Carotid artery stiffness with applications to cardiovascular pharmacology

Gen. Pharmac. Vol. 27, No. 8, pp. 1293-1302, 1996 Copyright © 1996 Elsevier Science inc. Printed in the USA.

ISSN 0306-3623/96 15.00 + .00 PII S0306-3623(96)00085-7 All rights reserved ELSEVIER

REVIEW

Carotid Artery Stiffness with Applications to Cardiovascular Pharmacology Michel E. Safar DEVARTMENT OF INTERNat MEDICINEAND INSERM (U337), BROUSSAtSHOSVlTAL,96 RUE DIDOT, PARIS, CEDEX 14 FRANCE ABSTRACT. 1. Increased carotid stiffness is a characteristic feature of cardiovascular aging, hypertension and, to a lesser extent, atherosclerosis. 2. The pharmacological approach, using nitrates, converting enzyme inhibitors, calcium entry blockers and blockers of the autonomic nervous system, may decrease carotid stiffness through 3 different mechanisms: passive decrease in blood pressure, active change in smooth muscle tone, and structural modifications of the carotid arterial wall. 3. In recent years, such changes have been studied in vivo, in both humans and rats, using original in situ carotid preparations and echotracking ultrasound techniques of high resolution, allowing to evaluate both static and dynamic stiffness. 4. This new approach for investigating arterial vessels through changes in both stiffness and thickness should provide a better evaluation of drug effects in cardiovascular pharmacology and new interpretations for cardiovascular events related to morbidity and mortality. Copyright © 1996 Elsevier Science Inc. OEN VHARMaC 27;8:1293--1302, 1996. KEY WORDS. Carotid stiffness, cardiovascular pharmacology INTRODUCTION There are two different functions of the large arteries in physiology (Nichols and O'Rourke, 1990): the conduit function and the buffering function. The role of arteries as conduits is to deliver an adequate quantity of blood from the heart to peripheral organs and tissues, according to their metabolic activity. This role has been extensively studied in the literature using the determination of blood flow. In contrast, the buffering function of the arteries (i.e., the ability to dampen the pressure oscillations resulting from intermittent ventricular ejection) has been investigated only recently. This function consists of instantaneously accommodating the volume of blood ejected from the heart, storing part of the stroke volume during systolic ejection and draining this volume during diastole, thus ensuring a continuous peffusion of organs and tissues. Because this function is strongly dependent on the viscosielastic properties of the vessels, it may be influenced by the pharmacological approach of arterial wall diseases, as observed nowadays in aging, hypertension and atherosclerosis (Nichols and O'Rourke, 1990; Safar et al., 1995; Safar and London, 1994) In both humans and rats, the most available model to evaluate the viscoelastic properties of the arteries and, therefore, arterial stiffness, is the common carotid artery. Indeed, the carotid artery is a straight, superficial vessel with several particularities. First, this artery may be considered as a cylinder, a model that may be easily used to investigate biophysical properties. Second, the mechanical properties of the vessel may be explored using noninvasive techniques that have been largely developed in recent years, particularly with ultrasound technology of high resolution (Nichols and O'Rourke, 1990; Safar and London, 1994; Hoeks et al., 1990; Tardy e t al., 1991; Kawasaki et al., 1987; Lichtenstein et al., 1995). Thus, it is possible to obtain information on aging and hypertension and, to a lesser exReceived 15 January 1996; accepted 3 February 1996.

tent, on atherosclerosis, which, in humans, predominates rather on the carotid bifurcation and the internal carotid artery. The purpose of the present study is to investigate carotid stiffness in humans and rats in 3 different ways: basic definitions of the carotid pressure-volume relationship and of the arterial mechanical properties, indices of carotid artery stiffness, and applications to cardiovascular pharmacology. Before developing these aspects, some limitations of the subject should be noted. First, only the function of the carotid artery in terms of capacity exchange and storage will be investigated and no attempt will be made to evaluate the viscoelastic properties of the wall material. Second, although the carotid artery is principally studied, no reference will be given to relationships with the baroreflex mechanisms in the high-pressure system. Finally, the changes of carotid stiffness in humans and rats will be limited to the principal drugs acting in cardiovascular pharmacology, with no attempt to develop the mechanistic approach of the underlying diseases. For these latter 3 subjects, the reader will have to consider more specific reviews (Safar and London, 1994; Laurent, 1995; Chapleau et al., 1995). THE C A R O T I D ARTERY PRESSURE-VOLUME RELATIONSHIP The ability of the carotid artery to instantaneously accommodate the blood volume ejected from the left ventricle ("Windkessel" effect) depends on the relationship between pressure and volume within this artery (Nichols and O'Rourke, 1990). The observed positive relationship is an oversimplification of the stress-strain relationship that defines the mechanical properties of the carotid. Stress is defined as P×r/h, P being the transmural pressure, r and h respectively, the radius and the thickness of the artery. Strain is defined as delta L/L (delta=change), where L is the length of the material at baseline (i.e., ideally in unstressed conditions) and delta L the change in length for a given stress. In cardiovascular physiology, the

1294 r/h ratio is often considered as low and nearly constant by comparison with P. On the other hand, delta L/L is replaced in a cylindrical artery by delta V/V, (i.e., the change in volume, delta V, from baseline volume V) or even by delta D/D, D representing the diameter of the artery. In this review, the understanding of the pressure-volume relationship will be limited to the definition of the 2 principal indices of carotid stiffness, compliance and distensibility. Basic definitions

Compliance is a term describing the amount of change in dimension (delta V or delta D) following a change in pressure (delta P). In physiology, compliance (C) is defined as C=delta V/delta P (or delta D/delta P) and represents the slope of the pressure-volume relationship at a specified point of the pressure-volume curve. Fig. 1 (upper panel) gives a typical example in which the carotid artery of normotensive Wistar-Kyoto (WKY) and spontaneously hypertensive (SHR) rats is studied in vitro (Caputo et al., 1995). Notice that, in this example, a constant and static pressure is applied and that only the ascending part of the curve is presented (i.e., the hysteresis of the curve is supposed to be neglectable) (Fig. 1). Because the structure of the artery influences its physical properties, the pressure-volume (or pressure-diameter) relationship is not linear (i.e., with a constant slope) but, rather, curvilinear. Because the arterial media is heterogeneous and composed of smooth muscle cells and connective tissue containing elastin and collagen fibers, at the lower distending pressures the tension is borne by smooth muscle and mainly elastin fibers, whereas, at the higher distending pressures the tension is predominantly borne by less extensible collagen fibers. Then, the arterial wall gets stiffer (i.e., less compliant). Finally, due to these particularities, the compliance can be defined only for a given transmural pressure. The static compliance measured at the usual (= set-point) pressure of a given animal is called "operating" compliance (Fig. I, upper panel). Normotensive and hypertensive populations have, by definition, different values of operating mean blood pressure (approximately 120 and 170 mmHg in each strain) (Fig. 1, upper panel) and therefore, of operating compliance, which is known to be decreased in hypertensive animals and humans (Nichols and O'Rourke, 1990; $afar and London, 1994). At operating values, static carotid compliance depends on two parameters: the set point (or operating) blood pressure level and the "intrinsic" viscoelastic properties of the arterial wall. In this review "intrinsic" is a word used to express the nonpressure-dependent parameters that influence carotid mechanical properties. Mathematically, this means that the curve has changed its intercept and/or its regulation coefficients. Biologically, such changes may reflect one or several alterations of the following parameters: smooth muscle tone, arterial geometry, arterial mass, or readjustment between the different components of the wall material, often called remodelling, etc. (Fig. 2). To compare the "intrinsic" properties of 2 different carotid arteries, these 2 arteries should be compared at the same transmural pressure, a situation that is often difficult to obtain in vivo (because normotensive and hypertensive populations have not, by definition, the same "set point" blood pressure), but is easy to obtain in vitro on static pressure-volume (or diameter) curves. Figure 1 (upper panel) shows, for instance, that for the same pressure, 150 mmHg, the carotid diameter is significantly higher in hypertensive than in normotensive animals, indicating different geometrical properties of the arterial wall. At 150 mmHg, compliance is nearly the same in normotensive and hypertensive animals, whereas it is clearly different between 50 to 100 mmHg (i.e., for the lower wansmural pressure ranges), indicating different buffering capacities of the carotid artery. To facilitate comparison of the viscoelastic properties of struc-

M.E. Safar SHR operatingpoint

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1000800 -

~(b)

600 -

400 I

50

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150

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F I G U R E 1. Diameter-transmural pressure curve studied in vitro

in normotensive Wistar-Kyoto (WKY) ([]) and spontaneously hypertensive (SHR) (0) rats (modified from Caputo e t al., 1995). Note that the hysteresis is neglected (see Table 1). In the upper panel, only the static curves are represented. In the lower panel, dynamic curves have been superimposed at different arbitrary values of transmural pressure. Note that the dynamic curve (b) might seem to prolong curve (a), although these two curves do not belong to the same static curve. On the other hand, curve (d) seems to prolong curve (c), although both belong to the same static curve.

tures with different dimensions and volumes, the compliance may be expressed relative to baseline volume as a coefficient of volume distensibility: (delta V/V)/dP, where delta V/delta P is compliance and V is baseline (i.e., ideally unstressed) volume. Because in a cylindrical artery, arterial volume per unit length is equal to arterial cross-sectional area, depending on the arterial diameter (D), the volume distensibility is often expressed as (delta D]D)[deha P, thus assuming a constant arterial length (Benetos et al., 1993b). For many years, a large number of markers of the viscoelastic properties of the arteries have been described and are summarized in Table 1 (O'Rourke, 1995). Because the "Young" modulus involves the thickness of the artery, it represents the slope of the stress-strain relationship and is widely recognized as the most adequate index to evaluate the stiffness of the arterial wall material itself. Arterial thickness has been difficult to evaluate, particularly in living humans (Roman et al., 1992; Girerd et al., 1994). For that reason, compliance and distensibility are the parameters that are most often used.

Carotid Artery Stiffness in Cardiovascular Pharmacology

1295

C CAROTID S T I F F N E S S )

Volume

S t a t i c curve ( ) FIGURE 2. Factors governing static and dynamic compliance (see text). Note that any pressure-volume curve (static or dynamic) is composed of an ascending (increase in pressure) ( / ) and a descending (decrease in pressure) part. The cross-sectional area between the 2 curves ( ~ ) called hysteresis, is usually considered as neglectable but determines the wall viscosity. There is yet little data studying the relation between wall viscosity and the static properties of the arteries [?] (Nichols and O{Rourke, 1990; Lichtenstein e t al. 1995).

Dynamic curve ( ........... )

P

Structure and function of the artery : - Arteriel thickness and geometry - Hypertrophyand remodeling - Smooth muscle tone

Mean arterial pressure ( = Set point bloodpressure) (= operating pressure)

f

Heart rate (frequency dependence)

Arterial wall viscosity

[?] Static vs pulsatile carotid pressure.volume relationship The role of blood vessels as conduits requires a continuous, steady blood flow for an efficient metabolic exchange (Nichols and O'Rourke, 1990). To maintain such a steady flow, there must be a steady (i.e., static) pressure head applied to the blood to overcome the energy losses related to blood viscosity and friction (i.e., the resistance to flow). On a blood pressure curve, this pressure is represented by mean arterial pressure. Using this simplified model of the circulation, the storage capacity of the arteries (= static compliance) is usually evaluated from a static pressure-volume relationship, as described in Fig. 1 (upper panel). In Fig. 1, mean arterial pressure is thus the operating (= set point) pressure. Whereas, in living animals and humans, this model of vascular function is sufficient to explore the venous system, the problem is much more complicated for the in vivo carotid artery, in which pressure and flow are pulsatile. As mentioned earlier, under dynamic conditions, the carotid artery instantaneously dampens the volume of blood ejected during ventricular ejection and this "Windkessel" effect requires, by definition, pulsatile pressure, volume and flow. For that reason, in the mathematical models of arterial vessels, the pressure-volume (or pressure-diameter) relationships of arteries must be represented as involving two different components: a steady component, representing the changes in the mean value of diameter and arterial pressure (= steady or static pressure-volume relationship), and a pulsatile

component, that describes the diameter and pressure fluctuations around the mean (= pulsatile pressure-volume relationship) (Nichols and O'Rourke, 1990; Safar and London, 1994; Glaser et al., 1995). Experimentally, Bergel (1961) made such observations in vitro on segments of thoracic aorta, and femoral and carotid arteries of dogs. At any given value of steady mean pressure, he stressed the vessels with sinusoidal transmural pressures, and measured the excursions of diameter by a photoelectric method. In the description of complex elastic modulus of the artery (Ec), he distinguished two components: the static modulus (Estat) and the pulsatile modulus (Edyn). The real part (Edyn) of the complex modulus (Ec) was significantly larger at all frequencies studied (2-18 Hz) than were measurements of the static elastic modulus (Estat), indicating stiffer arteries in dynamic than in static conditions at any given value of steady mean pressure. It is noteworthy that this finding is expected in any viscoelastic material, whether of physical or biological origin, because the wall has less time to reach its maximum potential strain in a test when the stress is sinusoidaI than it does in static tests. In Fig. 1, in addition to the static pressure-diameter relationship in WKY rats and SHRs (upper panel), a number of pulsatile (dynamic) pressure-diameter relationships (lower side) are indicated for different values of steady transmural pressure (exactly as described in the Bergel experiments). From this schematic representation, two important characteristics of the pulsatile (dynamic) relationship

1296

M.E. Safar TABLE 1. Indices of Carotid Arterial Stiffness

Elastic modulus

The pressure step required for (theoretical) 100% stretch from resting diameter at fixed vessel length. (AP.D)/AD (mmHg)

Arterial distensibility

Relative diameter (or area) change for a pressure increment; the inverse of elastic modulus AD/(AP-D) (mmHg -~)

Arterial compliance

Absolute diameter (or area) change for a given pressure step at fixed vessel length. AD/AP (cm/mmHg) or (cm2/mmHg)

Volume elastic modulus

Pressure step required for (theoretical) 100% increase in volume. Ap/(AV/V) (mmHg) = Ap/(AD/D) (mmHg) (where there is no change in length).

Young's modulus

Elastic modulus per unit area; the pressure step per square centimeter required for (theoretical) 100% stretch from resting length. AP.D/(AD.h) (mmHg/cm)

P indicates pressure; D, diameter; V, volume; h, wall thickness, A (delta), change. These indices are sitespecific and vary with distending pressure (from O'Rourke, 1995).

must be pointed out. First, below 150 mmHg, each pulsatile curve has a shallower slope than that of the corresponding static curve (Fig. 1, lower panel). Second, for a given static curve, such as that of WKY rats or SHRs in Fig. 1 (upper panel), the corresponding pulsatile curves are influenced by a small number of factors, which are: the frequency dependence of the slope, the set-point blood pressure, (called also mean blood pressure or operating pressure) and, finally, the viscosity of the arterial wall, which implies that the hysteresis of the pressure-diameter curve cannot be neglected (see Figure 2). In other words, any pulsatile curve, when analyzed alone and without consideration of the static curve, should be interpreted very cautiously. Fig. 1 (lower panel) provides several examples of these difficulties, which are indicated in the legend. In previous years, the static pressure-volume relationship has been widely investigated in vitro from samples of arterial strips or rings (Nichols and O'Rourke, 1990; Cox, 1977, Milnor, 1989; Dobrin and Rovick, 1969). More recently, in vivo static relationships have been studied in anesthetized animals using an original in situ carotid preparation (Levy et al., 1988) that will be described below. Actually, the investigation of the carotid artery in vivo in rats and humans is performed using pulsatile changes of diameter determined with high resolution echo-tracking techniques (Safar and London, 1994; Hoeks et al., 1990; Tardy et al., 1991; Kawasaki et al., 1987; Lichtenstein et al., 1995; Laurent, 1995). Thus it is possible to explore directly the dynamic pressure-volume relationship and to evaluate separately the static and the pulsatile components of carotid stiffness. Using this procedure, it has been verified in vivo in rats that, at any given value of mean arterial pressure, any pulsatile pressure-volume curve has a reduced slope by comparison with that of the corresponding steady curve (Glaser et al., 1995). A simplified representation of this result is given in Fig. 3. Following increasing doses of phenylephrine in WKY rats, a progressive resetting of the dynamic curves toward higher values of mean pressure and arterial cross-sectional area is observed, indicating that the constrictive effect of phenylephrine is offset by the increase in the distending pressure (Glaser et al., 1995). The static relationship indicated in Fig. 3 is mathematically deduced from a simple linear model relating mean arterial pressure and mean cross-sectional area measured in baseline conditions and following phenylephrine (Glaser et al., 1995). At any given value of mean arterial pressure, the slope of the static curve is obviously higher than that of the corresponding pulsatile curve, indicating that arteries are stiffer in dynamic than in static conditions at any given value of mean arterial pressure.

Active vs passive carotid pressure.volume r e l a t i o n s h i p The mutual relationships between the geometric characteristics of the arteries (diameter D) and the mechanical properties of the arterial wall (stiffness) at a given blood pressure are complex. In the typical case of the static pressure-volume relationship studied under circumstances of total arterial smooth muscle relaxation, at any given Cross-sectional area (mm2. 10"3) 1300

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FIGURE 3. Anesthetized WKY rats: effect of sodium nitroprusside and phenylephrine. The effect of the 2 drugs on the pulsatile cross-sectional area-arterial pressure curves are represented by comparison with the baseline curve (m-m). Note that (i) following sodium nitroprusside (SNP) ( D - Z ] ) , the carotid cross-sectional area (CSA) is maintained or even increased whereas blood pressure is significantly reduced, and (ii) following increasing doses of phenylephrine (PHE) ( © - O ) , the dynamic curves are reset toward higher values of pressure and CSA indicating that the phenylephrine constrictive effect is offset by the increase in the distending pressure. Two possible erroneous interpretations of the dynamic curves are stressed in this figure: (1) the PHE curve seems to prolong the SNP curve but they do not belong to the same static curve, and (2) under PHE, 2 pulsatile curves have different values of CSA for the same pressure although they belong to the same static curve. Regression relating mean cross-sectional area and mean arterial pressure ( 0 - 0 ) = static curve under phenylephrine.

Carotid Artery Stiffness in Cardiovascular Pharmacology value of transmural blood pressure (P), an artery studied in vitro becomes less compliant as it is dilated (Nichols and O'Rourke, 1990; Cox, 1977; Milnor, 1989; Dobrin and Rovick, 1969). This pattern is related to an increase in wall tension (P×D/2) as diameter increases, and to the transfer of this tension from elastin to the less extensible collagen fibers. However, in the presence of vasomotor tone, the situation is more complex. As shown in numerous animal experiments (Nichols and O'Rourke, 1990; Cox, 1979; Milnor, 1982; Dobrin and Rovick, 1969) difficulties arise from the interaction between: (1) the direct passive effect of blood pressure distention in relation with the nonlinear elastic behavior of the arterial wall material (Fig. 1); and (2) the active effects of smooth muscle contraction on carotid arterial stiffness and diameter. Thus, the different connections between carotid diameter and stiffness are more complicated to analyze under the various physiological conditions or after administration of vasoactive agents. Changes in diameter and compliance could be the passive consequence of blood pressure variations or could be the result of a direct action of the drugs and physiological stimuli on the arterial wall, principally on smooth muscle cells. Another major question of debate regarding the active action of drugs (and vasoactive agents) on structure and function of the carotid artery is whether the arterial smooth muscle relaxation is due to one or several of the following possibilities: direct in situ effect on vascular smooth muscle, indirect effect on vascular smooth muscle through change in endothelial function and release of vasoconstrictive or vasodilating compounds and, finally, mechanism of flow dilatation (Pohl et al., 1986; Safar and London, 1994). In the latter case, it should be recalled that any increase in carotid blood flow velocity may cause a resulting increase in diameter, with two important characteristics. First, the increase in arterial blood flow velocity is determined by a downstream increase in arteriolar diameter. Second, the increase in flow velocity and, consequently, in shear stress causes an increase in arterial diameter, again through a change in endothelial function. At the site of rats' carotid artery, the release of constrictive factors seems to predominate over that of relaxing factors (Levy et al., 1990). IN V/VO DETERMINATION OF CAROTID STIFFNESS In recent years, both static and dynamic indices of carotid stiffness have been described in rats and humans. All these indices may be measured in vivo.

Static indices of carotid stiffness In former studies described in the literature, the static carotid artery stiffness has been evaluated exclusively on strips or rings of arterial tissue as described in numerous reviews (Nichols and O'Rourke, 1990; Cox, 1977; Milnor, 1989; Dobrin and Rovick, 1969). More recently, investigations have been carried out in live anaesthetized rats. A novel preparation using 18-20 mm of nonexposed common carotid large artery in situ has been developed to establish the pressure-compliance relationship over a wide range of transmural pressures (Levy et al., 1988; Levy et al., 1990). With this preparation, the artery is submitted to a 25-mmHg pressure step from 0 to 200 mmHg. The reproducibility of the method approximates 5%. As summarized in Fig. 4a, different values of carotid compliance are obtained from zero to 200 mmHg (Levy et al., 1988; Levy et al., 1990; Benetos et al., 1993b). Because the carotid pressure-volume relationship approximates a sigmoidal curve, two different components of the curve may be described. Within pressures varying from 0 to 125-150 mmHg, the carotid compliance increases with transmuml

1297

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Static carotid compliance (nl/rnmHg) 15

WKY operating compliance SHR operating compliance

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50 75 100 125150175 200 Transmurat pressure (mmHg)

FIGURE 4. In situ carotid artery preparation: pressure-compliance curves in WKY rats and SHRs (Benetos et al., 1993b) See text. (a) Pressure compliance values in 3-month-old untreated WKY (IZ) and SHRs ([]). *P<0.001, SHR vs WKY. (b) Pressure-compliance values in before (D) and after ([]) local application of potassium cyanide. *P<0.001 before vs after.

pressure, but less in SHRs than WKY rats, so that the carotid arterial wall is stiffer in SHRs at each given value of transmural pressure. Above 125-150 mmHg, carotid compliance decreases with pressure, but the values are nearly identical in both strains. To evaluate the relative influence of smooth-muscle tone and structural changes on carotid stiffness, the arterial preparation may be filled with a solution of potassium cyanide (KCN) in saline (Fig. 3b). Abolition of vascular smooth-muscle tone with the KCN solution results in a significant increase in carotid compliance within the lower pressure ranges. Even after treatment with KCN, carotid compliance remains lower in SHRs than WKY rats, showing that structural changes are central for explaining the reduced carotid compliance in hypertensive animals. This finding in fully relaxed vessels is consistent with the increased thickness, smooth muscle mass and extracellular matrix observed within the arterial wall of hypertensive animals (Nichols and O'Rourke, 1990; Levy et al., 1988). Finally, using this methodology in anesthetized rats, it is possible to establish from 0 to 200 mmHg a highly reproducible volume compliance vs pressure relationship in vivo. The changes in compliance may be studied, not only in fully relaxing vessels using (KCN), but also after administration of vasoactive agents acting on arterial smooth muscle, enabling the role of smooth muscle tone within the lower pressure ranges to be evaluated (Levy et al., 1988; Levy et al., 1990; Benetos et al., 1993a,b). Furthermore, this procedure may be performed in the presence or absence of endothelium that may be

1298 per se responsible for changes in tone (Levy et al., 1990). However, the technique has two major disadvantages. First, only the pressurecompliance curve is established in vivo, and the pressure-diameter curve cannot be obtained in the absence of simultaneous diameter measurements. Second, flow is interrupted, so that the changes in vasomotor tone induced by pulsatile flow, particularly through changes in endothelium function, cannot be completely evaluated (Safar and Frohlich, 1995).

Dynamic indices of carotid stiffness Research in echography and pulsed Doppler velocimetry, particularly echo-tracking devices of high resolution, has been developed during the last decades so that we can now determine arterial diameter and wall motion transcutaneously (Hoeks et al., 1990; Tardy et al., 1991; Kawasaki et al., 1987; Lichtenstein et al., 1995). Using phase-locked echo-tracking, the Walltrack system (Hoeks et al., 1990) was developed to measure wall motion after echographic localization. The Diarad system (Tardy et al., 1991) was primarily described to obtain in humans the pressure-diameter curve of the radial artery by coupling the measurement of systolic-diastolicvariations of arterial diameter to those of digital blood pressure. With this procedure, no mode echography is required. A 10 MHz probe and a 1 l-ram focal length offer a high spatial resolution and an excellent reproducibility for the study of the human radial artery, provided that a stereotaxis apparatus is used. Now, the echo-tracking technique may be applied both to humans (Walltrack and Diarad systems) (Arcaro et al., 1991; Van Merode et al., 1990; Laurent et al., 1993; Hayoz et al., 1992) and rats (Diarad system) (Hayoz et al., 1992; Lacolley et al., 1995). In both cases, the major difficulty is to obtain a simultaneous determination of carotid pulse pressure (i.e., delta P) to calculate compliance. This is a critical point because, whereas mean arterial pressure is almost constant along the arterial tree, pulse pressure is known to increase markedly from central to peripheral arteries due to the progressive increase in arterial stiffness and to the summation of wave reflections along the arterial tree, as extensively shown in rats, dogs and humans (Nichols and O'Rourke, 1990; Tsoucaris et al., 1995; London et al., 1994). In conscious humans, transcutaneous applanation tonometry is used, making it possible to determine carotid systolic and diastolic blood pressure from the digitization of the blood pressure curve and the determination of brachial mean arterial pressure (London et al., 1994). In anesthetized rats, the echotracking measurements are performed on the right carotid artery, whereas intraarterial blood pressure is determined on the left carotid artery (Hayoz et al., 1992; Lacolley et al., 1995). Both in vivo and in vitro studies have validated the method (Glaser et al., 1995), indicating that the position of the two catheters and their length did not substantially alter determination of the pressure-diameter curve. With such dynamic curves, it is possible to characterize in vivo (under anesthesia in rats, and without anesthesia in humans) the pulsatile changes in diameter and blood pressure. Thus, the method gives the most adequate evaluation of the in vivo characteristics of the pressure-carotid diameter curve and, hence, of the effective buffering function of the artery, but only within the operating systolic-diastolic ranges. Several limitations should be noted for the interpretation of these curves (Safar and Frohlich, 1995). First, they cannot be extrapolated, (as evidenced from Fig. 1), and represent cross-sectional and not volumic compliance, thus, assuming that the artery length remains constant, an assumption that may be discussed (Lichtenstein et al., 1995) (Table 2). Second, because total abolition of vascular smooth muscle tone cannot be obtained in vivo, the passive properties of the arterial wall due to structural changes are

M.E. Safir impossible to evaluate. Finally, whether the changes in the dynamic curve and, hence, in the effective arterial function are mediated by a change in the set-point (= operating) blood pressure, and/or in the structure, and/or in the function of the static curve remains often difficult, or even impossible, to determine. Several examples of these difficulties are given in Figs. 1 and 3. A P P L I C A T I O N S IN C A R D I O V A S C U L A R PHARMACOLOGY A g e . r e l a t e d c h a n g e in carotid stiffness In mammals, either humans or rats, carotid arteries progressively stiffen with age due to intimal thickening, collagen fiber accumulation, calcium deposition and degeneration of the elastic laminae. The loss of distensibility is partly counteracted by progressive arterial dilation (due to the specific alterations in elastic tissue with aging) (Michel et al., 1994; Cox, 1977) and is accompanied by an increase in operational carotid stiffness. In WKY rats, significant changes in carotid vasomotor tone are associated with aging, as shown from studies of the in situ carotid preparation (Benetos et al., 1993a). Pharmacological stimulation of the cq-adrenoceptor with phenylephrine decreases carotid compliance in older, but not in younger, animals. Blockade of these receptors with prazosin or labetalol increases compliance in younger, and not in older, rats. Betareceptor stimulation with isoproterenol or blockade with propranolol has no effect in any studied groups. In parallel, it is important to note that aging does not affect cq-adrenoreceptor affinity and density, whereas it decreases beta density without changing affinity (Benetos et al., 1993a). In humans, carotid stiffness increases markedly with age, whereas no comparable finding is observed for peripheral muscular arteries, such as the radial or the femoral artery (Boutouyrie et al., 1992).

Decreased carotid arterial stiffness following nitrates Early works of Dobrin and Rovick (1969) and Gow (1980) on arterial tone, caliber and distensibility have suggested that an increase in arterial smooth muscle tone is associated with a decrease in arterial stiffness, whereas a decrease in smooth muscle tone is associated with an opposite effect. This subject was, however, beset by conceptual and methodological snags, including the value taken to be initial length (or diameter), whether comparisons should be made at the same diameter or at the same distending pressure, and the effects of change of wall thickness at different diameters. Moreover, the problem was studied exclusively in vitro, and untill recent years, there was no adequate measurement of all these parameters, in live animals or humans, in conditions of operational pulsatile pressure and flow. In vivo works on the human brachial artery have indicated that a reduction in arterial tone might have the opposite effect to that suggested by Gow (Levy et al., 1988), and actually decreased arterial stiffness (Safar et al., 1986; 1987). A more complete demonstration has recently been done on the carotid artery. Animal studies have shown that nitrates exert a preferential action in larger as opposed to the smaller, arteries (Nichols and O'Rourke, 1990; Safar 1990). Both in humans and rats, noninvasive echo-Doppler and echo-tracking techniques have extensively shown that a consistent dilation of the carotid artery occurs following intravenous nitroglycerin and sodium nitroprusside or oral isosorbide dinitrate (Van Zwieten et aI., 1995; Bouthier et al., 1986; Laurent et al., 1992; Safar, 1990). The vasodilating effect occurs even in the presence of a significant decrease in mean arterial pressure, suggesting a direct or indirect drug-induced mechanism acting on the arterial wall. The carotid artery dilatation is observed without any significant change in local vascular resistance and blood flow veloc-

Carotid Artery Stiffness in Cardiovascular Pharmacology ity, indicating that the mechanism of flow dilation is not operating with nitrate administration. Finally, nitrate derivatives are known to dilate the arteries in the absence of endothelium, thus pointing to a direct drug-induced smooth muscle relaxation (Basenge and Pohl, 1986; Van Zwieten et al., 1995). Echo-tracking techniques in hypertensive humans and normotensive rats have recently shown that operating carotid diameter and compliance are significantly increased following nitrates, whereas blood pressure decreases and distensibility is unmodified, as a consequence of the increase in carotid diameter (Glaser et al., 1995; Laurent et al., 1992). In WKY rats, it has been shown with acute experiments that, for the same operating mean arterial pressure and diameter (and, hence, thickness), operating carotid compliance is significantly higher under sodium nitroprusside than under acute dihydralazine (Fig. 5) (Glaser et al., 1995). This result showed for the first time in vivo that, following arterial vasorelaxation, a decrease in carotid arterial stiffness (as produced by nitroprusside) is associated with a decrease in arterial smooth muscle tone independent of the role of mechanical factors. Using intravascular ultrasound, Bank et al. (1995) also showed in healthy volunteers that nitroglycerininduced smooth muscle relaxation increased brachial artery compliance and decreased pulse wave velocity without significantly altering the viscoelastic properties of the wall material. Using the determination of carotid static compliance, similar resuits were noted with the converting enzyme inhibitor quinapril given acutely by gavage at a nonantihypertensive dose (Benetos et al., 1993b). For the same diameter and pressure, the carotid compliance of SHRs with this dose was increased in comparison with control SHRs. Again, in this example, a decrease in carotid stiffness was associated in vivo with a decrease in arterial smooth muscle tone. A similar finding has recently been reported with calcium entry blockers (Levy et al., 1994; Lacolley et al. 1995). Explanation of how dilation improves distensibility is difficult to explain. O'Rourke and Avolio, (in Nichols and O'Rourke, 1990), have suggested a mechanism whereby, at normal distending pressure, smooth muscle in the arterial wall is in series with some of the stiffer collagen components, but in parallel with the elastic lamellae. Contraction of smooth muscle tenses the collagenous components, whereas dilation transfers the stress to the elastic lamellae. Such an explanation appears to account for the findings referred to above, and is consistent with the arrangement of elastin, smooth muscle and collagen within t h e arterial wall. Glagov et al. (1992) have pointed out that the ¢ollagenous lattice within the wall would permit the wall to behave in this way by closing (and elongating) when muscle relaxes, and opening (and shortening) when muscle contracts. However, such a readjustment requires "intrinsic" modifications between the 3 different components of the arterial wall: smooth muscle, elastin and collagen. Recent basic studies have shown that establishment of appropriate cell-cell and cell-matrix contacts is crucial for the transduction of mechanical forces in smooth muscle tissues (Davies, 1995). As such, adhesive molecules like integrins (Hynes, 1987) might change the connections between arterial smooth muscle ceils and between these cells and extracellular matrix, thus leading to changes in the distribution of mechanical forces. Preliminary studies suggest that modifications in integrin subunits may be observed on arterial smooth muscle: in vitro on atherosclerotic plaques (Couffinhal et al., 1993) and in vivo following chronic treatment by guanethidine in WKY rats (Bezie et al., 1995).

Converting enzyme inhibitors and carotid stiffness In vitro, on perfused carotid arteries, converting enzyme inhibition significantly increases carotid diameter (Caputo et al., 1995). This

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FIGURE 5. Anesthetized WKY rats: operational static carotid compliance (mm2.mmHg-l.lO-3) (upper panel) and mean crosssectional area (mm 2.10 -3) (lower panel) under various vasoactive agents at different values of mean arterial pressure (Glaser eta/., 1995). Note that: (1) the curve relating compliance to pressure using phenylephrine ([]), L-Nitroarginine ( ~ ) and hydralazine (O) has the same shape as the static curve shown in Fig. 3, and (2), sodium nitroprusside (A) has a significantly higher compliance than hydralazine for the same pressure and MCSA (and, hence, wall thickness as acute experiments were performed).

increase disappears in the absence of endothelium (Levy et al., 1988) and seems to involve dominantly the contribution of AT1, angiotensin II, and not bradykinin, receptors (Caputo et al., 1995). In living rats, on the previously described in situ carotid artery preparation, the effects of various converting enzyme inhibitors (perindopril and quinapril by gavage; lisinopril using topical application) were investigated in a model of two-kidney, one-clip Goldblatt hypertensive (HT) rats and of spontaneously HT rats, which were compared to normotensive (NT) sham-operated animals (Levy et al., 1988; Levy et al., 1990; Levy et al., 1993b). Acute treatment with quinapril or lisinopril and long-term treatment with perindopril induced a significant increase in carotid compliance in both HT and NT rats. Within the lower pressure ranges, the increase in carotid compliance was observed for the same transmural pressure as in untreated animals, and occurred even after total relaxation of arterial smooth muscle tone. The compliance changes were related to both the structural {with long-term treatment) and the vasomotor tone-mediated components of the arterial wall, a result confirmed by concomitant histomorphometric studies. In parallel with animal experiments, clinical studies on con-

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ble-blind comparative studies and causing the same blood pressure reduction as converting enzyme inhibitors (Kool et al., 1995; Barenbrock et al., 1994).

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ARTERIAL PRESSURE (mmHg) FIGURE 6. Dynamic diameter-pressure curves (upper panel) and distensibility-pressure curves (lower panel) established i hr after treatment, in SHR treated with isradipine for 8 weeks: 0.6 mg/kg/day ( l l l l l l ) and 2.6 mg/kg/day (OQO) in comparison with control SHRs (OOO) and WKY rats (H[ZIZ) (Lacolley et al., 1995) Values are mean+SEM. Note that diameter-pressure curves of control SHRs seem to prolong those of control WKY rats, exactly as already shown in Figs. 1 and 2.

verting enzyme inhibition were performed in healthy volunteers and in HT subjects. Whereas a dilatation was obtained from the muscular brachial artery, no change or even a decrease, was obtained with the carotid artery (Safar and Levy, 1993). Because the latter result was observed in the presence of a significant reduction in blood pressure, it seems likely that the mechanical effect of the blood pressure reduction had offset the carotid arterial dilatation due to converting enzyme inhibition. On the other hand, clinical studies in patients with essential hypertension showed a significant increase in carotid arterial compliance, as observed with perindopril and lisinopril (Kool et al., 1995; Barenbrock et al., 1994). Because the carotid diameter was unchanged, or even decreased, the compliance increase might be due to blood pressure reduction alone. However, the role of drug-induced arterial smooth muscle relaxation cannot be excluded to explain compliance changes. Indeed, the increase in carotid arterial compliance following perindopril and lisinopril was not observed with hydrochlorothiazide or metoprolol given in dou-

Studies on the in situ carotid preparations were performed under 2 conditions: either acutely, using a diltiazem compound (Levy et al., 1993a) or with long-term treatment, using the dihydropiridine derivative isradipine (ISR), given orally (Levy et al., 1994). Both experiments indicated that carotid compliance increased (as for converting enzyme inhibitors) within the lower pressure ranges at any given value of transmural pressure. Similar findings have also been reported following verapamil in humans (Van Merode et al., 1990). With the acute administration of a diltiazem derivative, the results were similar in the presence or in the absence of endothelium. With long-term treatment, the response existed even after total abolition of smooth muscle tone, indicating that both a functional and a structural factor were involved in the compliance changes (Levy et al., 1994). Following chronic therapy with ISR, the role of structural factors on arterial stiffness was assessed on the basis of a reduction in arterial thickness, as well as in collagen and elastin contents. Within the aortic wall, there was a reduction in medial hyperplasia whereas, within the carotid artery, only hypertrophy was reduced (Levy et al., 1994). Thus, the remodeling of the arterial wall was not uniform, according to the vessel studied. In addition, such substantial structural changes with chronic treatment were observed although the antihypertensive effect of ISR measured in conscious rats was quite transient, reaching its maximal effect 1 hr after drug administration and disappearing after the 16th hour (Fig. 6) (Lacolley et al., 1995). With isradipine, the major interest of the echo-tracking technique and of the analysis of the dynamic pressure-volume relationship was to show that, with either acute or chronic ISR, the curve of SHRs was shifted toward the normotensive WKY curve, particularly when studied 1 hr after administration (Fig. 6) (Lacolley et al., 1995). Because acute and long-term treatment corresponded to different compositions of the arterial wall in SHRs, such findings indicated clearly that the effective arterial function was completely (although transiently) normalized with treatment, independent of the structural changes of the arterial wall. This finding, already expected from Fig. 1, confirms that, at any given value of mean arterial pressure, the static compliance is mainly dependent on smooth muscle tone and of the structural characteristics of the arterial wall. Pulsatile compliance, on the other hand, is governed by other factors, namely, the frequency dependence of the system, and, if sizable, the viscosity of the arterial wall (Fig. 1). These important and novel aspects of static and dynamic arterial stiffness may now be analyzed in vivo and, hence, are able to stimulate much fundamental research for a more comprehensive understanding of the mechanical factors that act on the arterial wall. This study was performed with a grant from the lnstitut National de la Santd et de la Recherche Mddicale (INSERM-U 337), Paris. We thank Dr. Gerard London and Bernard Levy for discussion and correction of the manuscript, and Anne Safar for its final form.

References

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M.E. Sa~r Van Merode T., Van Bortel L., Smeets F. A. Buhom R., Mooij J., Rahn K. H. and Reneman R. S. (1990) The effect of verapami| on carotid artery distensibility and cross-sectional compliance in hypertensive patients. J. Cardiovasc. Pharmacol. 15, 103-109. Van Zwieten P. A., Safar M., Laurent S., Pfaffendoff M., Maarten G. C. Hendricks M. G. C. and Bruning T. A. (1995) New insights into the role of endothelial dysfunction in hypertension. J. Hypertens. 13, 713-716.