall organization. Cell alignment and volume fraction were unaffected by hemodynamic perturbation, while overall vascular resistance and peak velocity were affected in both the short-term and long term. Data suggested that VSMCs adapt to their surrounding extracellular matrix by overexpressing RNA to overcome protein deficiencies triggered by flow-dependent signaling pathways. Ultimately, arterial maturation includes a compensatory response to hemodynamic changes and subsequent extracellular matrix deficiencies. VSMCs are capable of responding to their surrounding extracellular matrix. Further investigation of the link between hemodynamics and tunica media formation is required to better understand this process. Quantifying the relationship between hemodynamics and wall remodeling is an important step towards determining causal effects of abnormal cardiac morphogenesis.

Here, we provide quantitative evidence that WSS across the developing PAA system decreases as the embryo grows. A statistically significant difference exists between HH31 and HH34 in WSS. Early stage variations in arch artery to arch artery WSS levels are high. HH26 right PAAs experience greater WSS than the left PAAs, with the R AAVI experiencing the maximum shear stress. This suggests that in HH26 embryos, blood is primarily shunted to right side of the arches. This follows the disappearance of the L AAIV. Results show that from HH29 the L AAIII experiences more WSS than L AAIV and L AAVI. Although large variations in WSS exist between each PAA during initial stages, this distribution evens out on HH34 right PAAs. The variation in WSS is due to PAA diameter and blood flow velocity changes during development. The lower WSS values in HH34 embryos are due to rising blood flow velocities with development as shown in Figure 2.

Immunofluorescence experiments provide an in-depth view of cellular phenotypes during PAA remodeling over a critical period of cardiogenesis. Embryos as young as HH24 express α-SMA. The qualitative analysis of α-SMA, SM-calponin, and SM-MHC expression is quantified through fractional measures of cells positive for these markers. Cells expressing α-SMA spread steadily away from the lumen of the PAA towards the perimeter of the outer vessel walls from HH24 to HH36. Beginning at HH29 there is an increased striation and organization of PAA vessel walls. In order to achieve this organization, VSMCs must transition to a contractile phenotypic morphology, characterized by elongation of cell body and nucleus (Figure 10). Although α-SMA qualitative expression levels (Figure 5) seems to suggest that the fraction of cells expressing the contractile phenotype increases across stages, this is not the case. Quantitative results reveal no statistical significance in α-SMA expression between embryo ages (Figure 8). While the number of cells expressing α-SMA does increase over time, vessel walls thicken along this same time period, resulting in a “constant” α-SMA expression level.

Figure 10. Vascular Smooth Muscle Cell Phenotypes.

Figure 10.

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Summary of contractile marker expression levels and synthetic and contractile vSMC phenotype.

Analysis of SM-calponin expression in VSMCs provides a different account of phenotype development in the PAAs. Figure 6 shows that SM-calponin is expressed in VSMCs from HH29. This implies that before HH29, there is little or no expression. Indeed, there is no significant increase in SM-calponin expression (Figure 8) in HH29 PAAs, though right PAAs do express more SM-calponin than left PAAs. This jump in SM-calponin expression correlates with disappearance of the L AAIV from the PAAs. The SM-calponin expression drops after HH34, suggesting that an intermediate contractile phenotype consisting of α-SMA and SM-calponin is in effect between HH29 and HH34 consist with current findings (Rensen et al., 2007). The addition of high quantities of SM-calponin to the constant α- SMA expression levels indicate a shift towards contractile phenotype beginning at HH29, as opposed to a more synthetic VSMC phenotype in PAAs preceding HH29. From HH34, the decrease in SM-calponin expression in the PAAs suggests an increase in cell traction force, that subsequently alters VSMC morphology and increases migration capability by providing greater de- adherence to vessel walls (Hossain, Zhao, Woo, Wang, & Jin, 2016; Yamamura, Hirano, Koyama, Nishizawa, & Takahashi, 2007).

Immunofluorescence staining of SM-MHC in HH24 through HH36 PAA vessel walls increases over time, across stages. There is a significant increase of SM-MHC from HH26 to HH29, HH29 to HH31, HH34 to HH35, and HH35 to HH36. Right and left PAAs express similar levels of SM-MHC expression in initial stages. HH35 embryos show higher SM-MHC expression in right PAAs compared to the left PAAs, an opposite trend from SM-calponin expression. These expression patterns indicate that not all smooth muscle contractile phenotypes respond in the same fashion. Different smooth muscle phenotypes throughout the PAAs attribute contractility to the vessel through different signaling mechanisms, thereby restricting blood flow through the PAAs.

In summary, in early PAA remodeling (HH24 and HH26), only α-SMA is expressed in the PAAs and is localized to cells lining the vessel lumen. From HH29, SM-MHC and SM-calponin are expressed, indicating the transition to a more contractile VSMCs phenotype. HH29-HH34 marks an intermediate phenotype between full synthetic and full contractile morphology (Figure 10). From HH34, SM-calponin expression declines significantly while SM- MHC significantly increases. SM-MHC expression increases through HH36 when a fully contractile phenotype is achieved. SM-MHC and SM-calponin serve as determinants to phenotypic switching between synthetic, contractile, and intermediate phenotypes in the PAAs. One of the main aims of this study was to determine if WSS is a modulator of phenotypic switching seen in immunofluorescence experiments. Expression levels did not strongly correlate with WSS magnitude. Despite its reputation as a regular of vascular remodeling (Brownlee & Lowell, 1991; Kamiya & Togawa, 1980; Langille & Donnell, 1986), WSS alone does not appear to be a strong driver of PAA wall remodeling and maturation at this stage in development. Our results are in contrast to the findings Espinosa et al (2018), who hypothesized that the decrease in WSS, due to decreased flow, prompted a series of adaptation mechanisms. We believe that perceived changes may have been a combination of the new strain added to the vascular wall, as seen with ventricular ligations (deAlmeida, McQuinn, & Sedmera, 2007), as well as changes in wall shear stress levels. Maturation of PAA tunica wall media therefore involves complex and evolving interactions between cells and their local mechanical environment to coordinate morphogenesis and tissue maturation.

Conclusion

This study characterized VSMC phenotypes and WSS changes over time in the PAAs. Arch artery to arch artery variations in WSS were high in early (HH24–26) and mid-staged (HH29-HH31) embryos, before evening out in later staged (HH34) embryos, highlighting how variations in vessel diameter and flow velocity are accommodated through side specific flow dynamics. VSMCs in the PAAs transition from synthetic to contractile phenotype over time, with an intermediate phenotype present from HH29 to HH34. These phenotypic switches are initiated through variations in SM-calponin and SM-MHC expression over time. α-SMA maintains a constant level throughout PAA remodeling. While we did not find a strong quantitative relationship between WSS and VSMC phenotype switching, we were able to rule out WSS magnitude as the main driver of PAA wall maturation. We hypothesize that pressure and inherent wall tissue stress may play a larger role. Studies such as these that examine a hemodynamic factor in relation to morphological changes are important for the advancement of our understanding of morphogenesis.

Supplementary Material

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Acknowledgements

Cornell BRC Imaging facility, Kalay Seranthian for helpful discussions, AHA Summer Undergraduate Research Grant (JR), NSF Graduate Research Fellowship Program (SEL), Alfred P. Sloan Foundation (SEL), NIH HL110328, NSF CMMI-1635712, NIH S10OD016191 (Vevo-2100 ultrasound), NIH S10RR025502 (Zeiss LSM 710 Confocal) .

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