The Contribution of Vascular Smooth Muscle to Aortic Stiffness Across Length Scales

Abstract

The operation of the cardiovascular system in health and disease is inherently mechanical. Clinically, aortic stiffness has proven to be of critical importance as an early biomarker for subsequent cardiovascular disease; however, the mechanisms involved in aortic stiffening are still unclear. The etiology of aortic stiffening with age has been thought to primarily involve changes in extracellular matrix protein composition and quantity, but recent studies suggest a significant involvement of the differentiated contractile vascular smooth muscle cells in the vessel wall. Here, we provide an overview of vascular physiology and biomechanics at different spatial scales. The processes involved in aortic stiffening are examined with particular attention given to recent discoveries regarding the role of vascular smooth muscle.

INTRODUCTION

Cardiovascular disease, the leading cause of death worldwide, is a group of complex, multifactorial disorders of the heart and blood vessels, including coronary heart disease, myocardial infarction, angina pectoris, heart failure, and stroke [18,38,81]. Traditional risk factors for cardiovascular disease include age, gender, hypertension, cholesterol, tobacco smoking, alcohol consumption, family history, diet, obesity, physical activity, and diabetes mellitus [18]. An additional independent risk factor—aortic stiffness—has recently attracted the renewed attention of clinicians and researchers. Aortic stiffening has been shown in numerous studies to precede and predict negative cardiovascular outcomes, but its development is not well understood [30]. As aortic stiffening is an early event in aging-associated cardiovascular diseases, often before the onset of symptoms, its study may provide clues to the underlying cellular and molecular mechanisms of cardiovascular disease and lead to novel therapeutic interventions. This review presents a brief summary of elements of cardiovascular physiology and biomechanics (reviewed in depth elsewhere [27,32,71]) that are important in the context of aortic stiffness. These elements are organized by biological length scale, from organ to subcellular levels (Figure 1), with particular focus given to the contribution of contractile vascular smooth muscle to aortic stiffness.

Figure 1.

The aorta across length scales. An integrative consideration of structure and function at different biological levels of scale is necessary to understand cardiovascular physiology and biomechanics in health and disease. (A) System/organ level. Top: Contraction of the heart ejects blood into the arterial systemic circulation. The blood first enters the aorta, which distends and then recoils to propel the blood downstream. Bottom: With an aging- or disease-related increase in aortic stiffness, the aorta is less distensible and absorbs less of the energy of the pressure pulse. (B) Tissue level. Aortic stiffness dictates aortic function, and aortic stiffness is determined by the structural organization and composition of the vessel wall. The wall is arranged in three main layers (intima, media, and adventitia), each containing a characteristic cell type (endothelial cells, vascular smooth muscle cells, and fibroblasts, respectively). (C) Cellular/subcellular level. Vascular stiffness depends critically on both the extracellular matrix and the smooth muscle cells embedded in the matrix. VSMCs are physically connected to their surrounding matrix by transmembrane integrin receptors, which are connected via the actin cytoskeleton to contractile filaments responsible for force production and regulation of stiffness.

CARDIOVASCULAR PHYSIOLOGY

The Organ/System Level

The cardiovascular system functions to circulate blood throughout the body. The left ventricle of the heart pumps oxygenated blood through the successively smaller vessels of the bifurcating systemic arterial tree, through the proximal, large-diameter aorta, and subsequent elastic arteries, then the muscular arteries, and finally the distal arterioles, through the capillaries, where gas and nutrient exchange occur, and then through the venous tree [13,23]. The arteries most proximal to the heart are considered elastic conduits whose walls expand to accommodate the blood ejected by the heart and then recoil (Figure 1A), releasing their stored elastic energy and aiding in the propulsion of blood down the arterial tree. The flow of blood through the system is primarily impeded by the peripheral arteries of the microcirculation, which account for roughly 60% of the total resistance. By the time blood has reached the capillaries, the mean pressure has dropped substantially, the pulse pressure (the difference between systolic and diastolic blood pressures) has dampened out, and the flow is relatively steady.

The Tissue Level

The blood vessel wall, which is organized into three layers (Figure 1B), is composed of cells, extracellular matrix (principally the proteins collagen and elastin), and cell–cell and cell–matrix connections that link these components [5]. Blood vessels exhibit marked spatial heterogeneity between different wall layers and along the length of the vascular tree [5,21,22]. The outer adventitial layer contains fibroblasts, and the inner intimal layer contains endothelial cells. The endothelium produces potent vasoregulatory agents that modulate the tone of the dVSMCs found in the media, which is generally the thickest layer of the vessel wall. Smooth muscle contraction in microvessels can dramatically reduce lumen size, functioning to both increase vascular resistance and direct blood flow to tissues based on metabolic need. In large arteries the lumen reduction is modest, but stiffness is markedly increased in response to dVSMC contraction [5,19,23,55].

The Cellular/Subcellular Level—Vascular Smooth Muscle

Vascular smooth muscle contraction is initiated and regulated by various neurotransmitters, hormones, and paracrine factors, as well as mechanical stimulation and electrical stimulation, and a combination of these signals may be present at any given instant in vivo. Force generation derives from interactions between cytoskeletal proteins myosin and actin [23,33]. At least three kinds of pathways regulate vascular smooth muscle contractility: (i) those that regulate myosin activity; (ii) those that regulate actin availability; and (iii) those that regulate the nonmuscle actin cytoskeleton and FAs, integrin-based cell–matrix adhesions (traditionally called dense plaques or adhesion plaques in smooth muscle) (Figure 1C) [34,87]. This third regulatory scheme has come to light only recently from studies conducted mainly in airway smooth muscle [20,45,61,64,87] and runs contrary to classic models of a static ultrastructure for differentiated smooth muscle [5,60].

Although cytoskeletal plasticity is now recognized as an important factor in vascular smooth muscle as well, particularly in the cell cortex [6,34], most of what is known about FAs comes from studies of migrating cultured cells, where FA undergoes dynamic, tension-mediated formation, growth, and maturation, followed by release of the contacts with the matrix and recycling [9,14,28,78,84,88]. Tyrosine phosphorylation of FA proteins in migrating cells modulates FA size, location, and composition and is considered the biochemical signature of FA recycling and turnover [10,42,75,85]. FAs, which contain upward of 100 proteins, serve as the mechanical bridge between the outside of the cell and the actin cytoskeleton [15,16,37,49,86]. The FA performs mechanotransduction, wherein physical forces (hydrostatic pressure, shear, tensile stretch, etc.) are transformed into biochemical signals [4,53]. Aberrant mechanotransduction has been suggested to play a role in the pathogenesis and progression of cardiovascular and other diseases, but further investigation is necessary [29].

CARDIOVASCULAR BIOMECHANICS

The Organ/System Level

The nature of blood flow (pulsatile hemodynamics) is complex, determined not only by the time-variant pressure gradient established by contractions of the heart but also by the morphology (geometry), composition, and mechanical properties of the vessel walls, which vary along the length of the vascular tree [5,23]. Early attempts to model the arterial circulation and relate pressure to flow invoked the Windkessel model [46,76]. In its simplest form, this lumped parameter circuit model consists of a resistor representing the small arteries and microcirculation and a capacitor representing the compliance of the large arteries. This electrical analog captures the smoothing action of the aorta on flow from the heart, but incorrectly assumes that changes in pressure are felt instantaneously, without delays, throughout the entire system [40,41]. A more accurate model of the spatiotemporal variation in pressure and flow in the arterial tree treats the arterial system as a distributed load, using transmission line theory to account for transit delays, impedance mismatches between arterial segments, and wave reflections [2,40,65,66]. These models provide a valuable framework for describing and interpreting changes in cardiovascular function.

The Tissue Level

The biomechanical properties of the aorta wall depend on its material composition and organization. Vascular stiffness is commonly attributed to the major matrix proteins of the vessel wall, collagen, and elastin [71]. The basic notion is that at low pressures wall tension is born by distensible elastin, whereas at higher pressures the collagen fibers in the wall uncrimp and reorient to bear a greater portion of the load and effectively stiffen the vessel [26,52]. Elastin is responsible for the elastic recoil of the vessel in response to pulsatile flow, and the collagen protects the vessel from damage from overdistension.

The blood vessel wall exhibits mechanical behavior that is nonlinear, anisotropic (directionally dependent), and visco-elastic (exhibiting creep, stress relaxation, and hysteresis), with hysteresivity that is insensitive to strain rate [13]. In the unloaded state, the wall harbors residual stresses, and in vivo it exists in a prestretched, prestressed configuration [13]. While many quantitative models have been proposed to characterize this complex biomechanical behavior, most only possess a subset of these features [70]. In particular, most models primarily describe passive mechanical properties with very few attempting to account for active cellular behaviors.

The Cellular/Subcellular Level—Vascular Smooth Muscle

The contribution of the contractile smooth muscle cell to aortic stiffening with age has largely been overlooked. The dVSMC is believed to act as a short-term tension setter that does not directly contribute to the stiffness of the vessel wall, but rather contracts transiently to redistribute tensile forces between elastin and collagen, modulating stiffness only on short timescales [44,51,80]. However, this scheme has been challenged by recent studies demonstrating for the first time that the intrinsic stiffness of aortic smooth muscle cells increases with either age or hypertension, through mechanisms tied to the actin cytoskeleton, and may contribute directly to aortic stiffening [50,56]. Further studies of the mechanical behavior of individual differentiated vascular smooth muscle cells are necessary to bridge the gap in our understanding between vessel mechanics at the micro- and macroscales [39].

AORTIC STIFFNESS

Functional vs. Material Aortic Stiffness

Currently, the literature and the field have taken to referring to the various measures of aortic stiffness simply as “aortic stiffness,” which is imprecise and may cause confusion. A preferred nomenclature would invoke two varieties of stiffness—material stiffness and functional (or effective) stiffness. Material stiffness (e.g., the modulus of elasticity or Young’s modulus E) is geometry independent, whereas functional stiffness (e.g., PWV, impedance, etc.) depends on vessel geometry (e.g., diameter, thickness, in vivo prestrain, and tethering) as well as the material composition, and therefore material stiffness, of the vessel wall [19].

Functional aortic stiffness is assessed by noninvasive clinical procedures in vivo, and some researchers have tried to estimate material stiffness from the in vivo data using mathematical relations derived from hemodynamics (e.g., the Bramwell–Hill or Moens–Korteweg equations) [44]. The gold standard of clinical measures is PWV, which is determined by recording carotid and femoral pressure waveforms and then dividing the transit distance by the transit time between the two sites [40]. It is important to clarify that PWV is not the velocity at which blood travels through the lumen, but rather the velocity at which the pressure signal is conducted by the vessel wall. Another surrogate for material stiffness is aortic characteristic impedance Z_c, a measure of the vessel’s capacity for limiting the flow induced by a time-varying pressure gradient [2,40]. Aortic PWV and characteristic impedance have the same dependence (square root) on Young’s modulus and thickness [40]. Importantly, PWV has an inverse square root dependence on lumen diameter, while characteristic impedance is fivefold more sensitive to changes in diameter [40]. This distinction is critical for proper interpretation of functional stiffness measurements, especially as there are situations in which PWV and characteristic impedance change divergently [40].

Although material stiffness can be calculated from noninvasive measures of functional stiffness, which reflect not only the material stiffness but also the geometry, strains, and pressurization of the vessel in situ, in practice there may be substantial errors introduced due to the challenges of monitoring and controlling the relevant geometric and loading parameters during blood flow in vivo. For this reason, the study of material stiffness is better suited to direct measurement of suitable animal tissues in vitro (e.g., by uniaxial stretching of rings as in [55], by biaxial stretching of tissue strips as in [68], or by pressurization of intact vessels as in [72]). In some regards such experiments will allow investigators to more quickly and easily understand the biomechanical and morphological underpinnings of cardiovascular disease.

Implications of Increased Functional Aortic Stiffness for Cardiovascular Disease

It is thought that the increased functional stiffness of the aorta with age decreases the impedance mismatch between the aorta and the muscular arteries, allowing greater transmission of the pulse pressure to distal sites [40]. This excess energy causes end-organ complications by damaging the microvasculature in critical high-flow, low-impedance organs, such as the brain, retina, and kidney [25,35,69,73]. The increased pressure pulsatility may also promote hypertrophy or remodeling in the resistance vessels, which would affect resistance to flow and the blood supply to various tissues and could lead to subsequent development of hypertension [40,43]. Changes in aortic stiffness may also shift the return timing of reflected waves to earlier in systole, which could augment aortic pressure and increase the load on the heart [41].

Mechanisms of Changes in Aortic Stiffness

Aortic stiffening with age or disease is generally thought to be a consequence of modifications to the morphology and composition of elastin and collagen [19,71]. In the aorta, repeated loading cycles over time lead to the fatigue failure of elastin and a progressive shift of mechanical loads from damaged, fragmented elastin fibers to collagen, thereby stiffening the vessel. Elastin has a relatively long half-life in the body (>40 years); however, essentially all elastin synthesis is complete by adulthood, and there are no mechanisms to replace damaged elastin [8,47,58]. This long half-life makes elastin especially vulnerable to cumulative damage in the form of AGEs that can irreversibly crosslink elastin or collagen via nonenzymatic protein glycation [1,36,79]. In addition, elastic arteries are also known to undergo calcification [7,57], as well as enzymatic degradation via MMPs [74,82,83]. Increased collagen deposition, particularly in the collagen-rich adventitial layer, also leads to a stiffer aorta [12]. Oxidative stress [3] and inflammation [11,24] may further influence the structural changes that result in arterial stiffening.

The role of dVSMCs in aortic stiffening has been examined for the most part indirectly in studies of endothelial vasoregulatory dysfunction with age and disease (e.g., reduced bioavailability of nitric oxide) [17,59,62,63,77]. Recently, it has been shown that the stiffness of primary cultured aortic smooth muscle cells is increased in an aging primate model and in a hypertensive rodent model, representing a potential new mechanism for aortic stiffening, but the details of such a mechanism remain unclear [50,56]. Work from our laboratory employing a multiscale approach has now identified the cortical FA of vascular smooth muscle cells as a key subcellular regulator of aortic stress generation and stiffness [55]. Using a magnetic microneedle to apply piconewton forces to cell-bound, RGD-coated microbeads, we demonstrated that agonist stimulation induces stiffening of the VSMC cortex, and that the small molecule Src-inhibitor PP2 prevents this stiffening. Deconvolution immunofluorescence microscopy revealed that this cortical stiffening parallels agonist-induced increases in FA size, which are also Src dependent. Aorta tissue homogenates probed by Western blot with phosphospecific antibodies indicated increased tyrosine phosphorylation of FA proteins CAS, FAK, and paxillin in response to contractile stimulation, which is indicative of FA remodeling. Both PP2 and FI-14, a small molecule inhibitor of FAK, inhibited agonist-induced increases in tyrosine phosphorylation, as well as agonist-induced contractile force and stiffening, as measured with high-frequency, low-amplitude cyclic stretches that do not break cross-bridges. Taken together with previous studies, these results indicate that agonist stimulation induces actin polymerization, cortical stiffening, and Src-FAK–mediated FA growth and remodeling that strengthens the cortical cytoskeleton and FA to enable tissue stiffening [34,55] (Figure 2). Different FA proteins undergo varying degrees of redistribution in response to agonist stimulation, with proteins like VASP and zyxin that strengthen the link between the FA and cortical actin being most dynamic [48]. This shuffling of FA proteins is linked to actin- and microtubule-dependent endosomal pathways [48], which had previously been implicated in FA recycling in migrating cultured cells [31,54,67], and may help to reinforce high tension-bearing sites.

Figure 2.

Model of FA regulation of aortic stiffness. Agonist- and tension-induced growth and remodeling of FA in aortic VSMCs strengthen cell–matrix adhesion to facilitate force transmission and stiffening of the aorta. Differential redistribution of FA proteins is facilitated by endosomal recycling pathways. Modified from [55].

SUMMARY

The microcirculation, the main regulator of total vascular resistance, plays an important role in cardiovascular disease, but recent studies have indicated a substantial role for the large-diameter elastic aorta as well. Aortic stiffening, a known biomarker for subsequent cardiovascular disease, reduces the impedance mismatch between the aorta and the muscular arteries. As a result, a larger portion of the pulse pressure is transmitted distally to sensitive microvessels in critical organs, including the heart, brain, and kidney. Augmented transmission of the pressure pulse may induce hypertrophy and remodeling of the microcirculation and increase the afterload on the heart.

Clinical measures of aortic stiffness, while noninvasive, incorporate not only the mechanical properties of the vessel wall but also its geometry. It is important to distinguish functional measures of stiffness (PWV, Z_c) from measures of material stiffness (Young’s modulus). This will require thorough biomechanical investigations in vitro where loading conditions and vessel dimensions can be precisely tracked and controlled.

Recent inquiries into the multifaceted mechanisms of stiffness are forcing investigators to reconsider the aorta as more than simply a passive elastic conduit. Just as dVSMCs are integral to regulating the resistance of the microcirculation, these cells are also integral regulators of aortic stiffness. Further investigations of smooth muscle involvement will not only inform modeling efforts but may also lead to the development of new interventions to mitigate aortic stiffening and reduce the burden of cardiovascular disease.

Thus, the complexity of aortic stiffening with aging and disease warrants a multiscale approach to understand underlying mechanisms and their effects on cardiovascular function. Importantly, the success of this integrative approach will require interdisciplinary collaboration among clinicians, physiologists, biologists, and engineers.

PERSPECTIVE

Aortic stiffness is an early biomarker for cardiovascular disease. The mechanisms of aortic stiffness have long been presumed to be extracellular, although recent studies suggest that vascular smooth muscle cells are important regulators of stiffness. A multiscale approach that integrates insights across fields of study may lead to new therapies for aortic stiffness and cardiovascular disease.

Abbreviations used:

AGE

advanced glycation end product

CAS

Crk-associated substrate

dVSMC

differentiated (contractile) vascular smooth muscle cell

E

modulus of elasticity (Young’s modulus)

FA

focal adhesion

FAK

focal adhesion kinase

MMP

matrix metalloprotease

PWV

pulse wave velocity

Z

impedance