Abstract
Aging induces substantial morphological and functional changes in vessels. We hypothesized that due to morphological remodeling the total contractile forces of arteries increase, especially in older age as a function of age. Mean arterial blood pressure of rats and morphological and functional characteristics of isolated carotid arteries rats, from newborn to senescent, were assessed. The arterial blood pressure of rats increased significantly from 0.25 to the age of 6 months, and then it reached a level, which was maintained until age of 30 months. Wall lumen and wall thickness increased with age, mostly due to media (smooth muscle) thickening, whereas wall tension gradually reduced with age. Contractions of arteries to nonreceptor-mediated vasomotor agent (KCl, 60mM) increased in three consecutive age groups, whereas contractility first increased (until 2 months), then it did not change further with aging. Norepinephrine-induced contractions initially increased in young age and then did not change further in older age. These findings suggest that during normal aging due to remodeling of arterial wall (smooth muscle) the contractile capacity of arteries increases, which seems to be independent from systemic blood pressure. Thus, arterial remodeling can favor the development of increased circulatory resistance in older age.
Aging greatly affects the vascular system, yet there are only few experimental studies exist to assess—in a whole life span—the aging-induced morphological and functional (contractile) changes of arteries in a parallel manner. The importance of this issue is already brought up in 1958, in a “classical” study by Björn Folkow who proposed that the increased vascular smooth muscle activity can be a “trigger mechanism” for increased resistance to flow, vascular wall hypertrophy, and eventually to the development of hypertension. (1). Put it in a simpler way: Is it the remodeling-induced increased vascular smooth muscle activity can be a “trigger mechanism” for increased resistance to flow, vascular wall hypertrophy, and eventually to the development of hypertension. increased vasomotor tone of arteries (due to hypertrophy) occurring first, which is then followed by increases in mean arterial blood pressure (MABP) or vice versa?
Previous studies have shown that maturation and aging elicit substantial changes in various tissues from birth throughout adulthood and to old and senescence, and also changes occur in the morphological and functional properties of vascular wall (2–4), which could be due to—among others—changes in the hemodynamic environment (2,4). It is well established—in both humans and animal models—that the walls of large arteries (such as aorta, carotid, and femoral arteries) undergo structural changes with aging that are associated with histological alterations, independent of atherosclerotic mechanisms (5). For example, it has been shown that in carotid arteries of old rats lumen diameter and wall thickness increase compared with that of young and middle-aged rats (6–8), whereas in cerebral arteries changes in dimensional properties indicate an initial outward eutrophic in young, followed by inward hypertrophic remodeling in middle-aged and old rats (9,10). Bakker and colleagues reported that inward remodeling can occur in the presence of high level of vasoconstriction, whereas wall tension is reduced in vessels with low pressure, suggesting a link between vasoconstriction and inward remodeling (11) and high intraluminal pressure (12). Moreover, VanBavel’s group suggested that “wall tension may not only be regulated against disturbances in pressure, but also against changes in agonist concentration” (13).
It has been established that in early age MABP increases, whereas the values reported in older age are not equivocal (7,11,14–17). Because blood vessels respond to changes in pressure with functional and morphological changes, one can ask the question: Which change comes first, pressure or vasomotor tone? Studies on humans of various age groups, it was showed that with advancing age there were increase in wall mass, wall thickness, intima-media thickness, and lumen diameter of different arteries (carotid, branchial, radial, and aortas), and reduction in wall stress/tension, indicating that vessels undergo outward remodeling with aging (14,18–22). In another study, it was concluded that age is an independent risk factor for increased intima-media thickness (23,24). Thus, increase in wall thickness can lead to increase of systemic blood pressure during aging. It is of note that there are reports indicating that these two parameters could be correlated (21,24), whereas others suggest otherwise (20,25).
Importantly, most studies investigated only a limited age range or only two time points (8,9,14,17,26–28), precluding to characterize the overall effect of healthy aging from newborn to senescence regarding changes in morphological characteristics and contractile function of vessels and blood pressure, thus test Folkow’s hypothesis. Thus, we followed changes in morphological characteristics and arterial smooth muscle contractility of arteries in a whole life span of rats. We used the vasomotor agent potassium chloride, because it elicits arterial contractions without receptor mediation (2), thus changes in receptor availability would not interfere with the assessment of contractile capacity, as we found to be the case for angiotensin II (16). We have also used norepinephrine (NE) for comparison and internal control for assessing contractile function.
Methods
Animals
Ninety-eight male Wistar Kyoto (WKY) rats aged 8 days (0.25 month), 1, 2, 6, 9, 12, 19, 24, and 30 months (body weights: 14.3±4, 108.9±14, 205.6±15, 279.3±15, 322.6±11, 295.3±19, 348.9±26, 362.6±13, and 273±44g, n = 10 for each group) were used. These animals represented different age groups: newborn, juvenile, young, adult, middle-aged, late middle-aged, old, and senescent. Rats were kept in standard cages, exposed to 12 hours of light and 12 hours of dark. They had 24-hour access to water (tap water) and food (standard, provided by supplier). All procedures were conducted under protocols approved by Institutional Animal Use and Care Committee of the University of Pecs and were in accordance with the directives of the National Ethical Council for Animal Research and those of the European Communities Council (86/609/EEC).
Measurement of MABP of Rats
MABP was measured by two different methods, tail cuff (noninvasive) (29) and with direct manner, by carotid artery cannulation (16). Tail-cuff method: In brief, the tail of rat was heated to 37°C for 10 minutes before the measurement of blood pressure according to the instruction of Hatteras manual (www.hatterasinstruments.com), in order to dilate the main artery and make it more accessible for transluminal BP measurements with pulse sensor (Hatteras SC-1000; Hatteras Instruments, Cary, NC). The tail cuff was connected to a cylinder of compressed air through an arrangement of inlet and outlet valves that permitted inflation and deflation of the cuff at a constant rate. The animals quickly became familiar with the procedure and remained calm within the restrainer. In the rare cases, when signs of discomfort were present, the procedure was interrupted. Measurements were repeated three times and then averaged. In another group of animals, the arterial blood pressure values were measured directly by cannulating the carotid artery. Comparable data could be obtained by the two methods (see Supplementary Table 1).
Surgery
From rats, the common carotid artery was isolated by using a surgical microscope (SZX7; Olympus Inc., Japan) under intraperitoneal anesthesia induced by a ketamine–xylazine cocktail (78mg/kg calypsol [Richter, Hungary] + 13mg/kg [Eurovet, Belgium], respectively). Isolated segments were transferred to ice-cold Krebs solution. All other chemicals and drugs were obtained from Sigma-Aldrich (St Louis, MO), unless specified otherwise. After animals were anesthetized and carotid arteries exposed, one (usually left) carotid artery was ligated proximal and distal point to “keep” prevailing blood pressure inside the ligated section. This section was quickly removed and inserted to fixation solution. The second artery (usually right) was removed and segments were dissected into 2-mm-long rings. Four rings were then used in the experiments; thus, n represents the average of four measurements from one animal. After the removal of the arteries, the animal was euthanized with an intraperitoneal injection of pentobarbital (100mg/kg; Ceva Sante Animale, Libourna, France).
Vascular Wall Histology
After removal of artery, this section was quickly removed and inserted into 10% formalin fixation solution. After fixation of carotid artery, blood was carefully removed with fixative by needle and syringe and carotid artery was embedded in paraffin. Next, embedded samples were cut into 4- to 6-µm-thick sections and stained with hematoxylin and eosin (H&E) as described by Tulis (30). The sections were examined with video microscopy under different magnification. The following parameters were measured: wall thickness (media and adventitia layers) and artery lumen diameter (intima could not be measured due to resolution of light microscopy).
Calculation of Vascular Wall Tension and Wall-to-Lumen Ratio
According to Laplace’s law, the wall tension is equal to intravascular pressure divided by inner radius of vessel. In the modification of law by Otto Frank, wall tension (in living systems) is dependent also about wall thicknesses; therefore, we have TH = P/(ri * tC), where TH—wall tension (stained with hematoxylin); P—mean pressure; ri—inner radius of vessel; and tC—thickness of carotid wall. Wall-to-lumen (W/L) ratio was calculated by the equation: wall thickness/radius (WT/r), using the values of WT and r.
Isometric Force Measurements
Measurement of the changes in isometric force of the vessel rings was conducted as described before (16). In brief, in a four-chamber wire myograph system, vascular rings were mounted on tungsten wires (40 µm) connected to transducers. The chambers contained 5mL bath solution (DMT 610M; Danish Myo Technology A/S, Aarhus, Denmark). The diameter of the wire was 40 µm for artery. The bath solution was continuously perfused with 95% and 5% mixture of oxygen and carbon dioxide and the temperature of the solution was kept constantly at 36.9°C ± 0.1°C. Before the start of an experiment, normalization to blood pressure was performed on each vessel ring with the Myodaq 2.01 software (Danish Myotechnologies, Denmark) according to the protocol described in reference and then the rings were allowed to stabilize for 60 minutes. The Myodaq 2.01 software (Danish Myotechnologies, Denmark) was also used for data registration on a personal computer connected to the wire myograph (see Supplementary Material for more details).
Experimental Protocols
To assess the optimal concentration for vasomotor capability, we administered increasing concentration of KCl (1–80mM) to carotid arteries isolated from 0.25-, 2-, 12-, and 28-month-old rats. After establishing that KCl 60mM was the optimal concentration (Figure 4A), we used this concentration in the experiments. After vessels were stabilized for 60 minutes, 5mL of 60mM KCl (VWR International, Hungary) was administrated into each chamber. Maximal contraction to KCl was reached within 15–20 minutes. Then, after 10 minutes, the chambers were washed out three times with Krebs solution. The vessels were incubated for 20 minutes, then the procedures were repeated. Repeated administrations of NE 10−6 M were also used to test the receptor-mediated vasomotor capacity of arteries as a function of age and to functionally confirm the removal of perivascular nerves.
Normalization of isometric force generated to vascular wall thickness: Isometric forces generated by the vessels were normalized to wall thickness to assess the changes in contractility because arteries undergo remodeling as a function of age. Normalization was calculated by the following equation: N = F/T, providing ratio number, where F—force (mN) and T—thickness (smooth muscle thickness).
Statistical Analysis
All data are expressed as means ± SEM. The data were compared with one-way analysis of variance (ANOVA) followed by Tukey’s post hoc test. In addition to ANOVA, regression analysis was performed to obtain significant changes (if any) among various sections of regression lines by comparing their slopes. All analyses were performed using Sigma Plot 12.5 (Systat Software, Inc., Chicago, IL) for Windows software. Statistical significance was accepted at p < .05.
Results
Changes in the MABP of Rats as a Function of Age
The MABP of rats (Figure 1) increased from newborn until the age of 6 months, then decrease, but nonsignificantly until the age of 30 months (0.25 month: 81.3±10.3 mmHg; 1 month: 104.5±9.6 mmHg; 2 months: 108.4±8.4 mmHg; 6 months: 119.7±6.6 mmHg; 9 months: 117.9±4.9 mmHg; 12 months: 106.9±5.4 mmHg; 19 months: 106±5.3 mmHg; 24 months: 105.1±6.8 mmHg; 30 months: 103.8±6.3 mmHg; n = 10, p < .05 vs 0.25 month). Comparable results were obtained with tail-cuff method (see Supplementary Table 1).
Summary data show mean arterial blood pressure (MABP) of rats measured in carotid arteries significantly increased until age of 6 months, and then it was maintained until age of 30 months. *p < .05 versus 0.25 month (n = 10).
Changes in the Vascular Wall Thickness of Rat Carotid Arteries as a Function of Age
Figure 2 shows that total wall thickness of carotid arteries rapidly increased from newborn (0.25 month) until age of 2 months, then slowly until age of 19 months, then gradually increased to senescence (30 months). In addition, in Figure 2, changes in the thickness of media (smooth muscle) and adventitia (extracellular matrix) of arterial wall are depicted. Media increased gradually in three phases: phase 1 significantly increasing from 0.25 until 2 months, phase 2 then slowly until age of 12 months, phase 3 then again greater increase until the age of 30 months (significant changes occurred in all three phases), whereas adventitia increased gradually until age of 19 months and then remain unchanged until age of 30 months (significant changes occurred in the first two phases; see Supplementary Table 2, Wall thickness). Regression analysis shows that the thickening was significantly greater in the media then in the adventitia from 19 until age of 30 months (p < .05).
(A) Original images stained with hematoxylin and eosin in different age group; cross sections of isolated carotid arteries from 0.25-, 2-, 12-, 24-, and 30-month-old rats (mo; magnification ×400), scale bar represents 40 µm length; cartoon is shown to indicating that the type of growth of arteries with aging is an outward hypertrophy; (B) summary data show changes in the thickness of media (SM—smooth muscle, green line), adventitia (ECM—extracellular matrix, red line), and total wall (blue line) in rat carotid artery as a function of age (curves are the best fit for the data). * indicates significant change in slope (p < .05), NS = nonsignificant.
Diameter of Carotid Artery in Histological Preparations
Summary data (Figure 3A) show diameter (d = 2r) of carotid artery. Both vessel and lumen diameters showed three phases of changes. Vessel diameter rapidly increased until age of 2 months, then increased gradually until 30 months of age. Also, lumen diameter rapidly increased until age of 2 months and then slowly increased until age of 19 months, whereas in the last phase, there was no significant changes (see Supplementary Table 2, Dimensions).
Summary data show morphological measurement in rat carotid artery as a function of age. (A) Vessel inner and outer diameter; (B) wall-to-lumen ratio; (C) wall tension. * indicates significant change in slope (p < .05), † between the slopes of inner versus outer diameter (p < .05), NS = nonsignificant.
W/L Ratio
Summary data (Figure 3B) show change in W/L ratio. The W/L ratio shows a nonsignificant increase from newborn to senescence age (p < .088). Also, analysis shows three phases of changes in W/L ratio: First, there is a sudden increase until age of 2 months (due to an increase in wall thickness—outward hypertrophy), followed by inward eutrophic remodeling (when increases in wall mass are not accompanied with increase in lumen) until age of 19 months, then W/L ratio gradually increased until age of 30 months (outward hypertrophy; see Supplementary Table 2, Calculated parameters).
Calculation of Wall Tension
Summary data (Figure 3C) show reduction in calculated wall tension. It shows a rapid decrease until age of 2 months, then it did not change until age of 6 months, and then it slowly decreased until age of 30 months. The decrease was due to increase in vessel lumen and increase in vascular wall thickness (see Supplementary Table 2, Calculated parameters).
Changes in Isometric Force of Isolated Rat Carotid Arteries in Response to KCl as a Function of Age
First, the test concentration of KCl was determined. Summary data (Figure 4A) show the effect of increasing dose of KCl on the isometric force development of carotid arteries. In the present experiments, 60mM KCl concentration was used because it elicited close to maximal contraction (n = 4–6).
Original records and summary data (Figure 4B and C) show that the magnitude of KCl-induced contraction of carotid arteries exhibiting three phases: (i) a rapid increase from newborn (0.25 month) to the age of adult—2 months, (ii) then slow, but sustained increase to the age of 19 months, and then (iii) again rapid increase until age of 30 months of age. Changes were characterized with the slopes of the regression lines (see Supplementary Table 2, Vasomotor characteristics). NE-induced vasomotor response increased sharply until age of 6 months, and then it did not change further until age of 30 months (Figure 5B). Repeated administration elicited similar vasomotor responses as a function of age (see Supplementary Figure 1).
(A) Summary data show the cumulative dose-dependent contractile responses to KCl (1–80mM) of carotid arteries isolated from 0.25-, 2-, 12-, and 28-month-old rats; (B) original records show changes in contractile responses of rat carotid arteries to 60mM KCl, as a function of age. Black arrows indicate washout of KCl with Krebs solution; (C) summary data show changes in isometric force generated by isolated rat carotid arteries to KCl 60mM, as a function of age and that of normalized to the smooth muscle thickness (SMt). * indicates significant change in slope (p < .05), NS = nonsignificant.
(A) Original records show changes in the contractile responses of isolated rat carotid arteries to norepinephrine (NE) 10−6 M, as a function of age. Black arrows indicate washout of NE with Krebs solution; (B) summary data show changes in isometric force generated by rat carotid arteries to NE, as a function of age. * indicates significant change in slope (p < .05), NS = nonsignificant.
Assessing Contractility by Normalization of Vasomotor Responses of Carotid Artery to Media Thickness as a Function of Age
Because the media/smooth muscle layer of arteries undergoes morphological remodeling, the total isometric force generated by the vessels was normalized to media thickness. We have found that in normalized isometric force (contractility) first there was a significant increase from newborn until the age of 2 months and then it did not change until senescence (Figure 4C; see Supplementary Table 2, Vasomotor characteristics).
Discussion
The important novel findings of the present study are that from newborn to senescence: (i) MABP initially increased, then did not change, (ii) vascular wall thickness—especially the media (smooth muscle) layer—increased as a function of age, (iii) wall tension decreased from newborn to the senescence, (iv) the magnitude of nonreceptor vasomotor agent (KCl)-induced contractions gradually increased, whereas the contractility—after initial increase in early age—did not change further (v) magnitude of receptor-mediated vasomotor agent NE-induced contractions first increased, then did not change in older ages.
These findings suggest that arteries undergo substantial morphological and functional changes, that is, remodeling as a function of age, which seems to be independent from changes in arterial blood pressure, findings that can have important impact on our view on vascular remodeling during normal aging. It is of note that we have performed our study only on male rats. Nevertheless, it would be important to collect data on vessels of female rats as a function of age in future studies because there are studies suggesting that vasoconstrictor responses of male and female arteries are affected differently with aging (31).
MABP as a Function of Age
In the present study, we have found that the MABP (Figure 1 and Supplementary Table 1) of rats increased from newborn until the age of 6 months, and then it decreased to the age of 30 months. These findings are in agreement with findings of others, showing the similar trends of changes in blood pressure with age in rats (14–16,24) and humans (24,32), although others reported differing data (33).
Wall Thickness of Carotid Artery Increases as a Function of Age
The thickness of arterial wall increased from newborn to senescence (Figure 2), with an early rapid phase. Similar tendency was found from young (6 days) to old (70 weeks) normotensive rats (6,7,10) and in media thickness of carotid arteries from 6- and 23-month-old rats (8). This could be interpreted that hypertrophy compensates for the degeneration of elastic and fibrotic layers of smooth muscle to maintain or improve vasomotor function of artery (20,24). The slopes of curves showed that smooth muscle and total wall thickness increased in all three age groups, whereas in adventitia increased only in first two age groups, suggesting that smooth muscle is the main load-bearing component in wall (see Supplementary Table 2, Wall thickness). The present results show that both wall thickness and lumen radius (diameter; Figures 2 and 3A) increased as a function of age, without significant change in W/L ratio (Figure 3B), indicating an outward remodeling of arteries as a function of age (14,34). These findings correspond to those of humans showing that arterial diameters increase with age and a compensatory thickening of the arterial wall prevents the circumferential wall stress from increasing (18,21,35). The slopes of curves showed significant increase of inner and outer diameters except in last age group (19–30 months) when inner diameter did not change, indicating a hypertrophy of the wall (see Supplementary Table 2, Dimensions). It is of note that we have studied larger arteries, but it is likely that similar changes may occur in smaller resistance vessels, which could be an important topic for future studies (36).
Arterial Wall Tension
We have found that wall tension became reduced as a function of age, which is in agreement with findings of others (14). VanBavel’s group suggested that “increased wall tension (rise in blood pressure) leads to induced constriction, whereas low wall tension results in reduction in the magnitude of constriction (Laplace law)” (13). Based on this, in isometric conditions, agonists stimulation lead to vasoconstriction (increase in vasomotor response, therefore increase in force = increase in wall tension). In contrary, in isobaric condition, it leads to increases in wall tension resulting in vasoconstriction, thus reduction in wall tension (negative feedback loop). Our findings (Figure 3C) confirm this observation, that is, increases in vasoconstriction result in reductions in wall tension. This observation could have importance during aging, where increases in systolic and reductions in diastolic pressure (24,32,37) as a function of age could be—in part—due to increased contractile tone and reduced compliance (stiffness—contributes to reduction of systolic BP) of vessel in older age.
Arterial Contractile Capacity and Contractility as a Function of Age
To assess the contractile capacity of arterial smooth muscle, we have used KCl, because it acts without receptors, which could interfere with the interpretation of findings (2). From newborn to young age, the continuous increase in contractile ability of arteries (Figure 4C) as indicated by the slopes of curves (Supplementary Table 2, Vasomotor characteristics) is important, in order to provide the means to control vascular resistance, when systemic blood pressure and cardiac output also increase (38), which is then tempered in middle-aged and older arteries (39,40), then increases again in much older and senescent age (24–30 months old), which has not been investigated or shown previously (Figure 4C). This is most likely due to the aging-induced increase in smooth muscle mass and size, likely due to increases in actin/myosin synthesis (hypertrophy), collagen embedding, and reduction in elasticity (9,24), which leads to increase in effectiveness of contractile function (3). Folkow and colleagues postulated that according to Laplace’s law, resistant vessels of rats undergo structural changes, causing the medial layer (smooth muscle) to thickening and resulting in a geometrically related increased response to vasoactive stimuli (1,13).
There are many previous studies regarding aging-induced arterial structural remodeling (8,26–28). However, term “arterial remodeling” has been specifically used to refer changes in vessel parameters, such as cross-sectional area within the intima/media/adventitia, W/L ratio, etc. (41). Our results, by showing increases in vessel size (increased lumen) with unchanged W/L ratio, indicate an outward remodeling process eliciting reduction in wall tension (14,41), which is a normal physiological process, eliciting increased contractile capacity in older ages. Although vascular hypertrophy is usually connected to hypertension, these findings suggest that hypertrophy “compensating” for increasing lumen, stabilizing blood pressure control (14,26). Our findings are in agreement with that of Meininger and colleagues, showing the importance of changes in vascular wall (composition and organization of extracellular matrix) and primarily in smooth muscle as a function of age and that these changes contribute to maintaining the normal hemodynamics in old age (3,42).
Importantly, however, aside from the initial increase, the contractility of arteries did not change during aging (Figure 4C), suggesting that primarily morphological remodeling is responsible for the increased total arterial contractile ability as a result of aging. In order to further characterize the effect of age on contractile function, we used NE known to be mediated by adrenergic receptors (Figure 5). Interestingly, unlike KCl, NE-induced contractility increased until age of 6 months and then—in essence—remained the same. This could be due to reduction in β-adrenergic receptor density and aging-induced alterations in the adenylyl cyclase cascade beyond β-receptor level (43). Repeated administration of KCl and NE elicited similar vasomotor responses as a function of age (see Supplementary Figure 1). These findings suggest that total contractile force of arterial smooth muscle increases as function of age (but not the contractility), suggesting the importance of morphological remodeling. The finding that systemic blood pressure did not increased suggests that vascular remodeling is counterbalanced by other, for example, receptor-mediated mechanisms (NE, Ang II) to prevent increases in peripheral vascular resistance (16).
These findings support the idea that aging-induced structural and functional remodeling of arterial vessels are likely independent from changes in blood pressure and may predispose the circulatory system to the development of hypertension (44).
Conclusion
In conclusion, during healthy aging arterial wall thickness, especially smooth muscle layer is substantially increasing from newborn to the very old age, contributing to the increased contractile ability of arteries in older age. The arterial remodeling seems to be independent of changes in systemic arterial blood pressure suggesting roles for other mechanisms (genetic and epigenetic). This conclusion seems to support earlier concept of Bjorn Folkow: “an increase in wall-to-lumen ratio (due to increased smooth muscle layer, leading to increased contractility) could be important factor in pathogenesis of hypertension” (45), especially if other mechanisms fail to counterbalance the remodeling of arterial vessels.
Funding
This work was supported by the National Research, Development and Innovation (NKFI) Fund of Hungary, K 108444 and K 104984; FP7 Marie Curie project—Small Artery Remodeling (SmART); Developing Competitiveness of Universities in the South Transdanubian Region, ‘‘Identification of new biomarkers…’’ SROP-4.2.2.A-11/1/KONV-2012-0017 and ‘‘Complex examination of neuropeptide...’’ SROP-4.2.2.A-11/1/KONV-2012-0024; and the Hungarian Hypertension Society (MHT) 2013-2015.
Conflict of Interest
The authors declare no conflicts of interest.
Acknowledgment
The authors thank Robert Matics and Peter Degrell for their excellent advises and technical assistance.
References
Author notes
Address correspondence to Akos Koller, MD, PhD, Department of Physiology, New York Medical College, Valhalla, NY 10595. Email: koller@nymc.edu
Decision Editor: Rafael de Cabo, PhD
