The mechanical and morphological properties of systemic and pulmonary arteries differ in the Madagascar ground boa, a snake without ventricular pressure separation

Strong yet distensible arterial walls are critical for proper function of the vascular tree in animals. Strength is required to withstand high pressures when blood is ejected from the heart during systole, and distensibility is critical to ensure that the major arteries provide capacitance and pulse-pressure smoothing after each cardiac contraction (Shadwick, 1999). These mechanical properties derive from morphological features of the vessel walls, primarily thickness, elastin and collagen content, and the extent and mode of cross-linking and alignment of the elastin and collagen fibers (Dobrin, 1978; Wagenseil et al., 2009). These properties are established during embryological development in response to hemodynamic forces, such as wall shear stress, that continuously drive vascular remodeling (Jones et al., 2006; Reneman et al., 2006). In mammals, abrupt hemodynamic changes occur after birth, when the pulmonary and systemic circuits become fully separated (Langille, 1996). Intriguingly, reports in various mammals suggest that after birth, major structural changes occur in the aorta wall as compared with the pulmonary artery, the former becoming increasingly thicker-walled and stronger than the latter (Gerrity and Cliff, 1975; Leung et al., 1977). These changes probably reflect necessary adaptations to the considerably higher systemic blood pressure as compared with the pulmonary arterial pressure. However, it remains unclear whether intramural blood pressure indeed provides the causative link to differences in arterial wall morphology.

Intraventricular pressure separation describes the ability of the heart to eject blood into the systemic and pulmonary circulations at different pressures. This ability evolved independently in mammals and archosaurs (birds and crocodiles) through the establishment of a full ventricular septum that divides the ventricle into left (systemic) and right (pulmonary) chambers (Hicks, 1998). In contrast, with the exception of pythons and Varanus lizards, non-archosaur sauropsids typically lack a complete ventricular septum, resulting in similar systolic pressures in the systemic and pulmonary arteries (Jensen et al., 2014). Reptiles, therefore, represent an interesting possibility to investigate the relationship between pressure separation and arterial mechanical characteristics.

In previous studies, we analyzed the mechanical characteristics of the major arteries of ball pythons (Python regius), which have functional intraventricular pressure separation (Jensen et al., 2010a; van Soldt et al., 2015; Zaar et al., 2007), and the yellow anaconda (Eunectes notaeus) that lacks intraventricular pressure separation (Filogonio et al., 2018). These studies revealed that the aortas and pulmonary arteries of P. regius, as in mammals, differ in their mechanical properties, while they are more similar in E. notaeus. However, the similarity in mechanical properties between the aortas and pulmonary arteries of E. notaeus was not mirrored in their morphological features, as might have been expected. Thus, it is possible that, while intraventricular pressure separation has profound effects on the morphology of the great arteries, it may not always play the expected causative role.

In the present study, we therefore investigated the mechanical properties of the aortas and pulmonary arteries of the Madagascar ground boa (Acrantophis madagascariensis) and compared these with those of other species to better understand the relationship between intraventricular pressure separation and the mechanical properties of the great arteries. We first show intraventricular blood pressure data which suggest a lack of pressure separation in the ground boa, and then report our findings on the mechanical characteristics of the pulmonary artery and aorta walls. As in E. notaeus, we found that the pulmonary arteries are remarkably resilient to high strains, despite lacking apparent strength, and that this likely relates to their surprisingly small diameter, which may negate the need for increased wall strength. We conclude that differences in morphology and mechanical properties between the aorta and pulmonary arteries may be related to a combination of genetic or developmental factors in addition to pressure separation.

Nine captive-bred ground boas, Acrantophis madagascariensis (Duméril and Bibron 1844), with a body mass ranging from 244 to 655 g (344±46 g, mean±s.e.m., Table 1) were donated by a zoological garden and kept in accordance with §53 of Danish experimental animal welfare regulations (permit ID 2013-15-2934-00847). Snakes were fed rodents weekly, but fasted for several weeks prior to euthanasia.

Briefly, snakes were used for blood pressure measurements, histology, mechanical testing and biochemical analysis of vessel walls (Table 1). Prior to euthanasia, intraventricular blood pressure was measured in 6 snakes, of which 4 were conscious and at rest, while the other two were anesthetized. Blood pressure measurements were discounted from two snakes because of the poor quality of the recordings. Following euthanasia, 3–4 vessel rings from 7 unique locations were harvested from all 9 snakes (Fig. 1A) for morphological measurements (Fig. 1B) and tensile testing (Fig. 1A). Following tensile testing to rupture, vessel rings from all 9 snakes were retained for absolute collagen content determination. Excess vessel materials were collected and pooled, to enhance measurement reliability, for determination of collagen and elastin content as a percentage of dry mass (Table 2). Vessel samples were derived from one snake for histology. The experimental procedures are described in detail below.

Systemic and pulmonary blood pressure was measured in two anesthetized snakes by intraventricular and extracardiac cannulation (vertebral and right pulmonary arteries). Snakes were anesthetized by intramuscular injection of pentobarbital (30 mg kg⁻¹) and anesthesia was confirmed by lack of muscle tone. After subcutaneous application of Xylocain (20 mg ml⁻¹), the cardiac region was exposed through a 10 cm ventral incision. Right aortic and right pulmonary arterial pressure was measured by cannulating the vertebral and right pulmonary artery with PE60 catheters containing heparinized saline (50 IU ml⁻¹). Intraventricular blood pressure was measured in the cavum arteriosum and cavum pulmonale by creating a small incision in the respective ventricular walls and inserting PE90 catheters (see Wang et al., 2003, for details on experimental procedures). To measure systemic blood pressure in four fully recovered snakes, snakes were anesthetized by inhalation of isoflurane, and the dorsal aorta was cannulated in the tail with a PE60 catheter. Pressure measurements were taken 3, 5 and 24 h after recovery from anesthesia. Catheters were connected to Baxter Edward pressure transducers (model PX600, Irvine, CA, USA) placed at heart level of the snakes, and acquired using a Biopac MP100 data acquisition system (Goleta, CA, USA).

Images of histological sections of vessel segments were obtained from one snake as previously described (van Soldt et al., 2015). Briefly, the segments were fixed in formaldehyde, embedded in paraffin and sectioned (4 µm). The sections were stained with resorcin, Sirius Red F3B and Mayer's hematoxylin. Photographs were taken with an Olympus C-7070 WZ camera (Tokyo, Japan) mounted on a Leica DMRB microscope (Wetzlar, Germany) using both bright field and circular polarization.

All procedures were described previously (van Soldt et al., 2015). After blood pressure measurements were completed, snakes (n=9) were euthanatized (30–50 mg kg⁻¹ pentobarbital) and the heart, including great arteries, was excised. Aortic and pulmonary artery segments (∼1 cm) were collected from seven locations (left and right pulmonary artery, proximal and distal locations of left and right aorta, and dorsal aorta; Fig. 1A). Vessel segments were cut into 3–4 rings with a nominal length of 1 mm and submerged in 50 mmol l⁻¹ Tris-HCl solution (pH 7.4). Wall cross-sectional area (A; Fig. 1B), which corresponds to wall thickness as vessel ring height (h; Fig. 1B) is 1 mm, was measured by mounting the rings on a tapered glass rod at minimal strain, photographed using a Nikon microscope (Tokyo, Japan; Fig. 1B) with circular polarization filter and analyzed using ImageJ v1.47. A scale was photographed for calibration purposes. Vessel luminal diameter (d; Fig. 1B) was derived from tensile testing data. The rings were then placed at −20°C in 50 mmol l⁻¹ Tris-HCl solution (pH 7.4) until mechanical testing. Although no consensus has been reached on the effect of freezing on the mechanical properties of blood vessels (Akyildiz et al., 2014), freezing has not been found to affect mechanical properties significantly across a variety of freeze–thaw procedures (Akyildiz et al., 2014; Chow and Zhang, 2011; Graham et al., 2011; Kesava Reddy, 2004; Stemper et al., 2007).

All procedures were described by van Soldt et al. (2015). Briefly, after thawing to room temperature, a vessel ring was placed around two orthogonally bent hooks with a diameter (d_h) of 0.55 mm (aortas) or 0.35 mm (pulmonary arteries) and an initial linear distance between the hooks (l_h0) of 1.2 mm and 0.5 mm, respectively (Fig. 1C). One hook was connected to a load cell while a step motor moved the other. Travel distance and load cell readings were acquired continuously. Each ring was subjected to a cycle of five tests with a tension maximum of 0.25 N for the left or right aortas, 0.2 N for the dorsal aorta, and 0.075 N for the pulmonary arteries, because the pulmonary arteries were hypothesized to rupture at lower tension maxima as compared with the aortas. Subsequently, a tension test to rupture was carried out, with maximum travel distance set at the point of vessel rupture (x). Three to four (mean 3.7) rings from each vessel segment were tested. Ruptured rings were collected for hydroxyproline determination (Danielsen and Andreassen, 1988; Woessner, 1976) and absolute collagen content in milligrams was calculated as 7.46×hydroxyproline content (Neuman and Logan, 1950).

To determine the elastin and collagen fractions relative to dry mass, because of their small size, excess vessel material was pooled into one (pulmonary arteries) or two (aortas) batches. Duplicate determinations were performed per batch. Vessels were defatted with acetone and freeze-dried. Elastin content (percentage of dry mass) was determined after an extraction procedure according to Lansing et al. (1952). Aliquots of the extracts were used for hydroxyproline determination.

Stiffness and strain corresponding to pressures of 2, 5 and 10 kPa were then estimated by finding the combination of load and strain fulfilling the equation for P (Eqn 7).

Statistical analyses were performed as described previously (van Soldt et al., 2015). Briefly, we ran all statistical analyses in R 4.1.0 (http://www.R-project.org/) running in RStudio v1.4.1717 (https://www.rstudio.com/), using the packages lattice (Sarkar, 2008), lme4 (https://CRAN.R-project.org/package=lme4), car (Fox and Weisberg, 2011) and multcomp (Hothorn et al., 2008). For significance testing of mechanical and morphological properties, we used a mixed model [tested variable as a dependent variable, e.g. F_max, and individual animal (Table 1) as a random variable]. A post hoc Tukey test was used to identify significant differences between vessel segment pairs (P<0.05). Curves were calculated for each vessel section per snake and then combined, per vessel section, into mean curves for presentation in this work. Curve data for proximal and distal segments for both aortas were pooled using described equations (Baker and Nissim, 1963). One-way ANOVA was used for statistical tests on comparisons of morphological parameters between snake species using standard R commands.

We confirmed that A. madagascariensis lacks intraventricular pressure separation by measuring intraventricular and extracardiac (vertebral and right pulmonary arteries) systemic and pulmonary blood pressure (Fig. 2). Systemic and pulmonary pressure waveforms overlapped entirely for intraventricular pressure measurements, both during systole and diastole. Waveforms for extracardiac measurements overlapped during systole (Fig. 2A). Indeed, systemic and pulmonary systolic pressure were grossly similar at 4.9 and 4.7 kPa, respectively (n=2; Fig. 2B and Table 3). To ensure that anesthesia did not have significant effects on blood pressure, we also measured systemic blood pressure in conscious A. madagascariensis. Mean arterial blood pressure (MAP) values in anesthetized (4.0 kPa, n=2) and conscious (4.0±0.2 kPa, n=4) snakes were similar. Thus, these data suggest that A. madagascariensis does not have intraventricular pressure separation.

We quantitively determined vessel dimensions (wall thickness, cross-sectional area) by measuring basic morphological parameters of each individual vessel ring using brightfield microscopy (Fig. 1B), and performing biochemical determinations to assess vessel wall composition. The aortic vessels were consistently thicker walled (P<0.001, n=9; Fig. 3A–M) and of larger diameter than the pulmonary arteries (P<0.0001, n=9; Fig. 3A–L,N). More specifically, proximal and distal sections of the left (PLAo/DLAo) and right aorta (PRAo/DRAo) were not significantly different in either wall thickness (P=1, P=0.132; n=9; Fig. 3M) or diameter (P=1, P=0.979; n=9; Fig. 3N). However, the left aorta was thicker walled (P=0.0242; n=9) and wider (P<0.0001; n=9) than the right aorta (Fig. 3M,N). The dorsal aorta (DAo) was of larger diameter than either the left or right aorta (P<0.00001, n=9; Fig. 3N). Finally, the right pulmonary artery (RPA) was thicker walled (P=0.006, n=9) and wider (P<0.00001, n=9) than the left pulmonary artery (LPA; Fig. 3M,N).

In the vessel wall, the elastin-rich tunica media confers elasticity, while the tunica adventitia – which is composed predominantly of collagen fibers – confers strength. The histology (Fig. 3A–L) illustrates the difference in wall thickness, luminal diameter and tunica adventitia/tunica media ratio between aortic and pulmonary artery specimens. Microscopy using a circular polarization filter indicated that the aortic tunica media was thicker than the tunica adventitia (Fig. 3C,F). In contrast, in the pulmonary artery walls the tunica adventitia was either as thick as, or thicker than, the tunica media (Fig. 3I,L), suggesting that the aortic walls are more elastic than those of the pulmonary arteries. Indeed, determination of elastin and collagen percentage relative to dry mass (Table 2) appeared to suggest that aortic sections had higher elastin content (∼30%), but lower collagen content (∼20%) compared with pulmonary sections (∼9% and ∼40%, respectively), although note that these measurements represent pooled averages (Table 2).

Unit collagen (UC; Fig. 3O) is the collagen content of a vessel ring (in mg) divided by its circumference (in mm). Consistent with the higher collagen percentage of pulmonary artery walls (Table 2), pulmonary artery UC appeared higher than that of the aortas, despite the thinner walls of the pulmonary arteries. This high UC could be explained by the apparently thicker collagen-rich tunica adventitia of the pulmonary arteries (Fig. 3C,F,I,L), and the high collagen content of these arteries. The left aorta had higher UC than the right aorta (P=0.031), and the right pulmonary artery had higher UC than the left pulmonary artery (P<0.001). Altogether, these data suggest that the aortic walls are more elastic than the pulmonary artery walls.

We next investigated the mechanical properties of the aorta and pulmonary artery walls by subjecting vessel sections to 5 cycles of uniaxial mechanical tension testing, followed by a test to rupture. Loop curves demonstrated that steady state was reached by the fourth cycle for both aortic and pulmonary artery vessel segments (Fig. 4A). Although load limits in cycle tests were set according to the expected maximal load that the vessel segments could endure without premature rupture (see Materials and Methods), calculation of mechanical hysteresis (viscous damping, loss of elastic energy; loop area divided by area under the loading curve) showed that the pulmonary artery walls were significantly less elastic than those of the aorta (P<0.0001, n=9; Fig. 4B). Pulmonary arteries also experienced higher load and stress than aortic segments at low strains, and ruptured at significantly lower load and stress values, indicating that the pulmonary artery walls are weaker than the aortic walls (P<0.001, n=9; Fig. 4C–E). All aortic segments performed similarly. The left and right pulmonary artery segments also performed similarly initially, but at higher strains (ε>0.4) the right pulmonary artery displayed steeper load–strain and stress–strain relationships than the left pulmonary artery (Fig. 4C,D). Because left and right aortic distal and proximal sections did not differ in their mechanical properties (P=0.152, P=0.688, respectively; n=9), we pooled data for these sections and treated them as complete left and right aorta hereafter.

The load a vessel wall can endure is linked to gross morphological properties, such as cross-sectional area and collagen content. We therefore normalized maximum load values for these two variables to obtain maximum stress and maximum load/unit collagen, the latter of which may be regarded as a measure of ‘collagen quality’ (Fig. 5). In both cases, significant relationships between pulmonary artery and aorta sections persisted (P<0.001, n=9), suggesting that additional morphological factors, besides those investigated here, contribute to the differences in load that these vessels can endure.

Compliance curves calculated from load–strain curves corroborated the relative elasticity of the aortic sections (Fig. 4F). While both the aorta and pulmonary artery walls demonstrated a strong initial relative volume change, these were smaller for the pulmonary arteries (Fig. 4F). Surprisingly, as demonstrated by plotting systolic pressure with the compliance curves, all vessels appeared to operate within the shallow portion of the curve (Fig. 4F), affording them less flexibility in coping with increased blood pressure. Despite these differences in compliance, the elastic modulus, which quantifies the resistance of a vessel wall to deformation, was similar for all tested vessel segments over a broad range of pressure (Fig. 4G), although MAP normalization of pressure indicated that at higher values the elastic modulus of the pulmonary arteries deviated from that of the aortas. Altogether, mechanical tensile testing of aortic and pulmonary artery segments indicated that the pulmonary artery walls were weaker and less distensible than the aortic walls, which may relate to structural features other than those investigated here.

Our data indicate that, despite the lack of intraventricular pressure separation, A. madagascariensis pulmonary artery and aorta walls are morphologically different, and this is reflected in the mechanical properties of these vessels. To better understand the role of intraventricular pressure separation in defining aorta and pulmonary artery mechanical characteristics, we assessed comparative vessel mechanical function in the context of cardiovascular physiology of species with (P. regius; van Soldt et al., 2015) and without (A. madagascariensis) intraventricular pressure separation. We calculated vessel stiffness, modulus and strain at increasing intramural blood pressure of 2, 5 and 10 kPa (Fig. 6), coinciding with systemic and pulmonary systolic pressure of A. madagascariensis (both 5 kPa) and P. regius (10 and 2.7 kPa, respectively; Fig. 2B). Vessel strain captures deformation, while vessel stiffness describes resistance against deformation, in a manner where high stiffness correlates with low strain and vice versa. Finally, modulus normalizes stiffness for vessel cross-sectional area, facilitating inter-vessel comparisons.

Our calculations showed that with increasing intramural blood pressure, stiffness (representing absolute resistance to deformation), modulus and strain values significantly increased for all vessels regardless of species (Fig. 6A,B). However, the relative increases in stiffness, modulus and strain from 2 to 5 kPa and 5 to 10 kPa differed between species and between vessels. In A. madagascariensis, stiffness increased more steeply in aortic segments (from ∼0.025 to ∼0.06 and ∼0.235, P<0.0001; n=9, Fig. 6A) compared with pulmonary segments (from ∼0.025 to 0.04 and ∼0.1, P=0.0249 and P<0.001, respectively; n=9; Fig. 6A). Intriguingly, the reverse was true for P. regius, where stiffness increased more steeply in the two pulmonary arteries, though at different rates (on average from ∼0.025 to ∼0.15 and ∼0.3, P<0.0001; n=9, Fig. 6A), compared with the aortas (∼0.025 to ∼0.028 and ∼0.09, P=0.0042 and P<0.001, respectively; n=9, Fig. 6A). However, upon calculation of the modulus, which normalizes stiffness for vessel cross-sectional area, the statistically significant differences between aorta and pulmonary artery vessel walls in A. madagascariensis almost entirely disappeared, although relationships from 2 to 5 kPa, and from 5 to 10 kPa, were maintained in a similar fashion to stiffness (Fig. 6B). This suggested that in A. madagascariensis, differences in stiffness were explained by differences in vessel wall thickness, whereas in P. regius, they might additionally reflect differences in vessel mechanical properties.

In regard to strain, A. madagascariensis aortas and pulmonary arteries appeared to operate at significantly different levels of strain but strain increased similarly in response to pressure increases (∼2.5 times from 2 to 10 kPa, P<0.0001; n=9; Fig. 6C). In P. regius, however, the pulmonary arteries operate at higher strains than the aortas at 2 kPa (P<0.001, n=9; Fig. 6C) and only display, on average, a ∼20% increase in strain at 10 kPa. In addition, the relative increase in strain was comparatively steeper from 2–5 kPa (P<0.001, n=9; Fig. 6C) than from 5–10 kPa (P=0.0079, n=9; Fig. 6C). In contrast, the aortas displayed significant, linear increases in strain from 2 to 10 kPa (P<0.0001, n=9; Fig. 6C). Combined, these data show that in both species, the increase in vessel stiffness is steeper when blood pressure passes above species-specific systolic pressure, resulting in a polarization of vessel mechanical behaviors in P. regius, where systemic blood pressure and pulmonary blood pressure are significantly different.

We hypothesized that the observed inter-species differences were associated with differences in vessel morphology. We therefore compared morphological parameters (wall thickness, diameter and unit collagen) of vessel walls between these species (Fig. 6D–F). Intriguingly, while wall thickness did not differ (Fig. 6D), A. madagascariensis had significantly narrower pulmonary arteries (P<0.001, n=9; Fig. 6E) with a higher unit collagen (P<0.001, n=9; Fig. 6F) than P. regius. Intriguingly, these differences were mirrored by E. notaeus, highlighting a putative trend between species with and without intraventricular pressure separation.

Thus, A. madagascariensis pulmonary arteries are significantly narrower and weaker than aortas, but nevertheless respond similarly to increases in blood pressure. This stands in contrast to P. regius, where aortas and pulmonary arteries respond differently to increases in blood pressure.

Thoma (1893) was the first to propose that blood circulation impacts blood vessel morphogenesis, describing relationships between blood flow and vessel radius, as well as blood pressure and wall thickness (see Wagenseil and Mecham, 2009). Indeed, the wall of the mammalian aorta is thick and strong in comparison to that of the pulmonary artery, and these differences are associated with high systemic and low pulmonary blood pressure as a result of intraventricular pressure separation postnatally (Gerrity and Cliff, 1975; Leung et al., 1977). Here, we present measurements from Acrantophis madagascariensis demonstrating that morphological and mechanical characteristics of the aorta and pulmonary artery can diverge even in the absence of intraventricular pressure separation, corroborated by data from E. notaeus (Filogonio et al., 2018).

The absence of intraventricular pressure separation in A. madagascariensis is obvious when considering data from Python molurus (Fig. 2B; Wang et al., 2003). Here, systemic systolic pressure (10.01±1.13 kPa) is higher than pulmonary systolic pressure (2.67±0.51 kPa). Importantly, P. molurus systemic systolic pressure was higher (P=0.032) and pulmonary systolic pressure lower (P=0.049) than in A. madagascariensis.

Despite differences in morphology and mechanical characteristics between A. madagascariensis aorta and pulmonary artery walls, these vessels nevertheless responded similarly to increasing wall tension within physiological blood pressure ranges. In contrast, P. regius aorta and pulmonary artery responded differently, consistent with the intraventricular pressure separation in this species that polarizes systemic and pulmonary blood pressure. For example, the stiffness and modulus of all A. madagascariensis vessels increased more steeply from 5 to 10 kPa than from 2 to 5 kPa, in line with a blood pressure of 5 kPa (Fig. 6A,B). In contrast, P. regius pulmonary artery stiffness increased dramatically at pressures over 2 kPa, reflecting the systolic pulmonary blood pressure of 2.7 kPa. This aligns with stiffness/strain calculations in human and pig, where the pulmonary artery was also stiffer than the aorta at systemic blood pressure (Azadani et al., 2012; Matthews et al., 2010). Thus, the aorta and pulmonary artery are each optimized to handle respective physiological blood pressures, regardless of whether these are equal or divergent.

We were initially surprised that A. madagascariensis pulmonary arteries withstood the same blood pressure as the aorta, given their lower strength. However, the law of Laplace intuitively explains that narrow pulmonary arteries do not require strong, thick walls (high cross-sectional area) (Burton, 1965; Shadwick, 1999; Valentinuzzi and Kohen, 2011). Indeed, the pulmonary arteries of A. madagascariensis and E. notaeus were narrower than those of P. regius, but with similar wall thickness (Fig. 6D,E). Thus, the reduction of pulmonary artery radius may be a mechanism to withstand high pulmonary blood pressure in species that lack intraventricular pressure separation.

While theoretically compelling, the narrow pulmonary arteries of A. madagascariensis and E. notaeus were surprising, as narrow vessels implicate low blood volume. Studies in sheep demonstrated that abdominal blood flow and vessel diameter decreased concurrently postnatally (Bendeck and Langille, 1992; Bendeck et al., 1994; Langille, 1996; Langille et al., 1990). In rabbit, aorta and pulmonary artery diameter remain similar postnatally while aorta wall thickness increases, likely to accommodate increasing aortic blood pressure (Leung et al., 1977). Likewise, in P. regius, we previously found that aorta and pulmonary artery diameter were similar, but the aortic wall was thicker (van Soldt et al., 2015). Thus, one possible explanation for the narrow pulmonary arteries in A. madagascariensis and E. notaeus includes the capacity for right-to-left cardiac shunts, whereby systemic blood bypasses the lungs for systemic recirculation, potentially decreasing pulmonary blood flow (Hicks, 1998). Python regius has limited capacity for such shunts (Jensen and Wang, 2009; Jensen et al., 2010b; Wang et al., 2003), and in mammals, such shunts are impossible because of the physical separation of systemic and pulmonary circuits (Hicks, 1998). Thus, these animals may require a wider pulmonary artery to accommodate the volume of blood flowing through these vessels, instead gaining a thicker aortic vessel wall to accommodate the higher systemic blood pressure. Indeed, in the American alligator, Alligator mississippiensis, a species with complete ventricular separation but with an ability to promote right-to-left shunts through the foramen of Panizza, the left pulmonary artery is narrower than the right aorta, corroborating the hypothesis (Filogonio et al., 2021). However, further study is required to ascertain the level of intracardiac shunting in A. madagascariensis and E. notaeus, and the relationship between the separation of systemic and pulmonary circulation and the diameter of the aorta and pulmonary arteries.

We showed that pulmonary artery unit collagen was markedly higher in A. madagascariensis and E. notaeus compared with that in P. regius. Blood vessel wall structure is fundamentally similar across species, but collagen and elastin content are known to differ to change vessel mechanical properties (Shadwick, 1998, 1999). Wall stiffness is primarily mediated by collagen, suggesting that high unit collagen may complement reduced vessel diameter to strengthen the pulmonary arteries (Dobrin, 1978; Sage and Gray, 1979). Structural features of collagen that were not analyzed here, such as fiber diameter, type, cross-linking and alignment, could further contribute to a role for collagen in strengthening pulmonary artery walls (Ottani et al., 2001). Other components of the extracellular matrix, such as glycosaminoglygans, may also determine several morphological and mechanical characteristics of the arterial wall (Gandley et al., 1997).

Given that our data suggest that intraventricular pressure separation alone may not define gross morphological characteristics of the aorta and pulmonary artery, we suggest that ontogenetic factors may also contribute. The endothelium of the dorsal aorta arises through vasculogenesis from the splanchnic mesoderm (DeSesso, 2017), while that of the pulmonary arteries derives from mesodermal progenitors that originate at the posterior pole of the heart and give rise to a vascular plexus that later connects to the pharyngeal arch arteries (DeRuiter et al., 1993; Peng and Morrisey, 2013; Peng et al., 2013). This apparently divergent ontogeny may therefore establish diverging transcriptional programs that ultimately shape different vascular morphologies.

In conclusion, we showed that the absence of intraventricular pressure separation in A. madagascariensis does not equalize the morphologies and mechanical characteristics of its aorta and pulmonary artery. We propose that, to mitigate a higher wall tension as a consequence of increased pulmonary blood pressure, the pulmonary artery became narrower and the collagen content of its wall increased. However, this may only be possible in species with a capacity for right-to-left cardiac shunts as a mechanism to decrease pulmonary blood flow. Finally, we suggest that ontogenetic differences may be fundamental to the morphological differences between these arteries. Thus, in evolution, compensatory mechanisms to accommodate a range of intramural blood pressures may have followed the law of Laplace in different and sometimes unexpected ways.

We thank Jytte Utoft for cutting the vessel sections for histology, Eva K. Mikkelsen for her help in the elastin and hydroxyproline determinations, and Harald G. J. van Mil and Reuben Thomas for their expert help with the statistics. We also thank Catherine Williams for measuring blood pressure in anesthetized A. madagascariensis specimens.