Large artery biomechanical,geometrical,and structural remodeling elicited by long-term propranolol administration in an animal model

Abstract

Background:

Limited attention has been paid to the role of beta-adrenergic blocking agents on large artery function/structure, despite being clinically useful for treating many forms of cardiovascular disease.

Objective:

To assess long-term consequences of beta-blocker administration on the biomechanical properties, geometry, and histological structure of two major elastic arteries.

Methods:

Healthy male rats received water with their food, while beta-blockade was produced in rats by adding propranolol in their drinking water. The thoracic aorta and carotid artery were resected after three months for biomechanical (failure and inflation–extension) testing along with geometrical and histological evaluation.

Results:

The thoracic aorta presented increased strength longitudinally in propranolol-treated than untreated rats, resulting from increased adventitial collagen content. The distensibility of carotid artery increased in propranolol-treated rats at low-to-physiologic pressures, resulting from decreased medial collagen content. Structural remodeling was characterized by reduced lumen diameter, wall mass, and thickness-to-radius ratio. The latter, together with the greater resorption of the media than adventitia, related with the measured opening angle decrease in propranolol-treated rats.

Conclusions:

The geometrical/biomechanical remodeling was mediated by the hemodynamic effects of propranolol treatment, namely the reduced blood flow, and served to normalize in vivo hoop stresses as well as vessel compliance.

Keywords

Elastic arteries propranolol mechanical testing histomorphometry beta-blockade

1. Introduction

Propranolol and other beta-adrenergic blockers are effective and clinically useful for treating many forms of cardiovascular disease [1]. For treating heart failure, and as antianginal, antiarrhythmic, and antihypertensive agents, they reduce the heart rate and force of contraction, relieving the strain to the heart, and simultaneously lowering high blood pressure. Their hemodynamic effects have long been employed in the management of aortic dissection, and they have been used to retard the rate of aortic root dilation and the development of aortic complications in the Marfan syndrome [2–4]. Long-term administration of beta-blockers may also protect against abdominal aortic aneurysm expansion. Reports have provided evidence to suggest that the low expansion rate of abdominal aortic aneurysms among patients receiving beta-blockers was independent of a reduced mean, systolic, or diastolic pressure [5–7]. Other reports have suggested that specific beta-blockers may interact metabolically with aortic tissue. Propranolol suppressed the formation of aortic aneurysms in b-aminopropionitrile fed turkeys; this effect was not related to alterations in heart rate, blood pressure, or rate of pressure change [8]. It has been disclosed in fact that propranolol promoted collagen and elastin cross-linking, thereby increasing the tensile strength of aortic wall, so that the effect of propranolol may rely on the direct interaction with beta2-receptors situated in non-cardiac vascular tissues [9,10].

Surprisingly, limited attention has been paid to the role of beta-adrenergic receptors and their blocking agents on aortic function and structure [11], this being an important issue from the standpoint of cardiovascular patho-physiology. Despite the widespread use of beta-blockers, neither have their short-term effects on aortic mechanics been clarified [12,13], thus, remaining a subject of debate, and, even more importantly, nor have their long-term effects been well defined [14–18]. Specifically, clinical studies looking at in vivo measures of aortic wall stiffness in Marfan patients have reported inconclusive findings, namely reduced stiffness in some patients unlike the unchanged and impaired stiffness in others, and the findings reported cannot be extrapolated to the entire patient population treated with beta-blockers, including patients medically managed for chronic aneurysmal disease or acute aortic dissection and those without aortic disease managed for social and other anxiety disorders, migraine prophylaxis, glaucoma, hyperhidrosis, symptomatic control (tachycardia, tremor) in anxiety/hyperthyroidism, prevention of variceal bleeding in portal hypertension, etc. Still, such information is critical for understanding the consequences of beta-blocker therapy for the entire circulation, since the aorta and other large elastic arteries are not mere conduits of blood, but rather modulate cardiovascular homeostasis through the buffering of the pulsatile output of the heart and its translation into a steady flow in the periphery, while regulating left ventricular function and myocardial perfusion [19].

Toward this end, animal studies and in vitro testing may provide added insight into the geometrical, histological, and biomechanical aspects of large artery remodeling in response to chronic beta-blocker administration, since controlled experiments and detailed analysis can be done that are not accessible with clinical studies. Our group [20,21] has previously reported limited compositional and uniaxial tension data along the longitudinal direction for the aorta but, to our best knowledge, no multiaxial testing and failure data are available with reference to a properly-defined zero-stress state. The present study fills this gap by providing a comprehensive assessment of the chronic influence of beta-blocker administration on the biomechanical properties of two major elastic arteries, i.e. the thoracic aorta and carotid artery in an animal model, and by examining how modifications in these properties are associated with the geometrical and structural remodeling of the two vessels. Information on the multiaxial properties and strength of large arteries and on their zero-stress state is needed for a detailed biomechanical analysis related to propranolol-induced remodeling.

2. Experimental methods

2.1. Animals and beta-blockade

Beta-blockade was produced in fifteen male Wistar rats (3 months old, weighing 301 ± 6 g) by administration of propranolol hydrochloride in their drinking water, in a dose of 100 mg/kg per day. The rats were individually caged and had free access to rat chow and tap water. Male rats were selected in these experiments to minimize potential effects of sex hormones on arterial structure that may be more prevalent in females. Ten control rats matched by age, weight, and gender were housed under identical conditions, but were not given propranolol. Animal handling complied with the guiding principles of the American Physiological Society and the Greek Presidential Decree 160/1991, issued after the European Union Directive 609/1986.

Fig. 1.

Schematic diagram of the experimental procedures, showing the placement of the perivascular flow probe and the pressure transducer, as well as the segments of the descending thoracic aorta and left common carotid used for the failure and inflation/extension tests, respectively, and the rings used for geometrical and histological studies.

2.2. In vivo measurements and tissue preparation

Animals of both groups were euthanized 3 months after the commencement of propranolol or vehicle treatment. They were sedated with ketamine (100 mg/kg, ip) and xylazine (5 mg/kg, ip), and blood flow and pressure measurements were made under full anesthesia. Animals were connected to an anesthetic machine (MDS Matrx, Orchard Park, NY, USA) and to a volume control respirator (Inspira, Harvard, MA, USA), and isoflurane 1–2% (vaporizer setting) in oxygen (1 l/min) was administered for the maintenance of anesthesia. The chest was opened through a median sternotomy, and a 2.5-mm perivascular flow probe (2.5 PSB, TS420, Transonic Systems, Ithaca, NY, USA) was placed around the ascending aorta (Fig. 1) to provide continuous ultrasonic measurement of the mean cardiac output. Arterial pressure was measured using a 1.4F catheter-tipped transducer (SPC-450, Millar Instruments, Houston, TX, USA) inserted through the right common carotid artery and the tip placed into the ascending aorta (Fig. 1). Both hemodynamic parameters were monitored for 10 min, and the average systolic, diastolic, and mean pressure, together with the mean cardiac output, were recorded and analyzed using a digital acquisition system (Sonometrics System, Sonometrics Co, Ontario, Canada). The pressor response to isoprotenerol, an indirect agonist that releases endogenous norepinephrine from sympathetic nerve terminals, was used to confirm the efficacy of beta-adrenergic receptor blockade, as reported in our previous study [21].

Fig. 2.

(A) Photos of representative segments of the descending thoracic aorta from a propranolol-treated and an untreated rat. Note the markedly smaller dimensions of the vessel from the propranolol-treated rat. The smaller photos next to the larger ones show the longitudinally-opened specimens used for the failure tests and for measuring the distances between the successive pairs of intercostal branches; the latter are drawn with lines of yellow colour and numbered consecutively. (B) In situ and ex situ lengths, (C) longitudinal distance between the intercostals, and (D) in situ longitudinal stretch ratio for the descending thoracic aorta from propranolol-treated and untreated rats, and their statistical comparison. $NS$ denotes non-significant differences.

Euthanasia was induced with an intravenous overdose of pentobarbital sodium and the aortic segment was harvested from the left subclavian artery to the diaphragm, using eye surgery loupes (Heine HRP, Heine Optotechnik, Herrsching, Germany) to avoid damaging the wall, as was the left common carotid artery that was exposed via a midline neck incision (Figs 1 and 2). The longitudinal in situ length of these vessels was determined by measuring the distances between two superficial surgical knots. Loose peri-adventitial tissues were cautiously trimmed and the ex situ lengths measured. The longitudinal in situ stretches, defined as the ratio of in situ to ex situ segment lengths, were computed. 3-mm width aortic (at the level of the third intercostal arteries) and carotid (from the proximal edge of the excised vessel) ring specimens were fixed with formaldehyde. Two neighboring 1-mm width rings were taken at the level of the first intercostals and the diaphragm from the aorta as well as from the most proximal and distal parts of carotid artery, and kept for geometrical studies. The left-over aortic and carotid specimens were kept for biomechanical studies within 5 h after euthanasia, during which they were stored in Krebs-Ringer solution at 4°C and slowly warmed up to 37°C prior to testing.

2.3. Histological studies

Formaldehyde-fixed aortic and carotid artery cross-sections were processed according to routine histological procedure, paraffin-embedded, and cut into 5-µm sections. After deparaffinization and rehydration, adjacent sections were treated with hematoxylin-eosin for nucleus, orcein for elastin, and picro-Sirius red for collagen staining. Pictures were taken with an optical microscope (Olympus CX31, Olympus, Tokyo, Japan) connected to a video-camera (Altra20, Soft Imaging System, Munster, Germany), and processed with image-analysis software (Image-Pro Plus v4.5, Media Cybernetics Inc, Bethesda, MD, USA), as previously [22–24]. Measurements of vascular morphometric parameters were performed on hematoxylin-eosin stained sections by hand-tracing the luminal and wall areas, as a result of which the inner and outer diameters, wall thickness, and thickness to diameter ratio were calculated by the software upon the assumption of a circular cross-section. The thickness and area of media and adventitia were similarly measured, with the former delimited by the internal and external elastic lamina, and the latter by the external elastic lamina and the external edge of its dense part. Measured parameters were corrected by 32% for shrinkage artifacts, based on comparison of fresh tissue with that processed for histology, as conducted in pilot studies; resembling the 30% diameter reduction found in [25] for pressure-fixed aortic specimens on an otherwise similar processing protocol, unlike the smaller shrinkage disclosed for arterial tissues embedded in methyl methacrylate [26].

Elastin and collagen area densities were determined after color segmentation of the pictures, with orange for elastin and red for collagen. The area density of smooth muscle cells and remainder, e.g. ground substance, was determined as 100-area densities of elastin and collagen, assuming that the surface areas occupied by all components add up to the entire area of vessel media. Adventitia elastin, collagen, and remainder were similarly analyzed in terms of their relative area densities, assuming they all add up to 100%. According to the laws of stereology (“Delesse principle” [27]), the area density of a component in a biologic sample is equivalent to its volume density. The relative area densities of all measured components in each layer of the vessel wall were calculated with respect to the total area of that layer in the optical field. All measurements and quantifications were carried out blinded in optical fields every 10° around the vessel circumference and three sections to estimate values for each specimen.

2.4. Geometrical studies

After excision, the two 1-mm width rings from each anatomical location of the thoracic aorta and the carotid artery were placed inside a Petri dish containing Krebs-Ringer solution at 37°C, let to equilibrate for 30 min under no load, and photographed with a digital camera (model E400, Olympus Optical Co Ltd, Tokyo, Japan) coupled to a dissecting microscope (OP 5F, SFBN Mechanik, Bucha, Germany). They were then cut radially at their anterior aspect under the microscope, a procedure that caused them to open into sectors, supposedly relieving all stresses from the vessel wall [28]. After waiting for another 30 min for viscous phenomena to subside, pictures were taken of the open configuration. The inner and outer circumference of the rings, together with their thickness and cross-sectional area were measured from the pictures in the no-load (closed) and the zero-stress state (open configuration) with the aid of the Image-Pro Plus software. The opening angle was also measured as representative of circumferential residual strains, determined as the angle between the first extremity, the middle point, and the second extremity of the inner arc [28]. Results for each vessel were average measurements from the two rings and the two anatomical locations.

2.5. Biomechanical studies on aorta

2.5.1. Failure testing

From the aortic specimen retained for the biomechanical studies, one strip was prepared in the circumferential and another in the longitudinal direction (Fig. 2), and subjected to failure testing on a Vitrodyne V1000 Universal Tester (Liveco Inc, Burlington, VT, USA), as in past studies from our laboratory [22,29]. They were mounted in the grips of the apparatus using small pieces of non-slip paper. The lower grip was fixed while the upper was attached to the actuator that gradually stretched the strips at a 10-µm/s rate until rupture. To sustain normal tissue hydration, they were immersed in a saline bath regulated at 37°C via a heater coil (1130A, PolyScience, Niles, IL, USA). Their initial dimensions were recorded at no-load; their initial length by measuring the grip to grip distance, and their initial width and thickness by optical measurements using a laser beam micrometer (LS-3100, Keyence Corp, Osaka, Japan; 1-µm resolution) at four equidistant locations along the strips’ length and taking the average. The apparatus utilized load cells with 0.01-g resolution for the evaluation of load and a rotary encoder providing feedback on extension with 10-µm accuracy.

2.5.2. Data processing

Stretch was defined as the ratio of the strips’ deformed length at each load to their initial length and Cauchy stress as the ratio of load exerted to the strips times stretch to their initial width and thickness assuming tissue incompressibility. The stress–stretch data were regressed with polynomial functions of 7th–9th order using MicroCal Origin v8.5 (OriginLab^® Corp, Northampton, MA, USA), affording correlation coefficients >0.95, and elastic modulus (tangent) at each stretch value was calculated as the first derivative of stress with respect to stretch. Failure stress and stretch, representing tissue strength and extensibility respectively, were calculated as the maximum stress and stretch values. Peak elastic modulus, i.e. a measure of maximum tissue stiffness, was calculated as the highest slope of the stress–stretch curve prior to rupture.

2.6. Biomechanical studies on carotid artery

2.6.1. Inflation/extension testing

The carotid artery specimens were mounted at their in situ length in the experimental apparatus previously described from our laboratory [23,24]. They were cannulated on stainless steel catheters at both ends, filled with Krebs-Ringer solution (with 10⁻⁴ mol/l papaverine), and immersed horizontally in a bath filled with the same fluid, held at 37°C and aerated with a gas mixture of 95% O₂–5% CO₂ at pH 7.4. The catheter at one end was connected to a rigid support and that at the other end to a force transducer (Fort 100; World Precision Instruments, Hertfordshire, UK), measuring longitudinal force with 0.25-g accuracy. The force transducer was suspended from a micrometer (Tesa Technology, Renens, Switzerland), allowing longitudinal extension of the specimen. The lumen was inflated and deflated from 0 to 200 mmHg at a 0.15-mmHg/s rate using a syringe pump (model sp100i2; World Precision Instruments). Lumen pressure was monitored by a pressure transducer (BLPR; World Precision Instruments) with 0.5-mmHg accuracy and outer diameter by the LS-3100 laser micrometer with 1-µm accuracy. Ten cycles were conducted to reduce viscoelasticity and acquire reproducible data. The inflation part of the last cycle was kept for data analysis assuming negligible smooth muscle tone. All the data were amplified by a four-channel transducer (Transbridge 4M, World Precision Instruments), collected on a data acquisition card (DAQmx 6009, National Instruments, Austin, TX, USA), and stored by the Labview interface (v7.1, National Instruments) on an escorting computer.

2.6.2. Data processing

The circumferential and longitudinal stretch ratios $λ_{θ}$ and $λ_{z}$ were defined with respect to the zero-stress state as: $\begin{matrix} (1) & λ_{θ} = \frac{s}{S}, λ_{z} = \frac{l}{L}, \end{matrix}$ in terms of circumference S in the reference (zero-stress) state (see Section 2.4) and circumference s in the deformed (no-load or loaded) states; and in terms of ex situ and in situ lengths L and l (see Section 2.2). Circumferential residual strains $E_{θ}$ were determined at the inner (intimal) and outer (adventitial) wall boundaries, using Green’s definition: $\begin{matrix} (2) & E_{θ} = \frac{1}{2} (λ_{θ}^{2} - 1) . \end{matrix}$

The diameter–pressure-force data were regressed with polynomial functions of 7th–9th order using MicroCal Origin and the ensuing biomechanical parameters were calculated on the regressed data. Inner diameter $d_{i}$ of carotid artery was calculated from outer diameter $d_{o}$ , measured during inflation/extension, the in situ longitudinal stretch ratio $λ_{z}$ , and the cross-sectional wall area $A_{0}$ at zero-stress state (see Section 2.4), assuming tissue incompressibility for a cylindrical vessel: $\begin{matrix} (3) & d_{i} = \sqrt{d_{o}^{2} - \frac{4 A_{0}}{π λ_{z}}} . \end{matrix}$ The area compliance C and distensibility D were calculated from the lumen area $A = π d_{i}^{2} / 4$ and pressure P data: $\begin{matrix} (4) & C = \frac{\partial A}{\partial P}, D = \frac{1}{A} \frac{\partial A}{\partial P}, \end{matrix}$ and used as structural properties of the carotid artery. The mean circumferential and longitudinal Cauchy stresses $σ_{θ}$ and $σ_{z}$ were calculated as: $\begin{matrix} (5) & σ_{θ} = \frac{P d_{i}}{d_{o} - d_{i}}, σ_{z} = \frac{4 F + P π d_{i}^{2}}{2 π (d_{o}^{2} - d_{i}^{2})}, \end{matrix}$ where F denoted longitudinal force. The mean circumferential Green strain $E_{θ}$ was calculated via Eq. (2) in terms of mid-wall circumference at the reference (zero-stress) and deformed states, given by $S = (S_{o} + S_{i}) / 2$ and $s = (s_{o} + s_{i}) / 2$ . For a detailed exposition of the definitions presented in this section, the interested reader may refer to standard cardiovascular biomechanics textbooks, e.g. [28].

2.7. Statistics

Data are presented as mean ± standard error of the mean (SEM). For comparisons among the propranolol-treated and untreated (control) rats, the unpaired Student’s t-test was used on SPSS v20.0 (SPSS Inc, Chicago, IL, USA). Differences were considered significant at $p < 0.05$ .

3. Results

3.1. Hemodynamic parameters

Significant reductions of diastolic aortic pressure, cardiac output, and heart rate were found in propranolol-treated rats (Table 1). Heart rate remained virtually constant following isoprotenerol administration in those rats, while increasing in untreated rats, substantiating the efficacy of beta-blockade by the propranolol treatment used.

Table 1
Hemodynamic recordings from propranolol-treated and untreated rats, and their statistical comparison

Arterial pressure (mmHg) Cardiac output (ml min⁻¹) Heart rate (beats min⁻¹)

Systolic Mean Diastolic

Propranolol-treated rats 85.0 ± 3.3 68.6 ± 2.9 53.9 ± 2.9^* 37.5 ± 2.0^* 205.4 ± 5.6^*

Untreated rats 87.1 ± 2.3 74.3 ± 2.4 64.0 ± 2.1 45.6 ± 3.3 233.1 ± 14.9

	Arterial pressure (mmHg)	Cardiac output (ml min⁻¹)	Heart rate (beats min⁻¹)
Propranolol-treated rats	85.0 ± 3.3	68.6 ± 2.9	53.9 ± 2.9^*	37.5 ± 2.0^*	205.4 ± 5.6^*
Untreated rats	87.1 ± 2.3	74.3 ± 2.4	64.0 ± 2.1	45.6 ± 3.3	233.1 ± 14.9

$p < 0.05$ vs. untreated rats.

Fig. 3.

Effect of chronic propranolol treatment on histomorphometric parameters of (A and E) outer and inner diameters, (B and F) thickness of vessel wall, media, and adventitia, (C and G) ratio of wall thickness to inner diameter, and (D and H) area of vessel wall, media, and adventitia in the no-load state for (A–D) the descending thoracic aorta and (E–H) carotid artery from propranolol-treated and untreated rats. p-values are shown for between-groups comparisons. $NS$ denotes non-significant differences.

3.2. Morphometry and residual strains of the thoracic aorta and carotid artery

Figure 3 shows the morphological parameters evaluated on the histological sections; see Figs 4 and 5 for representative sections. The inner diameters of both vessels were smaller in propranolol-treated rats and their outer diameters were even smaller, by virtue of their thinner wall. Smaller was also the ratio of wall thickness to inner diameter in propranolol-treated than untreated rats. These findings were corroborated by the respective parameters measured from the digitized pictures of the thoracic aorta and carotid artery in the no-load state (data not shown). A decrease in the area and thickness of the media and entire wall was further observed via histo-morphometry in propranolol-treated rats. Note in Fig. 3 that the same trends were observed in both vessels, yet differences were less significant in the carotid artery.

Fig. 4.

Representative histological sections of the descending thoracic aorta from an untreated (A) and a propranolol-treated rat (B) stained with hematoxylin-eosin. Higher (×40) magnifications of the rectangular regions of interest are shown in the insets, positioned to the right of the low (×2.5) magnifications, which are stained with hematoxylin-eosin (C and D), orcein (E and F), and picro-Sirius red (G and H).

Fig. 5.

Representative histological sections of the left common carotid artery from an untreated (A) and a propranolol-treated rat (B) stained with hematoxylin-eosin. Higher (×40) magnifications of the rectangular regions of interest are shown in the insets, positioned to the right of the low (×4) magnifications, which are stained with hematoxylin-eosin (C and D), orcein (E and F), and picro-Sirius red (G and H).

The results for the opening angle and residual strains at the intimal and adventitial surfaces of the thoracic aorta and carotid artery are listed in Table 2. The mean opening angle from the two thoracic segments examined and that from the two carotid segments decreased significantly in propranolol-treated rats. However, the compressive intimal residual strain in the former and the tensile adventitial residual strain in either vessel did not vary significantly between propranolol-treated and untreated rats, nor did the longitudinal stretch ratio at the in situ condition, as the two vessels underwent analogous shortening from the in situ to the ex situ condition (Table 2). Non-significant was also the difference in longitudinal distance between the intercostal arteries among propranolol-treated and untreated rats, despite the significantly smaller length of the thoracic aorta in the former (Fig. 2).

3.3. Histological composition of thoracic aorta and carotid artery

The histological composition of the thoracic aorta and carotid artery is presented in Fig. 6 for propranolol-treated and untreated rats. Concerning the media components, no change was observed in the area density of elastin, but we observed in propranolol-treated rats a significant decrease in the area density of collagen and a concomitant increase in that of smooth muscle cells and remaining components; the latter reached significance only in the thoracic aorta. Concerning the adventitia components, a significant increase in the area density of collagen, no change in the area density of elastin, and a decrease in that of the remaining components were noted in the thoracic aorta of propranolol-treated group, whereas no differences in the adventitia components were noted in the carotid artery. Collectively, the relative area densities of elastin, collagen, and remaining components of the entire wall did not differ among propranolol-treated and untreated rats.

3.4. Thoracic aorta biomechanical properties

Shown in Fig. 7 are the individual results of the failure experiments for all thoracic aortic specimens resected from propranolol-treated and untreated rats. The typical exponential shape of the stress–stretch curves before failure was apparent in both animal groups and specimen directions, but significant group differences were noted in longitudinally-oriented specimens. The central body of the curves from propranolol-treated rats was situated in the upper part of the diagram compared to that from untreated rats (Fig. 7(B)), unlike the curves of circumferentially-oriented specimens that were coincident (Fig. 7(A)). In either group of animals, the curves of longitudinally-oriented specimens were on average directed on the upper left part of the diagrams relative to those of circumferentially-oriented specimens.

Table 2
Morphometric parameters and residual strains in the no-load state for the descending thoracic aorta and carotid artery from propranolol-treated and untreated rats, and their statistical comparison

Opening angle (deg) Inner residual strains (−) Outer residual strains (−) In situ longitudinal stretch ratio (−) Distance between intercostals (mm)

Descending thoracic aorta

Propranolol-treated rats 29.1 ± 3.5^* −0.031 ± 0.009 0.036 ± 0.008 1.289 ± 0.022 2.816 ± 0.049

Untreated rats 42.7 ± 3.2 −0.045 ± 0.007 0.048 ± 0.011 1.281 ± 0.024 2.870 ± 0.059

Carotid artery

Propranolol-treated rats 75.0 ± 5.5^* −0.014 ± 0.034^* 0.063 ± 0.050 1.647 ± 0.059 –

Untreated rats 93.2 ± 7.6 −0.131 ± 0.039 0.169 ± 0.039 1.663 ± 0.028 –

	Opening angle (deg)	Inner residual strains (−)	Outer residual strains (−)	In situ longitudinal stretch ratio (−)	Distance between intercostals (mm)
	Descending thoracic aorta
Propranolol-treated rats	29.1 ± 3.5^*	−0.031 ± 0.009	0.036 ± 0.008	1.289 ± 0.022	2.816 ± 0.049
Untreated rats	42.7 ± 3.2	−0.045 ± 0.007	0.048 ± 0.011	1.281 ± 0.024	2.870 ± 0.059
	Carotid artery
Propranolol-treated rats	75.0 ± 5.5^*	−0.014 ± 0.034^*	0.063 ± 0.050	1.647 ± 0.059	–
Untreated rats	93.2 ± 7.6	−0.131 ± 0.039	0.169 ± 0.039	1.663 ± 0.028	–

$p < 0.05$ , propranolol-treated vs. untreated rats.

Empty entry – no data available.

Fig. 6.

Effect of chronic propranolol treatment on the relative area density of elastin, collagen, and vascular cells in the entire wall, and its medial and adventitial layers for the (A) descending thoracic aorta and (B) carotid artery from propranolol-treated and untreated rats. p-values are shown for between-groups comparisons. $NS$ denotes non-significant differences, SMC denotes smooth muscle cells, and Other denotes media and adventitia components apart from elastin, collagen, or SMC.

Fig. 7.

Stress–stretch data of all specimens with (A) circumferential (CIRC) and (B) longitudinal (LONG) orientation from the descending thoracic aorta of propranolol-treated and untreated rats. (C) Effect of chronic propranolol treatment on the biomechanical parameters of failure stress, failure stretch, and peak elastic modulus determined from failure testing. p-values are shown for between-groups comparisons. $NS$ denotes non-significant differences.

Data analysis disclosed that the failure stress and peak elastic modulus of the aortic wall were significantly higher in propranolol-treated than untreated rats for longitudinally-oriented specimens, while not differing for circumferentially-oriented specimens (Fig. 7(C)). No statistical difference among propranolol-treated and untreated rats was found in failure stretch of specimens with either orientation. For each animal group, failure stress and peak elastic modulus of longitudinally-oriented specimens were significantly higher than those of the respective circumferentially-oriented specimens, while the opposite was true for failure stretch.

3.5. Carotid artery biomechanical properties

Shown in Fig. 8 are the results of inflation/extension experiments on cannulated common carotid arteries at the in situ stretch ratio, incubated in Krebs-Ringer solution with papaverine to preserve a passive smooth muscle tone. The inner radius vs. lumen pressure data from both propranolol-treated and untreated rats exhibited a bi-phasic curve shape, specifically a preliminary low-pressure phase from 0 to 80 mmHg corresponding to a wide diameter range and a terminal phase at physiological and high pressures with diameter locking. Despite the similar curve shapes, inner radius was smaller over the entire range of lumen pressures in propranolol-treated than untreated rats (Fig. 8(A)), and longitudinal force and wall thickness were greater (Fig. 8(B) and (C)). Furthermore, pressure inflation generated a larger diameter enlargement at low pressures in propranolol-treated rats, namely a wider preliminary phase and a similar appearance of the terminal phase, implying a more distensible wall than that of untreated rats at low pressures (Fig. 8(D)). Figures 8(F) and (G) depict the cumulative stress vs. stretch data, where it is noted that the circumferential data for propranolol-treated rats were positioned lower than that of untreated rats, indicating a less stiff wall in the former relative to the latter.

Fig. 8.

Effect of chronic propranolol treatment on the average (A) inner radius, (B) longitudinal force, and (C) wall thickness vs. lumen pressure data determined from inflation-extension testing of the carotid artery. The biomechanical remodeling elicited by propranolol is also presented in terms of (D) distensibility and (E) compliance vs. lumen pressure curves, and in terms of (F) longitudinal and (G) circumferential stress vs. circumferential stretch curves. Data points were averaged at specific values of lumen pressure from 0 to 200 mmHg in steps of 10 mmHg.

4. Discussion

4.1. Biomechanical remodeling of large arteries by propranolol treatment

Virtually all available information on the biomechanical remodeling of large arteries elicited by propranolol treatment has relied on human in vivo studies, having direct clinical relevance [11–18]. Basic non-invasively measurable quantities have been reported, i.e. aortic pressure and diameter, that have been reduced in terms of gross measures of overall structural stiffness; direct ones such as aortic distensibility and pulse wave velocity, and indirect such as augmentation index. Such measures are useful for comparisons among untreated and propranolol-treated Marfan patients, but the mechanisms involved in their alterations are difficult to analyze because of the complexity and number of their determinants, including the set point of distending pressure, passive tissue elements, smooth muscle tone, and support of perivascular tissue, most of which cannot be delineated by in vivo experiments and may thus be responsible for the inconsistent findings reported. The need of in vitro experimentation is evident, since it is in this setting that the many parameters of interest can be controlled and detailed analysis of both the circumferential and longitudinal response of arterial tissue can be done, which is essential for complete biomechanical characterization; see the discussion by Humphrey [28].

Our group has previously documented the long-term effects of propranolol [20], reporting that the thoracic aorta underwent progressive stiffening with the duration of treatment. Based on those time-course data, the 3-month time point was selected herein for estimating the effects of propranolol. In analogy to that communication, this work demonstrated that maximum aortic stiffness in propranolol-treated rats was significantly greater than that in untreated rats but only longitudinally (Fig. 7(C)), while circumferential properties are physiologically more relevant, as they determine aortic compliance and pulse pressure. Importantly, the present failure stress data are consistent with early studies [9,10] that evidenced increased tensile strength of the aortic wall by propranolol treatment through stimulation of lysyl oxidase activity, and promotion of collagen and elastin cross-linking.

Note, however, that even though the present uniaxial tension data may be superior compared with our previous findings in that the biomechanical properties were determined along both vessel axes and over a wide range of stresses from zero up to the level of material failure, this testing methodology does not correspond to optimal physiological conditions. The strip tests performed are limited to one-dimensional stress fields, and the structural integrity of the vessel wall is not preserved, making it difficult to apply the acquired information on the biomechanical properties to intact vessel segments, since there is decoupling of the circumferential and longitudinal loads and deformations as experienced physiologically. Accordingly, the presently-reported inflation/extension testing data for carotid artery (Fig. 8) are considered as more physiologically pertinent for large artery biomechanical analysis.

4.2. Geometric and structural remodeling in association with biomechanical remodeling

Together with the remodeling of biomechanical properties, significant morphological and structural changes developed in the thoracic aorta and carotid artery. Both underwent thinning, characterized by a decrease in diameter and wall thickness, and in lumen and wall areas (Fig. 3), but their longitudinal stretch ratio was not affected by propranolol treatment (Table 2). We monitored the length of the thoracic aortic segment at euthanasia and found that it was always shorter in propranolol-treated rats (Fig. 2). Dobrin et al. [30] reported that the longitudinal retractive force in arteries is mainly exerted by elastin, and Langille et al. [31] argued that a reduced stretch ratio may be ascribed to vessel growth, effected both circumferentially and longitudinally. Overall, the non-varying stretch ratio in this study may be an outcome of vessel diminution and the invariant elastin content.

We have reported for the uniaxial tension experiment that passive stiffness is determined by the area density and the ultrastructure of elastin at low stresses, of collagen at high stresses, and of both elastin and collagen at physiologic stresses [32]. Notwithstanding possible promotion of collagen and elastin cross-linking [9,10] that was not assayed herein, changes in collagen densities may be chief factors leading to the biomechanical remodeling in response to chronic propranolol treatment. The increase in adventitial collagen density of thoracic aorta (Fig. 6(A)), having a substantial longitudinal component, may be related to the increased longitudinal strength of the aortic wall in propranolol-treated rats (Fig. 7(B) and (C)). Using similar reasoning, the lower medial collagen density in the carotid artery of propranolol-treated rats that is circumferentially oriented (Fig. 6(B)) may be associated with the increased distensibility at low-to-physiologic pressures (Fig. 8(D)) and the lower circumferential stresses at all stretch levels (Fig. 8(G)). The invariant adventitial collagen density that is oriented mostly longitudinally may account for the non-varying longitudinal stress–circumferential stretch curves (Fig. 8(F)). Note, however, that light densitometry allows at best semi-quantification of elastin and collagen contents. Errors may have entered by co-localization of various proteins, deviations in fiber density at various levels of resolution, and ambiguous demarcation of the external edge of adventitia.

4.3. Zero-stress state remodeling of large arteries by propranolol treatment

In association with the biomechanical and structural remodeling, the zero-stress state of large artery tissue was modified. The opening angle is a measurement characterizing the residual stresses existing in the tissue at the no-load state. Their existence is profitable for vessel function by way of reducing the stress concentration at the inner (intimal) part of arterial wall under physiologic loads [28,33,34]. We found that the opening angle varied with location on the arterial tree and structural remodeling. In particular, the decrease in the opening angle of specimens from the first intercostals and the diaphragm, and from the proximal and distal common carotid artery resulting from propranolol treatment (Table 2) seems to associate with the decrease in wall thickness to inner diameter ratio (Fig. 3). The reduced opening angle in the propranolol-treated than untreated rats corroborates Fung’s hypothesis, that is of the inner wall suffering greater resorption than the outer, as we found the media of the thoracic aorta and carotid artery from propranolol-treated rats to be thinner than that of the arteries from untreated rats, unlike the adventitia that was equally thick (Fig. 3). Changes in opening angle may be ascribed as well to changes in the material properties (see for instance [35]), so that the reduced opening angle in propranolol-treated rats may be related to the circumferentially softer carotid artery (Fig. 8).

4.4. Consideration of the hemodynamic effects of propranolol

Following prolonged propranolol administration, there was a significant reduction in heart rate and cardiac output (Table 1), which is in line with early [36] and recent [37] studies, disclosing 30% fall in conscious rats given doses of 20–100 mg/kg/day.

Table 3
Morphometric and biomechanical parameters, calculated at the mean in vivo arterial pressure, for the descending thoracic aorta and carotid artery from propranolol-treated and untreated rats, and their statistical comparison

Inner diameter (mm) Wall thickness (mm) Circumferential stress (kPa) Distensibility (mmHg⁻¹) Compliance (mm²/mmHg)

Descending thoracic aorta

Propranolol-treated rats 1.668 ± 0.035^* 0.184 ± 0.012 325 ± 25 – –

Untreated rats 1.930 ± 0.132 0.197 ± 0.016 391 ± 51 – –

Carotid artery

Propranolol-treated rats 0.961 ± 0.029^* 0.086 ± 0.009 44 ± 5 0.01406 ± 0.00116 0.0104 ± 0.00104

Untreated rats 1.063 ± 0.052 0.067 ± 0.007 69 ± 13 0.01109 ± 0.00117 0.00998 ± 0.0016

	Inner diameter (mm)	Wall thickness (mm)	Circumferential stress (kPa)	Distensibility (mmHg⁻¹)	Compliance (mm²/mmHg)
	Descending thoracic aorta
Propranolol-treated rats	1.668 ± 0.035^*	0.184 ± 0.012	325 ± 25	–	–
Untreated rats	1.930 ± 0.132	0.197 ± 0.016	391 ± 51	–	–
	Carotid artery
Propranolol-treated rats	0.961 ± 0.029^*	0.086 ± 0.009	44 ± 5	0.01406 ± 0.00116	0.0104 ± 0.00104
Untreated rats	1.063 ± 0.052	0.067 ± 0.007	69 ± 13	0.01109 ± 0.00117	0.00998 ± 0.0016

$p < 0.05$ , propranolol-treated vs. untreated rats.

Empty entry – no data available. The inner diameter was calculated by Eq. (3) from the outer diameter, measured during inflation/extension for the carotid artery and prior to resection, i.e. in the in vivo state, for the thoracic aorta.

It is tempting to speculate that the presently-reported arterial remodeling is at least partially an outcome of chronically-reduced blood flow and not independently controlled. The effects of altered blood flow have been examined to a considerable degree in arteries; those through which flow has been reduced undergoing diminution of their lumen radius, unlike those through which flow has been enhanced by e.g. distal arteriovenous anastomoses that tend to enlarge in size [24,38]. Interestingly, it appears that the chronic adjustment in vessel caliber after propranolol treatment (see Figs 3(A) and 8(A), and Table 3) is similar to that reported for reduced flow and so is the chronic adjustment in wall thickness at in vivo pressure (Fig. 8(C) and Table 3) that is increased in flow reduction ([39] and our unpublished data), despite the smaller thickness at no-load (Fig. 3(B) and (F)), similar to observations in rat mesenteric arteries after 4-week exposure to reduced flow [40]. Rudic et al. [41] previously deduced that the active length-tension response was identical and displaced to smaller diameters in large arteries adapted to long-term flow reduction, yet the shape of our curves was modified apart from being shifted to smaller diameters (Fig. 8(D)–(G)). A fall in the opening angle of carotid arteries exposed to flow-overload has also been reported by Lu et al. [42] and Kritharis et al. [24], which is counterintuitive to the reduced opening angle found herein (Table 2). However, much like the present data, earlier studies showed no change in wall scleroprotein contents in flow reduction [31].

An important, much related issue is whether the presently-reported remodeling is independent of sympathetic nerves. Two different large elastic arteries were considered, the descending thoracic aorta and common carotid artery of the normotensive male Wistar rat. Both are conduit arteries that are poorly innervated but with beta-receptor activity; see for instance [43] for an early and [44–46] for recent studies, so that it is not likely that remodeling was driven by sympathetic nerves. Note, too, that the structural remodeling of porcine thoracic aorta induced by surgical thoracic sympathectomy in our recent work [47] included increased inner aortic diameter, wall thickness, and scleroprotein contents, i.e. the exact opposite to the present data. Therefore, the interaction with beta-receptors appears to be the principal factor responsible for the remodeling induced by propranolol. However, future studies with cardio-selective beta-blockers without blocking activity in the vascular system could ascertain whether the mechanism of action stems from beta receptors located in the heart.

Our study shows that the geometrical remodeling elicited by propranolol-treatment resulted three months after the initiation of treatment in restoration of wall stress and compliance at levels similar to those found in untreated rats (Table 3). Note that similar restoration of wall stress and compliance, as well as of shear stress and smooth muscle tone was attained in pressure overload (see the seminal contributions by Hayashi, Matsumoto, Stergiopulos, and colleagues [48,49] and the review article by Hayashi and Naiki [50]), whereas the geometrical and biomechanical adaptation in flow overload led to restoration of only wall and shear stress [24].

4.5. Limitations and clinical implications

Unfortunately, we only measured aortic pressure and cardiac output in the current experiments, although flow through each vessel would have been needed to measure shear rates. Evidence from an animal study [51], however, suggests that there may be no significant redistribution of the cardiac output so that changes in flow to the various regional circulations (and particularly to the left common carotid and descending thoracic aorta) may result primarily from the reduced cardiac output caused by propranolol. Then, the decreased inner diameter of both vessels at in vivo pressure shown in Table 3 would lead to normalization of the reduced cardiac output and thus to shear rate restoration at levels similar to those in untreated rats. Note also that the invasive flow measurements were performed using a perivascular flow probe, while use of non-invasive ultrasound to measure cardiac output might have produced more reliable results, and the pressure probes used for the invasive pressure measurements have been recently shown to considerably affect central artery hemodynamics in mice [52]. A final note regards the biomechanical characterization of the aorta and large arteries which is complex and presents many challenges. No single arterial segment exhibits the same properties or may be anticipated to have identical remodeling capabilities, so that it is unreasonable to extrapolate segmental properties to the entire arterial tree, not even to all elastic arteries.

Preliminary clinical importance may be ascribed to the unchanged large artery stiffness at in vivo pressure after chronic beta-blockade. It is hypothesized that when normotensive patients are treated with beta-blockers for a sufficiently long time, as in the case of essential tremor, glaucoma, migraine prophylaxis, phaeochromocytoma (in conjunction with alpha-blockers), symptomatic control (tremor, tachycardia) in anxiety and hyperthyroidism, theophylline overdose, prevention of variceal bleeding in portal hypertension, possible mitigation of hyperhidrosis, social and other anxiety disorders, and, controversially, for reduction of preoperative mortality, etc, their large arteries might undergo structural remodeling but not stiffening, although confirmation waits for clinical studies that will determine in normotensive patients the long-term effects of beta-blockers on in vivo large artery mechanics. It is additionally unknown whether the remodeling would in fact have unfavorable consequences for the cardiovascular system, had the duration of treatment been longer. Future studies should examine large artery biomechanical and structural remodeling elicited by prolonged propranolol treatment in animal models of aneurysmal disease and Marfan syndrome. Another important question from the standpoint of cardiovascular pathology is whether the different blocking agents that are used in the medical management of abdominal aortic aneurysms [53], Marfan syndrome [54], and aortic dissection [55] have differential effects on large artery structure and function, and whether these are mediated by their hemodynamic effects.

In summary, evidence is submitted of biomechanical and histological remodeling of the thoracic aorta and carotid artery in response to chronic pharmacological blockade of beta-adrenergic receptors, indicating that normal status of beta-receptors is necessary for the maintenance of proper large artery structure and function in rats. Propranolol administration generated lumen diameter and wall mass reduction, and reduced thickness to radius ratio. The latter result, together with the greater resorption of the media compared with the adventitia, related with the measured decrease in opening angle. The biomechanical properties of large arteries changed, with the thoracic aorta in propranolol-treated rats presenting increased strength longitudinally, as a result of the increased adventitial collagen content compared to untreated rats. The diameter–pressure curves of carotid arteries were shifted to smaller diameters and distensibility increased in propranolol-treated rats at low-to-physiologic pressures, as a result of the reduced medial collagen content. Our findings show that the geometrical/biomechanical remodeling is likely mediated by the hemodynamic effects of propranolol treatment, i.e. the reduced blood flow, and serve to normalize hoop stresses as well as vessel compliance at in vivo conditions.

Footnotes

Acknowledgement

The authors thank Mr. Constantinos A. Dimitriou for superb assistance with inflation/extension testing.

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