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Biomechanical theories of atherosclerosis 

Biomechanical theories of atherosclerosis
Chapter:
Biomechanical theories of atherosclerosis
Author(s):

Jolanda J. Wentzel

, Ethan M. Rowland

, Peter D. Weinberg

, and Robert Krams

DOI:
10.1093/med/9780198755777.003.0012
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date: 05 March 2021

Introduction

Atherosclerosis is predominantly seen as an inflammatory disease driven by lipid accumulation within the arterial wall. The endothelium plays a key role in regulating the uptake of lipids and inflammatory cells. It is often overlooked that the disease develops at certain predilection sites and this suggests that localizing factors regulate the critical endothelial properties. These localizing factors have been studied extensively over the last hundred years; biomechanical factors have emerged as key candidates and are the subject of a number of reviews (14).

This chapter will critically evaluate the role of biomechanical factors in atherosclerosis, and in order to assist readers with a less pronounced background in biomechanics, we start by defining biomechanical parameters.

Biomechanical definitions

Mechanics is the study of the behaviour of materials under a mechanical load, and biomechanics is the sub-speciality of biological materials. A load is defined as the force acting on a perpendicularly oriented surface (Biomechanical theories of atherosclerosis Fig. 12.1a). To normalize for dimensions, load is converted to stress, which is force divided by the area over which it acts. Material deforms on the application of stress and again a correction is made for the dimensions of the material, for example by dividing deformation by initial length—this is the strain (Biomechanical theories of atherosclerosis Fig. 12.1a). The stress required to produce a certain strain is determined by tissue properties and for isotropic, non-viscous material can be summarized by tissue stiffness or Young’s modulus (E; Biomechanical theories of atherosclerosis Fig. 12.1b).

Fig. 12.1 Definitions of mechanical metrics are displayed in this figure. (a) The normalization of force by area to obtain a stress (σ‎ N/m2), and of deformation (L[m]) by initial length (Lo[m]) to obtain strain (ε‎). The slope of the stress-strain relationship is tissue stiffness (Young’s modulus: b). For blood vessels curvature has to be taken into account (c). (See text for further details.)

Fig. 12.1
Definitions of mechanical metrics are displayed in this figure. (a) The normalization of force by area to obtain a stress (σ‎ N/m2), and of deformation (L[m]) by initial length (Lo[m]) to obtain strain (ε‎). The slope of the stress-strain relationship is tissue stiffness (Young’s modulus: b). For blood vessels curvature has to be taken into account (c). (See text for further details.)

Under normal physiological conditions the artery wall is constantly subject to mechanical loads. The primary externally-applied loads are caused by blood pressure and flow (Biomechanical theories of atherosclerosis Fig. 12.1c). For vessels, being circular in cross section by nature, some forces balance out and blood pressure results in circumferential stress in the vessel wall, which can be calculated from Laplace’s law:

σθ=P*rh

where P is blood pressure, r is lumen radius, h is vessel wall thickness and σθ is circumferential stress. A typical value of P is 13.3 kPa and that of the resultant circumferential wall stress is 100–150 kPa. In addition to the normal stress of blood pressure, the luminal surface of the artery also experiences a tangential externally-applied stress caused by friction from the flowing blood—this is termed haemodynamic wall shear stress. This shear stress is normally denoted by tau (τ‎, Pa), and a typical value of shear stress is ∼1 Pa, which is four orders of magnitude lower than blood pressure. Thus, while shear stress impacts on numerous aspects of vascular mechanobiology, it is usually of insufficient magnitude to alter arterial wall integrity directly. Most arteries also experience external loads due to the surrounding tissue and/or due to the motion of the tissues to which they are attached; for example, epicardial coronary arteries experience stress related to torsion of the heart during cardiac contraction, which may also influence atherosclerosis progression (5).

Finally, it is important to note that the internal stress of an artery wall is not only dependent on the externally applied loads, but also on residual stresses of its constituents. Residual stress is defined as the stress that exists within a material body in the absence of an externally applied load.

For an elastic material such as the artery wall, the presence of stress (or, more generally, a load) entails the presence of strain (or, more generally, a deformation), which is dependent on the stiffness of the material. Strain is a function of, but not equivalent to, stretch (for instance, in one-dimension, Green strain, E= 12λ21, where λ‎ is stretch and stiffness is a mechanical property of a material that describes the resistance to deformation for a given load (defined as dσ/dE).

Stress and strain are used in biomechanics instead of force and deformation for the following reasons: when one compares two similar materials (same Young’s modulus) but with one having twice the width of the other, then in order to deform the thicker material similarly one has to apply twice the force, while the stress in both materials is similar. When one compares two similar materials but now one is twice as long as the other, a similar force will induce twice the deformation, but strain will be similar. As a consequence, when one calculates stress over strain for an unknown material, then their ratio is the stiffness of that material. Importantly, stress, strain, and stiffness can all be defined point-wise (i.e. locally) within a material body. This latter phenomenon has led to the concept of stress or strain fields which can be estimated nowadays from finite element methods.

Several theories aim to provide an explanation for the observed predilection sites in atherosclerosis. These theories may be divided into transport theories, shear stress theories, and mechanical strain theories.

Transport theories

History of transport theories

The term atherosclerosis derives from two Greek words, ἀ‎θ‎ή‎ρ‎α‎, meaning gruel, and σ‎κ‎λ‎ή‎ρ‎ω‎σ‎ι‎ς‎, meaning hardening. Gruel refers to the lipid-rich material at the core of the lesions. As emphasized by Rindfleisch (6), histological studies show that this material lies within the arterial wall rather than on it. Where does the lipid come from? Although some lipid may be synthesized by the wall itself and a fraction of the lipid in advanced plaques may derive from the membranes of red blood cells, there is overwhelming evidence that the majority of the lipid in atheromata derives from lipoproteins, particularly low density lipoprotein (LDL), circulating in the blood.

This ‘insudation theory’, sometimes called the ‘lipid hypothesis’, was developed in its modern form by Anitschkow (7); it was stimulated by his observation that an atherosclerosis-like disease occurred in rabbits made hyperlipidaemic by adding cholesterol to their diet. Indirect and direct support for the concept has accumulated over the years, and may be summarized as follows:

  • Tracer and immunofluorescence studies show that circulating LDL does enter the arterial wall (8).

  • The proportion of different fatty acids in early lesions is similar to their proportion in LDL (9).

  • Patients with heritable hyperlipoproteinaemias, and hence markedly increased concentrations of lipid in their plasma, have an accelerated form of the disease (10).

  • Even in the normal population, plasma concentrations of total cholesterol and of LDL are highly correlated with the risk of cardiovascular disease (11).

  • Statins lower plasma cholesterol and LDL concentrations, and reduce the disease (12).

  • Atherosclerosis-like diseases are induced in animals made hyperlipidaemic by feeding them cholesterol, and disease progression is stopped or reversed by removing the dietary supplements (13).

  • Genetically hyperlipidaemic animals (Watanabe Heritable Hyperlipidaemic rabbits, and Apo-lipoprotein E or LDL-receptor knockout mice) develop atherosclerosis-like disease even on a normal diet (14, 15).

Perhaps the most important evidence, studied for over 100 years, is the association between anatomical variation in the arterial wall uptake of plasma macromolecules and anatomical variation in lesion prevalence. If plasma concentrations of macromolecules are uniform, this association suggests that transport properties of the wall may themselves play a key role in how quickly disease develops at a particular site. Variations in uptake around branch points are of particular interest because they have led to a debate about the transport properties that predispose to disease.

Anitschkow drew particular attention to the fact that diet-induced lesions develop in an arrowhead-shaped region surrounding the downstream half of branch ostia in the rabbit aorta. Experiments with intravital dyes such as Evans’ Blue and its isomer, Trypan blue, have suggested that such regions are particularly permeable to circulating macromolecules, before the disease develops. When injected intravenously, these dyes bind to plasma proteins, some of which enter the wall and stain it blue. The intensity of staining is highest downstream of branch mouths in pigs (16), supporting the idea that high permeability to macromolecules predisposes to disease. However, Mitchell and Schwartz (176) showed that the distribution of fatty streaks in adult human aortas was different from those in rabbits: regions downstream of branches were spared the disease rather than being the most susceptible. Since the experiments with intravital dyes seemed to show that such regions were highly permeable to macromolecules, Caro et al. (17) suggested that disease in people is caused by a restricted egress across the endothelium of material made or modified within the wall, rather than an enhanced influx. Although this hypothesis could explain why human lesions seemed to occur in regions of low rather than high permeability, it is hard to reconcile with the strong relation between plasma LDL concentration and disease prevalence.

An alternative explanation for the discrepancy between rabbit and human disease is that lesion patterns change with age; according to this view, the discrepancy arises because immature rabbits have inappropriately been compared with mature people (18). Animals tend to be used when young, for reasons of cost, whilst post-mortem arteries are more often available from mature than immature people. When mature rabbits are fed cholesterol, the regions downstream of aortic branch points are spared disease (19); conversely, in immature human aortas, the downstream regions are susceptible to lipid deposition (20). Furthermore, although regions downstream of branch points have high permeability in immature aortas, that is not the case in mature vessels (21). Hence, when age is taken into account, there is a good spatial correlation between rabbit lesions, human disease, and regions of high uptake; it becomes possible to apply the insudation theory/lipid hypothesis to people.

A final point is that as well as determining where lesions develop, the level of uptake of plasma macromolecules by the wall may also determine the nature of the disease. Two types of lesion can be induced in hyperlipidaemic mice by placing a tapered cuff around their carotid arteries: lesions resembling stable human plaques develop at the narrower, downstream end of the cuff; whereas lesions resembling the unstable thin-cap fibroatheroma (TCFA) develop at the wider, upstream end (22). If the same cuff is used in normocholesterolaemic mice, to avoid complications caused by the development of disease, uptake of plasma macromolecules is elevated at both ends of the cuff, but significantly more so at the upstream end, where the TCFA develops (23). This result is consistent with a particularly high uptake of circulating lipoproteins leading to the development of the TCFA, perhaps by causing the formation of the large lipid-rich core that is characteristic of these lesions.

Mechanisms of shear-dependent transport

Given the key roles of arterial wall transport properties suggested by these studies, it is important to understand the underlying transport processes; however, our knowledge is surprisingly incomplete in several areas. Studies in which LDL was labelled in both its protein and lipid moieties, and in which labelled, non-metabolized analogues of LDL were used, have shown that the particle enters the wall intact (8). There is a continuous transport of water and solutes from the lumen to the adventitia; this transport involves both convection and diffusion, as pressure is lower in the adventitial microcirculation and concentrations are lower in adventitial lymphatics than in the arterial lumen. The entry of LDL is dependent on pressure; there appears to be a strong influence of increased stretch of the wall (24). Interestingly, there may be a limit on the upper size of molecules that are able to penetrate the entire thickness of the wall. Labelled albumin, introduced into the lumen of perfused arteries, can later be detected on the adventitial side (25), but there has not been a similar demonstration for particles of the size of LDL.

The endothelial cell layer appears to be a major barrier to transport. Evidence for this includes increased transport in the vicinity of individual endothelial cells undergoing mitosis (26). Unfortunately, the dominant route for LDL transport across the endothelium has not been established. Solute transport occurs through the endothelial cell (transcellular) and through the space between cells (paracellular). Paracellular transport is regulated by the formation between cells of tight junctions, which consist of a series of molecules: junctional adhesion molecule (JAM 1 and 2), platelet endothelial adhesion molecule (PECAM 1), vascular endothelial (VE)-cadherin, occludin, and claudins. They are tightly controlled and their relative expression varies between the different barriers in our body (endothelial, epithelial, and blood–brain barriers). For instance, E-cadherin’s are expressed in epithelial cells, while VE-cadherin is expressed predominantly in endothelial cells. Stable adherens’ junctions are thought to be required for the formation of tight junction and wall shear stress plays an important role in their formation. Endothelial cells under shear stress reorient their cytoskeleton and form ‘stress fibres’: thick-cabled cytoskeletal fibres oriented in the direction of force. Stress fibres have been shown to be coupled to adherens’ junctions and through that mechanism stabilize tight junctions (27, 28).

A more molecular explanation of how shear stress affects tight junctions has also been developed. The phosphoinositide-3 kinase (PI3-K)-protein kinase B/Akt pathway is known to be under the control of shear sensitive G-PCR and RTK-receptors in endothelial cells (2931). After their activation, PI3Ks activate phosphatase and tensin homologue (PTEN), leading to Akt phosphorylation. In the presence of β‎-catenin, VE-cadherin induces forkhead box factor 1 (FoxO1), allowing it to activate expression of the junctional protein claudin-5. Interestingly, the PI3K–Akt pathway is regulated by at least two mechanosensors, each activated by different shear stress patterns. The mechanosensitive RTK receptor is predominantly activated by disturbed shear stress, while the G-PCR is activated by high shear stress (2931). As a consequence, high shear stress leads to stabilization of tight junctions, while low and disturbed shear stress leads to apoptosis, cell turnover, and remodelling of junctions and lipid uptake.

Shear stress also activates Wnt signalling, translocation of β‎-catenin into the nucleus of endothelial cells, and expression of the junctional protein claudin-3 (3234). In addition, Troy and Dr6, which are downstream targets of the Wnt signalling cascade, may be involved in regulating expression of Zonula Occludin (ZO-1) in endothelial cells. Other factors such as the alternative Frizzled-4 ligand Norrin, which is a co-activator of the canonical Wnt/β‎-catenin signalling, also contribute to the induction of junctional molecules such as claudin-5 (3234)

The transport of water across endothelium is about 100-fold faster than the transport of LDL. This will lead to concentration polarization—LDL will have a higher concentration at the endothelial surface than in the blood as a whole. Convective transport of LDL towards the endothelium, and the relatively low permeability of the endothelium to LDL, lead to a build-up of the solute at the surface; this increased concentration leads to diffusion of LDL away from the wall, until an equilibrium is reached. The LDL concentration near the wall at this equilibrium is a matter of debate. Some simulations have suggested increases as large as 10–20%, and have also shown that the degree of elevation depends on local blood flow characteristics, perhaps accounting for local variation of LDL transport into the wall (35). However, studies that take into account the fact that water enters the wall predominantly between endothelial cells, rather than uniformly across them, have contradicted this finding when using physiologically realistic parameters (36). Furthermore, although the glycocalyx layer that coats the endothelium will restrict diffusion, leading to increased concentration polarization, it also shields the polarized layer from the flow, making it less likely that this mechanism can account for local variations in transport (36).

Clinical evidence for transport theories in atherosclerosis

Some research groups have studied LDL transport into the vessel wall as an underlying cause of atherosclerosis through computational studies. It is hard to decide on the correct parameters in these simulations. Often a fluid-wall, single-layered model incorporating shear-dependent transport parameters like hydraulic conductivity and permeability for macromolecules is used. Further assumptions relate to the flow across the arterial wall, for which Darcy’s law is employed. The mass balance of LDL can be governed by the convection-diffusion mechanism. Reaction of the LDL with particles in the vessel wall should be considered.

Simulations performed in 3D reconstructions of human coronary arteries showed co-localization between macromolecule accumulation and low shear stress regions (37, 38). Although LDL concentrations are correlated to low shear stress, regions of high luminal surface concentration do not always co-locate with the sites of lowest WSS (38). The degree of elevation of luminal surface LDL concentration is affected by water flux into the vessel wall. Pulsatility of flow leads to mixing of LDL with the blood and thus has also a great influence on the LDL transport (37).

Application of these models also explains the higher prevalence of plaque with hypertension. Hypertension is associated with higher transmural pressure, and leads to global elevation of LDL concentration in the arterial wall by facilitating the passage of LDL through wall layers (37).

Shear stress theories

History of shear stress theories

Anitschkow (7) suggested that mechanical forces might explain the development of fatty streaks downstream of aortic branch points in cholesterol-fed rabbits, but it was left to Fry (39) to propose a haemodynamic mechanism—he suggested that disease occurs in regions of high WSS. The elevated stress was thought to damage endothelial cells, accounting for the higher permeability seen in these regions. This intuitively satisfying theory was contradicted by the discovery that in the human aorta, regions downstream of branch points are spared disease. Caro et al. (17) proposed that high WSS is protective and that disease occurs instead in regions of low WSS. A subsequent study by Ku et al. (40) compared the distribution of wall thickness in the human carotid bifurcation with WSS measurements in transparent models of the bifurcation and found that the wall was thickest in areas where blood flow changed direction to the greatest extent during the cardiac cycle. A new index, the Oscillatory Shear Index (OSI) was developed to capture this pattern of stresses (40). Because high OSI is associated with low average WSS, the latter appearing in the denominator of the former, the theories of Caro et al. and of Ku et al. have become combined to some extent.

In addition to high, low, and oscillatory WSS, a large number of other WSS metrics have been proposed, including transverse WSS (41), the WSS spatial gradient (42), the WSS angle gradient (43), the WSS angle deviation (44), and the peak WSS temporal gradient. Related suggestions include the dominant harmonic (45), the relative residence time (46), and the Low Shear Index (47). Correlations exist between many of these metrics (48, 49), implying that they capture different features of essentially the same type of flow; the phrase ‘disturbed flow’ has been widely employed to describe it, but the term is imprecise and so cannot be related to any specific biomechanical mechanism.

Mechanisms underlying shear stress theories

The effect of shear stress on endothelial cell genotype and phenotype has been studied extensively and is the subject of a variety of reviews (2, 5052). On the basis of a recent genome-wide study, we have identified 24 pathways, which for clarity are organized in three categories: inflammation, oxygen-radical formation, and apoptosis. (Mechanisms underlying lipid uptake and fluid flux into the vessel wall have already been discussed in transport theories.)

Shear stress and inflammation

A few pathways are specifically upregulated in low and disturbed shear stress (NFkB, Akt, and MAPK signalling pathways). Some signalling pathways that are associated with atheroprotection exert a lesser effect. The most important is the G-PCR-MAPK5 pathway, which has been shown to play a crucial role in controlling the KLF2 and KLF4 transcription factors via MEF2c. Both transcription factors play an essential role in anti-atherosclerotic gene expression, and are increased under high shear stresses (5355).

The nuclear factor kappa B (NFkB) pathway is known to be affected by shear stress: it is upregulated under both low, and low, disturbed shear stress and down regulated at high shear stress (Biomechanical theories of atherosclerosis Fig. 12.2) (5659). The pathway is activated through the tyrosine kinase (TRK)–IP3K–Akt pathway and through PECAM1 (6062). The IKK–NFkB pathway is known to be sensitive to spatial and temporal gradients of shear stress (5659). While PECAM1 has been shown to be involved in the reaction to pulsatile shear stress, it is currently unknown how spatial gradients are detected. One possibility is that the RTK-receptor could form heterodimers (Tie1-Tie2) that activate NFkB under spatial shear gradients (6264).

Fig. 12.2 Three major signalling pathways involved in the regulation of inflammation by shear stress. On the left side is the IKK–IkB–NFkB pathway, which is regulated by low shear stress and pulsatile shear stress. After activation by PECAM1, a complex is formed of VEGFR2 and VE-cadherin, which activates the kinase (IKK). IKK ubiquinates IkB in the NFkB–IkB complex, thereby releasing NFkB in the nucleus. NFkB encodes for ∼200 inflammatory genes, including IkBα‎, thereby initiating a feedback loop. In the middle section is the IP3–Akt pathway. The phosphoinositide-3 kinase (PI3-K)-protein kinase B/Akt pathway is known to be under the control of shear sensitive G-PCR and RTK-receptors in endothelial cells. After their activation, PI3Ks activate PTEN, leading to Akt phosphorylation. Akt inhibits FOXO1, activates NFkB, eNOS, NRF2, and cellular metabolism. Another important, shear-controlled proinflammatory pathway is depicted on the right side: the mitogen-activated protein kinases or MAPK-pathway. The MAPK-pathway is activated by RTKs and G-PCR and after Ras-Raf activates a three-tier phosphorylation cascade, where each kinase needs to be double phosphorylated. Phosphatases dephosphorylate the cascade and, as they are produced by the transcription factors regulated by these kinases, one or more negative-feedback loops exists in these pathways

Fig. 12.2
Three major signalling pathways involved in the regulation of inflammation by shear stress. On the left side is the IKK–IkB–NFkB pathway, which is regulated by low shear stress and pulsatile shear stress. After activation by PECAM1, a complex is formed of VEGFR2 and VE-cadherin, which activates the kinase (IKK). IKK ubiquinates IkB in the NFkB–IkB complex, thereby releasing NFkB in the nucleus. NFkB encodes for ∼200 inflammatory genes, including IkBα‎, thereby initiating a feedback loop. In the middle section is the IP3–Akt pathway. The phosphoinositide-3 kinase (PI3-K)-protein kinase B/Akt pathway is known to be under the control of shear sensitive G-PCR and RTK-receptors in endothelial cells. After their activation, PI3Ks activate PTEN, leading to Akt phosphorylation. Akt inhibits FOXO1, activates NFkB, eNOS, NRF2, and cellular metabolism. Another important, shear-controlled proinflammatory pathway is depicted on the right side: the mitogen-activated protein kinases or MAPK-pathway. The MAPK-pathway is activated by RTKs and G-PCR and after Ras-Raf activates a three-tier phosphorylation cascade, where each kinase needs to be double phosphorylated. Phosphatases dephosphorylate the cascade and, as they are produced by the transcription factors regulated by these kinases, one or more negative-feedback loops exists in these pathways

NFkB is under the control of IKK and amongst its target genes are IkBα‎ and Cézanne, which inhibit the formation of NFkB and IKK, respectively (6568). Negative-feedback loops make the pathway less sensitive to external influences, but might lead to oscillations (6568). Indeed, oscillations have been reported both after ligand and shear stress stimulation, and the oscillation frequency seems to be regulated by the level of shear stress. As the frequency of oscillations affects target gene expression, this pathway is a truly frequency modulated signalling pathway. The target genes of the NFkB pathway regulate cytokines/chemokines, cell adhesion factors, acute phase proteins, and cell surface receptors. Consequently, the pathway plays a central role in attracting and capturing inflammatory cells, but also in their differentiation in the subendothelial space.

Other important, shear-controlled proinflammatory pathways include the mitogen-activated protein kinases or MAPK-pathways. The MAPK-pathways consists of a three-tier phosphorylation cascade, where each kinase needs to be double phosphorylated. Phosphatases dephosphorylate the cascade and, as they are produced by the transcription factors regulated by these kinases, one or more negative-feedback loops exists in these pathways (6972). Theoretical studies have confirmed that this cascade can react gradually, in an oscillatory mode or in a switch-like response, depending on the degree of stimulation (6972). The complexity of the dynamics of the MAPK-pathways makes it difficult to interpret single time-point measurements of single molecules. Indeed, increased activation of members of the signalling pathway has been reported both after low and high shear stresses (29, 59, 69, 7378). More specifically, studies have shown that ERK1/2, ERK5, Jun, p38, are upregulated in response to (high) shear stress, while Jun and p38 are known proinflammatory molecules, which become phosphorylated at higher shear stress values. Other studies have indicated that low shear stress and/or prolonged shear stress activate the MAPK pathways (75, 79, 80). These apparently contradictory findings may be reconciled by the dynamic behaviour of the MAPK pathway to shear stress. Another explanation for the discrepancy may reside in the fact that the MAPK pathways regulate proinflammatory (JNK) and anti-inflammatory (KLF2-KLF4) transcription factors. It is known that proinflammatory transcription is regulated by low shear stress, while anti-atherogenic pathways are regulated by high shear stress.

A few pathways were recently identified, after combining microarray studies from several sources, thereby increasing the power of the statistics. The NOD-like pathways were involved, regulating the inflammasome and chemokines like CXCL1, MIP3, and CCL2 (69). Interestingly, Toll-like receptors were shown to react to shear stress and through their activation (CD14) stimulated AKT-dependent NFkB activation and production of IP-10 and IFN-α‎ (69).

Shear stress and oxygen radicals

Endothelial cells possess an intricate mechanism to control formation of reactive oxygen species (ROS). ROS is a collective term that refers to oxygen radicals such as superoxide, O2, and the hydroxyl radical, OH, and to non-radical derivatives of O2, including hydrogen peroxide (H2O2) and ozone (O3). ROS are determined by the activity of a variety of sources, including NADPH-oxidases, xanthine oxidase, mitochondria, and uncoupled eNOS (8184), but also by inhibition of anti-oxidants such as superoxide dismutase, catalase, glutathione peroxidase, thioredoxin, peroxiredoxins, and heam oxygenase-1. Note that reactive nitrogen species (RNS), such as nitric oxide (NO), nitrogen dioxide (NO2-), peroxynitrite (OONO), dinitrogen trioxide (N2O3), nitrous acid (HNO2), etc., also play a complex role in endothelial disorders.

Oxygen radical production in endothelial cells seem to follow a model of balance, where low, disturbed shear stress is associated with increased ROS-production and high shear stress with decreased ROS production. The precise mechanisms are depicted in Biomechanical theories of atherosclerosis Fig. 12.3.

Fig. 12.3 Shear stress also regulates oxygen radical production by controlling all aspects of their production (NADPH-oxidases), binding (eNOS and NO), and regulation (NRF2).

Fig. 12.3
Shear stress also regulates oxygen radical production by controlling all aspects of their production (NADPH-oxidases), binding (eNOS and NO), and regulation (NRF2).

In resting and dividing endothelial cells, ROS are mainly produced by mitochondria, but as shear stress regulates NADPH-oxidases and anti-oxidant system expression and activation, the ‘redox state’ is drastically changed after exposure to shear stress. During sustained high shear stress, the NADPH-oxidases, NOX-1, NOX-2, and NOX-4, are downregulated, and consequently ROS production is downregulated (8184). In addition, after sustained high shear stress, KLF-2 and KLF-4 transcription factors are upregulated, thereby increasing eNOS expression (8587). In parallel, the IP3K–Akt pathway is activated by shear stress, which enhances phosphorylation of eNOS. As a consequence, NO production—a ROS scavenger—is increased and peroxynitrite is formed. Peroxynitrite is thought to exert physiological functions in low concentrations and pro-atherogenic functions at high concentrations (Biomechanical theories of atherosclerosis Fig. 12.3) (87).

The transcription factor nuclear factor erythroid 2-related factor 2 (NRF-2) is the most important regulator of the expression of molecules that have anti-oxidant functions within the cell (8890). Under resting conditions, NRF-2 is constitutively bound by the Kelch-like ECH-associated protein 1 (KEAP1)–Cullin 3 (CUL3) E3 ligase complex. NRF-2 is the sole controller of the enzymes that are responsible for producing glutathione (GSH), which is the most abundant anti-oxidant cofactor within the cell, but it also upregulates other anti-oxidant regulators, like TXN production, quinone detoxification, iron sequestration, and GSH production. Sustained high shear stress upregulates NRF-2, thereby increasing anti-oxidant genes and proteins, and thus reducing ROS. As sustained high shear stress also reduces ROS production this combined effect is a strong stimulus for controlling oxygen radical concentrations (Biomechanical theories of atherosclerosis Fig. 12.3) (8890).

Sustained disturbed flow reduces KLF-2 and KLF-4 levels, and thereby eNOS expression and NO production. On the other hand, it increases NOX-1 and NOX-2, and enhances ROS production. As NRF-2 is reduced in regions of disturbed shear stress, its regulated genes (HO-1, thioredoxin reductase-1 (TrxR1)) are reduced, thereby reducing ROS scavenging. Sustained high ROS concentration in cells will enhance expression of adhesion molecules (VCAM-1 and ICAM-1) through activation of NFkB and the MAPK pathways and it will enhance the oxidation of LDL in the subendothelium, thereby creating a milieu where macrophage phenotype is directed towards M1 and foam cell formation. At high concentrations, ROS will induce sustained DNA damage and endothelial cell apoptosis, and promotes leakage of lipids into the vessel wall.

Shear stress, apoptosis, and cell turnover

Several pathways determine endothelial cell turnover. The PI3K–Akt pathway plays an essential role in this process, especially when stimulated through Tie1-Tie2 (62, 9194). Newly identified pathways are the JAK–STAT and the WNT pathways, which may play an indirect/direct role in endothelial cell turnover. High ROS production also facilitates endothelial cell apoptosis. It has been suggested that these pathways regulate death-associated protein (DAPK) as a final protein (95, 96). We found evidence for shear stress regulation of the TGF-β‎ signalling, which also plays a role in apoptosis (69). Another interesting finding was the activation of the JAK–STAT pathway by shear stress (69). As these conditions occur at sites of flow disturbance induced at or near side branches, it has been postulated that local endothelial cell apoptosis may enhance LDL uptake and thereby initiate and sustain atherosclerosis at these sites (9597).

Clinical evidence of shear stress theories

De novo early plaque and plaque progression

Early observations on the relationship between shear stress and atherosclerotic plaque localization were based on autopsy material (17, 98100), and consequently no information on the influence of shear stress on plaque progression over time was available. Application of computational fluid dynamics in 3D reconstructions of coronary arteries of humans and laboratory animals have tended to confirm the earlier observed co-localization between low shear stress regions and de novo atherosclerotic plaque (101103), although a recent review has challenged this concensus (104). Plaque progression was also shown to be related to low or oscillatory shear stress and this relationship was modulated by the level of hypocholesteraemia (100, 105, 106). Interestingly, a combination of shear stress, plaque phenotype, and plaque burden was shown to have a better predictive value for plaque progression than each of these parameters alone (107, 108). In a very large clinical trial (500+) of the Japanese population, lumen narrowing distal to the stenosis and plaque burden, but not plaque growth, were related to low shear stress (<1Pa). The combination of plaque burden (>58%) and low shear stress increased the positive predictive value from 25% to 41% (109, 110).

Plaque composition

Shear stress is not only involved in plaque initiation and progression, but also in modulating the composition of the atherosclerotic plaques. Early studies using histopathology for plaque characterization showed that regions presumed to experience low shear stress were associated with advanced plaques features like inflammation, a large lipid-rich necrotic core, a thin fibrous cap, and positive outward remodelling (111, 112), while presumed high shear stress regions were associated with a more stable plaque phenotype. Later studies confirmed this observation by VH-IVUS (113) or OCT (114) in combination with biplane angiography. Also, an increase in plaque vulnerability was observed at the low shear stress regions, reflected by an increased size of the necrotic core.

The causative role of low shear stress in vulnerable plaque formation was elegantly shown in several animal studies of plaque development in pre-defined low shear stress regions (115, 116). For that reason a tapered cuff was developed that creates a well-defined haemodynamic environment: low shear stress upstream from the cast and low oscillating shear stress downstream of the cast. Plaques with characteristics of plaque vulnerability were observed particularly in the low shear stress region (115117).

Vascular remodelling

Vascular remodelling occurs during development of arteries and organs, and is beneficial in maintaining sufficient blood flow to the organs during growth. Early studies showed a convincing relationship between shear stress and vessel growth. More recent studies have revealed the role of shear stress during plaque remodelling (118). During plaque development arteries can undergo compensatory expansive remodelling, presumably to maintain the local shear stress at a constant level (119121). In general, outward remodelling will lead to a persistence of shear stress in straight vessel segments, and to low shear stress in the inner curvature of curved vessel segments. The persistence of a low shear stress region may be associated with a continuous uptake of lipids and inflammatory cells, which may contribute to further development of vulnerable plaques. These postulates predict that plaques with a large necrotic core are found at low shear stress locations, which was recently confirmed (111, 113, 122124). Plaques may also overcompensate for plaque growth, the so called ‘excessive outward remodelling’ (111), leading to a lumen radius increase. This type of remodelling is observed in a small percentage of arteries (125) and might lead to a persistence of low shear stress or may even lead to a viscous circle in which low shear stress leads to even lower shear stress (126). The opposite reaction of the vessel wall has also been observed during atherosclerotic plaque formation. The precise mechanisms of vascular shrinkage are unknown, but scar formation has been proposed. Unfortunately, information regarding the relationship between vascular shrinkage and shear stress is currently lacking.

In the later phases of the disease, positive remodelling seems not to be sufficiently effective to compensate for plaque growth, resulting in narrowing of the vessel lumen. In general, lumen narrowing initiates when plaque burden exceeds 40% (120). Although the precise mechanism limiting outward remodelling is unknown, intraplaque bleeding (127, 128), multiple plaque ruptures (129), and a circumferential extension of endothelial dysfunction at the side of the plaque have been put forward as possible explanations (120, 130). Once atherosclerotic plaques encroach into the lumen, ECs experience a change in local shear stress, i.e. high shear stress at the upstream part and low/oscillatory shear stress at the downstream side of the plaque, where initially low shear stress was present (110). It is, however, unclear whether ECs covering the advanced atherosclerotic lesion remain responsive to changes in local shear stress, as the shear stress-dependent transcription factor KLF2 seems downregulated, cross-talk between ECs via connexins is diminished, and eNOS expression is decreased at plaques (132, 133). In contrast, ECs overlapping stented arteries retain their ability to respond to flow (134, 135).

In more advanced stages of the disease the necrotic core can be found at high shear stress locations (124). The fact that plaque ruptures/ulcerations are often observed at the upstream side of advanced plaques has strengthened the idea that high shear stress is involved in upstream plaque destabilization (112, 136138). Plaque composition at the upstream side of the plaque is markedly different from the downstream side. The region upstream of the plaques is often associated with more macrophage accumulation and apoptosis, lipid accumulation, intraplaque haemorrhage, and thinner fibrous caps (69, 112, 137). Interestingly, upstream plaque regions exposed to high shear stress show an increased strain—a local measure for plaque weakness—implying that those regions are more vulnerable to rupture (139). A recent study in human coronary arteries confirmed an increased vulnerability of the plaque regions after exposure to high, but also to low, shear stress (113). However, more detailed analysis on the association between shear stress and plaque rupture indicated a linear relationship between shear stress and plaque rupture (140). As studies have reported low and high shear stress as predictors for plaque vulnerability, further studies are needed to investigate the potential causative role of shear stress in plaque destabilization.

Strain theories

History of strain theories

Strain is induced when a stress is applied to a mechanical body (see section “Biomechanical definitions” for details). In a simplified geometry, such as a cylindrical artery, circumferential strain is in equilibrium with blood pressure. However, in realistic geometries, strain would also vary as a result of a non-uniform curvature (bends, side branches, bifurcations, and taper), changes in wall composition (such as the switch from a more elastic to a more muscular architecture on proceeding peripherally), and through the influence of supporting structures—the passage of vertebral arteries through the foramen transversarium of cervical vertebrae is an extreme example of the latter but, more commonly, actions of muscles and effects of tethering are also likely to be important.

Strain is much less discussed than WSS in relation to arterial disease. Drawing on classical pressure vessel stress analysis, Thubrikar has proposed several examples of sites where atherosclerotic lesions could be associated with locally elevated stress and strains (141, 142). Although pressure vessel theory is strictly only applicable to stiff, isotropic and linearly elastic materials, it is possible to gain qualitative insight into regional strains; arteries display nonlinear stress-strain responses, but within the physiological pressure range a linear approximation of the curve is reasonable (143).

The vertebral arteries exhibit a periodic distribution of atherosclerosis: lesions occur in the segments that are free to expand, but are absent where the artery passes through the bone canal (144, 145). Similarly, the internal carotid artery is less prone to atherosclerosis where it passes through the carotid canal at the base of the skull (146). At myocardial bridges, where an epicardial coronary artery passes under a band of heart muscle and then re-emerges, the tunnelled segment is generally free of atherosclerosis. And lesions abruptly cease where the coronaries enter the myocardium, even when the proximal segments are severely affected (147).

Experimental studies have provided additional evidence for the importance of intramural stress and strain. Thubrikar et al. (148) showed that lesion formation can be prevented in cholesterol-fed rabbits by restricting vessel expansion. Under anaesthetically induced hypotension, a liquid dental acrylic was poured around the junction between the left renal artery and aorta, and, upon setting, created a rigid perivascular cast; mean arterial pressure recovered post-surgery (148). The branch was spared of fatty streaks, whereas control ostia became diseased. Lesions did develop when casting was performed at normal blood pressure. Tropea et al. (149) induced hypertension and hypercholesterolaemia in rabbits by aortic banding and a cholesterol-enhanced diet, respectively. Wall motion and intimal plaques were reduced proximal to the banding in regions of the aorta that had been loosely or firmly encircled with an external wrapping.

A problem with both the observational and interventional studies is that reducing wall motion will affect WSS, despite claims to the contrary. If cyclic strain is restricted, then luminal diameter averaged over the cardiac cycle will be reduced, so long as wall thickness is not drastically decreased. Even if wall thickness diminished sufficiently to maintain the normal average WSS, variation in WSS during the cardiac cycle would increase in the constant-diameter segment. For a given volumetric flow rate, WSS depends on diameter raised to the third power, so even small differences could have significant influences on shear stress. Similar issues may apply to data derived from patients with chronic aortic coarctation—blood pressure and lesion prevalence are increased proximal to the occlusion and decreased distal to it (150)—and to results from the numerous experimental studies that have confirmed this finding (151): the same volumetric flow rate must occur in both segments but pulse pressure differs and wall remodelling is likely to occur, so WSS may not be the same upstream and downstream of the restriction. The same criticism can also be applied to mechanistic studies in which cyclic strain is applied to endothelial cells by culturing them on an elastic membrane that is periodically stretched. The bulk of the overlying medium will remain stationary when the cells move, so they also experience fluid dynamic shear stresses. It would be necessary to control for these fluid effects before attributing changes in cell behaviour solely to mechanically induced strain. To date, effects of mechanical strain and fluid shear stresses may not have been separated sufficiently well for lesion development and pro-atherogenic mechanisms to be unambiguously attributed to the former.

Mechanism of strain theories

Far less is known about the effect of strain/stretch on signalling pathways than about the effect of haemodynamic wall shear stress. It is known that some of the mechanosensors—G-PCR, stretch-activated calcium channels, integrins, and PECAM-1—are sensitive to stretch (152155). And it is known which signalling pathways are regulated by these sensors: MAPK, NFkB, the cytoskeleton, and cAMP, PI3K–Akt pathways. Indeed, several studies confirmed that members of these pathways react to stretch (156158). This has led to the finding that endothelial cells after stretch increase COX-2 levels, activate eNOS, and secrete ET-1, IL-6, and MMP (156158). Thus endothelial cells are ‘primed’ under physiological strain, as they are under shear stress.

Pathological strain, defined as either too high (>15%) or too low (< = 5%), is associated with an increase in NFkB signalling, reduced NO production, but increased ROS production and increased MAPK activity (Biomechanical theories of atherosclerosis Fig. 12.4). These findings suggest that pathological strain could aggravate the atherogenic state induced by low shear stress (156158). However, regions of pathological strain do not always coincide with regions of low shear stress and it is necessary to discriminate between side branches and curved segments.

Fig. 12.4 Endothelial strain (for explanation see text), activates three important pathways: ROS, NFkB, and the MAPK. And while far less is known than for shear stress regulation, both high strain and low strain have been shown to regulate pro-atherogeneic signalling pathways. As high strain is present at predilection sites, emphasis is on the regulation of high strain signalling pathways. The three signalling pathways depicted here are similar to those identified for low shear stress and it seems if high strain regions coincide with low shear stress regions, the pro-atherogenic effects could be reinforced.

Fig. 12.4
Endothelial strain (for explanation see text), activates three important pathways: ROS, NFkB, and the MAPK. And while far less is known than for shear stress regulation, both high strain and low strain have been shown to regulate pro-atherogeneic signalling pathways. As high strain is present at predilection sites, emphasis is on the regulation of high strain signalling pathways. The three signalling pathways depicted here are similar to those identified for low shear stress and it seems if high strain regions coincide with low shear stress regions, the pro-atherogenic effects could be reinforced.

Near bifurcations and side branches, there are regions where the near-wall blood velocity is low, and strongly changing in direction and strain is pathologically increased (159161). Strain is also increased above a large necrotic core, but here the shear stress is high. At or near side branches, the PI3–Akt pathway is activated through mechanosensitive RTK receptors (Tie1 and Tie2), and the resulting increased Akt kinase activity leads to formation of NFkB and, in combination with increased ER-stress, to NRF-2 activation, and HO-1 production, and ROS inhibition, which forms a negative-feedback loop to control ER-stress. NFkB is further activated by spatial and temporal shear stress gradients often occurring at or near bifurcations, which have a large angle with their mother vessel (T junctions). Here inhibition of Foxo1 is low (162, 163), and endothelial cells are driven to increased turnover, cobble stone formation, reduced tight and adherens’ junctions, and increased permeability and lipid uptake into the vessel wall. During those conditions, Akt is also activated through VEGFR2, leading to a low-level chronic inflammation. This further favours lipid uptake and due to the persistence of vortices, the expression of adhesion factors, it also enhances inflammatory cell uptake into the vessel wall.

In the inner curvature of moderately curved vessel segments like the coronary arteries, sustained low, helical velocity patterns, and low shear stress is present; the shear stress vector varies minimally in direction, while strain is increased pathologically (Biomechanical theories of atherosclerosis Fig. 12.5) (164). During these conditions, the dominant effects are those of a reduced anti-atherogenic to atherogenic balance. This imbalance is characterized by reduced KLF-2 and KLF-4 expression, leading to a reduced anti-atherogenic gene repertoire (1, 50), and a reduced NRF-2 expression and increased NOX expression, resulting in sustained ROS production. Furthermore, the reduced transit times and reduced stability of tight junctions leads to an increased lipid uptake, which will oxidize due to the increased ROS production. The increased ROS production in curved segments also activates NFkB, in contrast to the temporal and spatial gradients near side branches, leading to an increased adhesion factor expression (ICAM-1 and VCAM-1) and uptake of inflammatory cells. The more central role of ROS in curved vascular segments over side branches has recently been confirmed in genome-wide profiling studies (165).

Fig. 12.5 Imaging of a coronary artery of a patient by a combination of non-invasive Multislice CT (MSCT) and NIR-IVUS. The NIR-IVUS provides the vascular composition while the lumen reconstruction based on X-rays permits computational fluid dynamics simulations and the prediction of wall shear stress. This novel approach allows decipherment of the role of haemodynamics in lipid accumulation.

Fig. 12.5
Imaging of a coronary artery of a patient by a combination of non-invasive Multislice CT (MSCT) and NIR-IVUS. The NIR-IVUS provides the vascular composition while the lumen reconstruction based on X-rays permits computational fluid dynamics simulations and the prediction of wall shear stress. This novel approach allows decipherment of the role of haemodynamics in lipid accumulation.

(Reproduced from Wentzel, J., Van der Giessen, A., Garg, S, et al; In Vivo 3D distribution of lipid-core plaque in human coronary artery as assessed by fusion near infrared spectroscopy-intravascular ultrasound and multislice computed tomography scan, Circulation: Cardiovascular Imaging; Vol.3, No.6, (2010) with permission from Wolters Kluwer.)

Finally, we note that more direct molecular effects may be involved. An interesting concept championed by Stephens is that repeated cyclic strain vibration of the vessel wall leads to fatigue failure of fibrous proteins and hence to lesions (166).

Clinical evidence of strain theories

There has been less clinical interest in the evaluation of mechanical theories than in haemodynamic theories of atherosclerotic plaque formation. However, the recent developments in fluid-structure modelling have revitalized this interest and new clinical studies, including strain, have now been conducted. The majority of these studies have focused on studying the role of wall stress in plaque rupture.

Plaques ruptures if the local wall stress (i.e. stress within an atherosclerotic lesion) exceeds the strength of the fibrous cap (167). Stresses in the arterial wall are influenced by a variety of factors, including the blood pressure, local geometry, and the plaque composition (168). The wall stress is 10,000 times higher than the blood flow induced shear stress at the endothelium and, thus, it is hypothesized that shear stress modulates the plaque composition and that wall stress is responsible for the final plaque rupture (134). Interestingly, peak cap stresses in symptomatic patients are higher than those in asymptomatic patients, suggesting that plaques with higher stresses may be more prone to rupture, thus leading to cardiovascular events. Accordingly, if plaques are sorted according to plaque phenotype, plaques classified as thin cap fibroatheroma showed higher peak cap stress than plaques with pathological intimal thickening (169, 170).

Biomechanical stress metrics could, therefore, potentially be used to assess the risk of plaque rupture. However, the threshold value of wall stress that needs to be applied for risk prediction is currently under debate (171), since the cap strength might vary, depending on its collagen content. The highest wall stress is typically found (a) at the thinnest parts of the fibrous cap (172, 173), (b) in regions with increased macrophage density, (c) in regions with intraplaque haemorrhage (174), and (d) in the presence of local microcalcifications (175).

Selected reading

Anitschkow N: Experimental atherosclerosis in animals; in Cowdry EV (ed): Arteriosclerosis. New York M, 1933, pp 271–322. Anitschkow N: Experimental atherosclerosis in animals.Find this resource:

Caro CG, Fitz-Gerald JM, Schroter RC. Atheroma: a new hypothesis. Br Med J 1971; 2(5762): 651.Find this resource:

Ku DN, Glagov S, Moore JE, Jr, et al. Flow patterns in the abdominal aorta under simulated postprandial and exercise conditions: an experimental study. J Vasc Surg 1989; 9(2): 309–16.Find this resource:

Mohri Z, Rowland EM, Clarke LA, et al. Elevated uptake of plasma macromolecules by regions of arterial wall predisposed to plaque instability in a mouse model. PLoS One 2014;9(12): e115728.Find this resource:

Pedrigi RM, de Silva R, Bovens SM, et al. Thin-cap fibroatheroma rupture is associated with a fine interplay of shear and wall stress. Arterioscler Thromb Vasc Biol 2014; 34(10): 2224–31.Find this resource:

Peiffer V, Sherwin SJ, Weinberg PD. Does low and oscillatory wall shear stress correlate spatially with early atherosclerosis? A systematic review. Cardiovasc Res 2013; 99(2): 242–50.Find this resource:

Slager CJ, Wentzel JJ, Oomen JA, et al. True reconstruction of vessel geometry from combined X-ray angiographic and intracoronary ultrasound data. Semin Interv Cardiol 1997; 2(1): 43–47Find this resource:

Wada S, Karino T. Theoretical study on flow-dependent concentration polarization of low density lipoproteins at the luminal surface of a straight artery. Biorheology 1999; 36(3): 207–23.Find this resource:

Weinberg PD. Rate-limiting steps in the development of atherosclerosis: the response-to-influx theory. J Vasc Res 2004; 41(1): 1–17.Find this resource:

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