Mechanics and Composition of Human Subcutaneous Resistance Arteries in Essential Hypertension

Methods

Results

Demographics of the study subjects are shown in Table 1⇓. Seven normotensive and 14 hypertensive individuals were investigated. The normotensive group had proportionately more women; otherwise, there were no significant differences between the groups in age, body mass index, serum creatinine levels, and serum lipid profile. Blood pressure was significantly elevated in the hypertensive subjects, who were untreated at the time of the study.

The structure of subcutaneous small arteries of the subjects studied is depicted in Table 2⇓. Media width and media-to-lumen ratio of the vessels of hypertensive subjects were increased (P<0.01), whereas media cross-sectional area and lumen diameter did not exhibit significant differences between the groups. The growth index, that is, the percent difference in cross-sectional area of the media between vessels from hypertensive and normotensive subjects, was 32.8%.

Vascular mechanics were studied by progressively increasing intraluminal pressure. The left panel of Figure 1⇓ shows that wall distensibility plotted against intraluminal pressure was similar in arteries from both groups. The right panel of Figure 1⇓ depicts the media stress-strain relationship, which was significantly shallower in the vessels from hypertensive subjects, indicating that isometric stress was lower in hypertensive vessels. From the stress-strain relationship, incremental elastic modulus was calculated and plotted against intraluminal pressure and circumferential stress (Figure 2⇓). The left panel of Figure 2⇓ shows that incremental elastic modulus plotted against intraluminal pressure was lower in vessels from hypertensive subjects. The right panel demonstrates that when plotted against circumferential stress, the slope of the incremental elastic modulus (β), which demonstrates the stiffness of wall components, was significantly lower in vessels from hypertensive subjects than in those from normotensive subjects. Since in the present study there was a marked imbalance in the number of male and female hypertensive subjects, the mechanics of vessels from male and female normotensive and hypertensive subjects were evaluated separately. Figure 3⇓ shows that vessel mechanics in male and female hypertensive subjects were identical. No significant differences were found between vessels from male and female normotensive subjects. Differences between mechanics of all normotensive and hypertensive subjects were thus maintained within men and women.

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Figure 1.

Left, Incremental distensibility plotted against intraluminal pressure in relaxed subcutaneous small arteries from normotensive and hypertensive subjects. The curves did not differ significantly. Right, Media stress plotted against strain at progressively higher intraluminal pressures. Curves were significantly different (P<0.01). Error bars indicate SEM.

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Figure 2.

Left, Incremental elastic modulus plotted against intraluminal pressure in relaxed subcutaneous small arteries from normotensive and hypertensive subjects. **P<0.01. Right, Incremental elastic modulus plotted against media stress at progressively higher intraluminal pressures. The slope of both curves (β) was significantly different (P<0.05). Error bars indicate SEM.

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Figure 3.

Left, Incremental elastic modulus plotted against intraluminal pressure in relaxed subcutaneous small arteries from male and female hypertensive subjects. Right, Incremental elastic modulus in vessels from male and female hypertensive subjects plotted against media stress at progressively higher intraluminal pressures. The slopes of both curves (β) were similar. Error bars indicate SEM.

Electron microscopic evaluation of the structure and composition of the media of pressurized small arteries from 5 of the 7 normotensive subjects and 9 of the 14 hypertensive subjects showed a trend toward a greater number of smooth muscle layers (P=0.07) and higher collagen volume density (P=0.08) in vessels from hypertensive subjects and a significant increase in the collagen-elastin ratio (P<0.05) (Table 2⇑).

Discussion

This study evaluates the vascular mechanics of subcutaneous small resistance size arteries from hypertensive subjects for the first time using a pressurized system, which may represent a more physiological approach than other methods previously used for examining vascular structure and mechanics.¹⁷ The composition of the media of vessels was also studied to determine whether changes in the volume density of collagen (less distensible) and elastin or smooth muscle (more distensible) could explain the differences in mechanical properties. We show that subcutaneous small arteries of the hypertensive subjects studied present an altered structure, as previously reported by others¹³ and in our own studies.¹⁴ In this particular group of subjects, media-to-lumen ratio of small arteries was increased, as was media width, whereas media cross-sectional area was not. However, although differences between the media cross section of small arteries did not achieve statistical significance, the growth index was 32.8%, which suggested a trend toward what has been designated as hypertrophic remodeling.¹⁰ This finding is in contrast to previous studies from our own group¹⁴ and others¹⁸ in which the predominant finding was that of eutrophic remodeling, but is in agreement with the first results obtained from the study of human subcutaneous small arteries.¹³ The heterogeneity of individuals with essential hypertension suggests the need for caution in making our conclusions in view of the range of results that may be encountered.

The lumen diameter of a vessel will depend on opposing forces of transmural pressure and compliance of the vessel wall. Compliance, that is, the ability of a vessel to buffer changes in pressure, depends on the geometry and stiffness of the wall components of the vessel. Decreases in lumen size (resulting mainly from remodeling in hypertension¹⁰ ) may reduce compliance, which is geometry-dependent. However, vascular compliance may be normalized by decreases in elastic modulus. Incremental elastic modulus is geometry-independent and depends on the stiffness of the vessel wall components. Elastic modulus is determined by the combined elastic modulus of the structural components of the vascular wall: connective tissue, elastin and collagen fibers, smooth muscle cells, and endothelial cells. Previous studies of the mechanics of large arteries have demonstrated that vascular wall compliance is not necessarily reduced in hypertension both in animal models and in human essential hypertension.⁶ The mechanism for the absence of reduced vascular compliance and distensibility despite increased media thickness and collagen deposition has been obscure. In very small arteries from the brain, the pioneering studies of Baumbach et al¹⁹ showed for the first time that wall mechanics in hypertension might not be altered as expected. These investigators first reported that compliance and distensibility could actually be increased and that the slope of the incremental elastic modulus in relation to circumferential stress, that is, the stiffness of wall components, could be reduced in pial arterioles of approximately 80 μm in lumen diameter from stroke-prone spontaneously hypertensive rats (SHRSP).⁸ They found similar results in small arteries (<150 μm in lumen diameter) but not larger small arteries (150 to 300 μm in lumen diameter) in the brain. Their studies demonstrated that the composition of vessels with reduced stiffness was altered, with increased elastin content,¹¹ which could explain the direction of the alteration in mechanics. We have found in some groups of SHR,⁹ and in Dahl-salt sensitive rats²⁰ and deoxycorticosterone acetate (DOCA)–salt hypertensive rats,²¹ similar or lower stiffness of wall components of mesenteric small arteries than in equivalent vessels from normotensive rats. Together with previous⁶ and more recent^{22 23} findings in large arteries from animal models and humans, this may suggest that a reduced stiffness of wall components is indeed a common characteristic in hypertension. The mechanism may not be the same for all vascular beds, however, because in contrast to pial arterioles, in which elastin is increased, in large arteries in SHR most studies have shown an increase in collagen deposition²⁴ (although the volume density of collagen was not increased²⁵ ). In mesenteric small arteries of SHR we have recently demonstrated increases in collagen as well.¹² In the present study we found that even though the stiffness of wall components is decreased in vessels from hypertensive subjects, the volume density of collagen in the media tends to be increased, although not achieving statistical significance, and the collagen-elastin ratio is significantly enhanced. This agrees with our previous findings in mesenteric arteries of similar dimension in SHR.¹² It would be expected that an increased collagen-elastin ratio might increase stiffness.¹⁹ Thus it is likely that in these vessels other factors are involved, and it is possible that changes in extracellular matrix–smooth muscle cell attachments may play a role in the change in mechanical properties found in both human arteries and those of experimental models of hypertension. Recent studies have demonstrated that in aorta of SHR, fibronectin and α5-integrin are increased.²⁵ It has been speculated that increases in cell–extracellular matrix attachment sites may contribute to the maintenance of equivalent mechanical properties even though SHR vessels, or human vessels in the present study, are subjected in vivo to higher pressures. The mechanisms involved in the reduction of the elastic modulus in relation to wall stress and pressure in resistance arteries from hypertensive individuals remain unclear. Since differences in the volume density of more- versus less-distensible components of the media (smooth muscle and elastin versus collagen) do not appear to play an important role, the difference in mechanical properties could be related in part to the characteristics and/or organization of these components in the vascular wall, particularly the arrangement, alignment, and adhesion of fibrillar and cellular elements. Models of arrangement of these components in the vascular wall in general assume that collagen is arranged in series to smooth muscle cells and in parallel to elastin.²⁶ The linking of collagen to smooth muscle cells and the degree to which the collagen jacket is tensed may thus play an important role, since collagen is less distensible than elastin.²⁷ Its contribution is supposed to occur in the latter portion of the pressure curve, since collagen fibers may be coiled and not under tension until the smooth muscle cells in series and the elastin in parallel have been stretched. In the remodeled small artery, with more closely aligned cellular and fibrillar components due to changes in adhesion of cellular and fibrillar structures, the collagen fibers may be recruited at higher distending pressures in vessels from hypertensive subjects than in vessels from normotensive subjects. Thus, although in small arteries from hypertensive subjects vascular compliance may be reduced in part because of their smaller lumen and greater collagen-elastin ratio, this may be compensated by the engagement of collagen fibers, and resulting tensing of the collagen jacket may occur in a later portion of the pressure curve. This has already been proposed by Bank et al²⁸ to explain changes in compliance and stiffness in the human brachial artery during contraction and relaxation. If for each level of media stress, elastic modulus (stiffness) is lower, this will contribute to buffering the effects of greater intravascular pressure in hypertension.

Elastic properties of subcutaneous small arteries from hypertensive and normotensive individuals have been the subject of one previous study.¹⁵ That study was carried out with the wire myograph technique and showed that elastic modulus was not increased in hypertensive vessels. The present results extend the previous observations, showing comparable results with the newer and more physiological approach of examining small arteries in a pressurized system and distending them progressively by increasing intraluminal pressure rather than by stretching the vessels with two wires inserted in the lumen. We have previously demonstrated that structural data obtained with the pressurized system are also comparable to data from observations using wire myography.²⁹ With respect to the study of mechanical properties, both techniques may have limitations. Stretching a vessel with two wires may result in vessel shortening, which may affect the transversally measured elastic properties of the shortened vessel; and pressurization of vessels may result in elongation, which necessitates adjustment of the microcannulas to maintain parallel walls. This could also affect the mechanical characteristics measured. In the present study, however, very little or no adjustment was necessary in the distance between the microcannulas. Thus, it is unlikely that this played a role as a confounder in the results obtained. The loss of tethering by surrounding tissues results in different conditions when the vessels are studied in vitro relative to the in vivo situation. However, the present study does not pretend to examine the in vivo behavior of these vessels, but rather the in vitro characteristics of the vessel wall under similar conditions in arteries from normotensive and hypertensive subjects to help understand the changes that have occurred in the vessel wall. Furthermore, because the original length in vivo of the vessels studied cannot be known, axial stiffness cannot be evaluated whereas circumferential stiffness may be quantified. The arterial wall is anisotropic, that is, direction-dependent.³⁰ Axial and circumferential compliance have been evaluated in carotid arteries in situ in SHR and shown to be similar.³¹ However, these studies may be performed only when the in vivo length of vessels can be evaluated, such as with the use of ultrasound techniques, which are not yet applicable to the study of human subcutaneous small arteries.¹⁷ Because mechanical properties may be altered biaxially,^{31 32} uniaxial stiffness as investigated in the current study has to be interpreted cautiously.

A limitation of this study is that the proportion of men and women differed in the normotensive and hypertensive groups. This resulted from the difficulties inherent in the recruitment of subjects for an invasive study such as this one, in which a biopsy of subcutaneous tissue was performed. This could potentially have contributed to the suggestion of hypertrophic growth in resistance arteries from this group of hypertensive subjects, who were predominantly male. However, the stiffness of small arteries from normotensive men and women was not significantly different, and the same was found for hypertensive men and women (Figure 3⇑). Also, within men, the stiffness of vessels was greater in normotensive than in hypertensive individuals, and in women, the direction of the findings was similar, although not achieving statistical significance in the latter because of the small numbers. Thus, although this imbalance in the number of subjects from each sex is a recognized limitation, it may not have influenced the results.

The clinical relevance of these findings may result from the fact that altered mechanics of the vascular wall may affect the velocity of the pulse wave, wave reflection, pulsatility of vessels, blood flow velocity, and accordingly shear stress. Vascular wall damage, and with it cardiovascular risk, may be modified by changes in wall mechanics. Decreases in the stiffness of wall components may protect the vessel wall. The extent to which these changes in mechanics are protective and whether their absence may be an indicator of increased vascular risk remain to be determined.

In conclusion, the components of the wall of subcutaneous small arteries of hypertensive subjects are less stiff than in comparable vessels from normotensive subjects. The mechanism for the increased compliance of small arteries of hypertensive individuals remains unclear. The changes found in the composition of the media of the wall of small arteries, an increased collagen-elastin ratio, cannot explain the differences in mechanical properties between vessels from normotensive and hypertensive subjects. It is possible that changes in extracellular matrix architecture or in cell–extracellular matrix attachments may play a role, but this remains to be established. Whether similar changes occur in other vascular beds at the same level of the vasculature requires further study.