Hemodynamic forces and artery architecture
The endothelium monolayer being the dividing factor between the vascular wall and blood flow is subjected to fluid forces of greater magnitude than any other tissue (Davies, 1986). The magnitude can be estimated by Poiseuille’s law, which states “that shear stress is proportional to blood flow viscosity and inversely proportional to the third power of the internal radius” (Malek et al., 1999). Pulsatile blood flow and pressure produce hemodynamic forces such as cyclic stretch, hydrostatic pressure and fluid shear stress on the EC (Tseng et al., 1995). Hemodynamic parameters such as disturbed and oscillatory flow and low shear stress have been implicated in the initiation and development of cardiovascular diseases (Anssari-Benam et al., 2013). Hemodynamic forces impact regions of curvature such as coronary arteries and bifurcations such as the carotid artery (Back et al., 2013).
“Endothelial shear stress (ESS) is a biomechanical force on the endothelial surface that is determined by blood flow, vessel geometry and fluid viscosity that is computationally estimated using fluid dynamics models and is expressed in units of dynes/cm” (Cunningham and Gotlieb., 2005). “It is a product of shear rate and blood viscosity” (Chatzizisis et al., 2007). Shear rate is the change in blood flow from the arterial wall to the centre of the lumen. Blood viscosity is the ability of blood to flow; it measures the internal friction that causes blood to resist flow.
In an equation, shear stress is expressed as τ = 4μQ/πr2 where m is the viscosity, Q is the flow rate and r is the vessel radius. The Reynolds number (Re) measures the stability of flow: Re = 4ρQ/πμD (g/cm2) where ρ is blood density, D is vessel diameter, μ is blood viscosity and Q is flow rate. “This represents a ratio of inertial fluid momentum to viscous frictional forces” (Cunningham and Gotlieb., 2005).
Shear stress of ≥15 dyne/cm2 induces endothelial quiescence and a gene expression profile which is atheroprotective, shear stress of ≤4 dyne/ cm2, stimulates an atherogenic phenotype and is therefore present at sites of atherosclerosis (Malek et al., 1999). Shear stress differs between arteries and veins. In the venous system, shear stress ranges from 1-6 dyne/cm2 while the arterial network experiences a greater and higher range 10-70 dyne/cm2. However, this range is subject to arterial architecture as a shear stress as low as 0.5 dyne/cm2 can occur in areas of curvature and bifurcation (Malek et al., 1999).
Shear gradients are divided into two categories- Temporal gradients are the change in shear stress that occurs in the same location over a short period of time. A spatial gradient is the difference in shear stress between two points of a cell at the same time. White et al 2001 developed an in vitro model that separated these gradients which previously hadn’t been achieved. The findings suggested temporal gradients acted as stimulants for EC proliferation and therefore considered significant in atherosclerotic-susceptible regions. Spatial gradients had no greater influence on EC proliferation than steady uniform shear stress (White et al., 2001). Significant stresses are created in the vasculature by the mechanical environment.
Types of shear stress and flow
The type of flow depends on the region of the vasculature; the carotid bulb varies in flow types. High shear occurs in the medial wall of the bulb whilst oscillatory stress occurs on the lateral wall.
It has been suggested that undisturbed laminar flow is atheroprotective and stimulates cellular responses which are vital for endothelial cell function (Traub and Berk, 1998) and a healthy vascular system. Undisturbed laminar flow promotes an anti-inflammatory, antithrombotic, anticoagulative, profibrinolytic and antihypertrophic state (Cunningham and Gotlieb, 2004). This type of blood flow pattern is mainly seen in linear areas of the vasculature and has a high shear stress and rate of average 12 dyne/cm2 (Yoshizumi et al., 2003).
Laminar flow transforms into disrupted flow, which is classified as a decrease in shear stress with forward and retrograde flow specifically at regions of curvature such as the carotid artery. Shear stress rate is typically <4 dynes/cm2. Areas of disturbed flow are associated with temporal and spatial shear gradients and experience recirculation, flow separation and reattachment (Estrada et al., 2011). This alters the homeostatic environment of vascular cells and down-regulates eNOS, endothelial repair and disturbs cellular alignment. Leukocyte adhesion, ROS and lipoprotein permeability are all up-regulated, contributing to the formation of a site of inflammation which starts as a fatty streak, progresses to an atheroma and finally a complex or vulnerable plaque which may rupture (Cunningham and Gotlieb, 2005).
Oscillatory stress is “periodic flow reversal with time-averaged shear stress approaching zero” that varies with the cardiac cycle and causes recirculation vortexes. Due to significantly low shear stress, cells subjected to oscillatory flow do not orientate perpendicular to direction of flow. This flow pattern occurs at the outer wall of the carotid sinus and has a shear stress range of approx -7 to +4 dynes/cm2. Studies have shown correlation between fatty streak and lesion development and oscillatory flow resulting in a pro-atherogenic environment (Ku et al., 1985).
Pulsatile / Pulsatile ESS
Due to PSS the vessel wall undergoes a cyclic stress composed of radial and circumferential factors and the EC experiences a hemodynamic shear stress (Imberti et al., 2002). In vivo ECs are exposed to pulsatile pressure (normal: 120=80 mm Hg) and cyclic stretch (normal: 6–20%) at approx 80 bpm (Estrada et al., 2011). Depending on the area of vasculature shear
stress varies. Pulsatile flow creates a region of flow separation and reversal at the sinus’s outer wall.
Steady shear stress
Steady shear stress is a flow pattern which is temporally and spatially uniform and used in many experimental preparations. This usually induces many of the same EC responses as pulsatile shear stress but has qualitative and quantitative differences. Steady shear stress causes cells to reorientate their axis parallel to the direction of blood flow; this decreases the cells resistance, lowering the shear stress (Traub and Berk, 1998).
Multiple roles of low ESS in atherosclerosis have been suggested. This is why low ESS may be a main causative factor in coronary artery disease. Low ESS promotes LDL permeability, uptake and synthesis, it promotes oxidative stress and the production of ROS which oxidise LDL, and it reduces NO bioavailability which is atheroprotective (Li et al., 2003). Low ESS most importantly induces inflammatory mediator expression and is capable of activating transcription factors such as NF-ĸβ. When activated NF-ĸβ upregulates gene expression of adhesion molecules VCAM- and ICAM-1 and chemotactic factors such as MCP-1 and pro-inflammatory molecules TNF-α, IFN-γ and IL-1 (Chiu et al., 2004).
Low ESS promotes ECM degradation in the vascular wall and fibrous cap which are both made of collagen and elastin fibres. Low ESS upregulates MMP gene expression, in particular MMP2/9, these matrix degrading proteins are released from macrophages, VSMCs and endothelial cells upon activation of transcription factors (TFs) which upregulate pro-inflammatory cytokines TNF-α, IL-1, IFN-γ. Low ESS may play a potential role in plaque calcification and neovascularisation as VEGF is shown to be upregulated (Catzizisis et al., 2007). Sakamoto et al 2006 reported migration of SMCs was decreased by shear stress of 1.5 Pa, however 0.1 Pa shear stress did not suppress migration (Sakamoto et al., 2006).
The most cited factors toward initiation of atherosclerotic plaque in the current literature are abnormalities in shear stress and flow patterns. However, clinical findings suggest that plaque still forms at arterial sites that are associated with steady and high shear stress rates (Anssari-Benam and Korakianitis, 2013). A review of 27 studies on low and oscillatory shear stress revealed in fact that the evidence supporting its part in atherosclerosis was less robust than assumed (Peiffer et al., 2013).
Cells sense their physical environment by translating mechanical forces into biochemical signals which results in a mechanosensitive feedback loop known as mechanotransduction. Mechanotransduction regulates cellular functions including differentiation, proliferation, migration, apoptosis and homeostasis. Disturbing this feedback process alters cell signalling events, such as gene and protein expression, and leads to pathophysiological changes, in this case atherosclerosis (from the editors, Nature 2009).
There are many forms of endothelial responses to fluid shear stress. Mediators of mechanotransduction can be divided into two groups: In cell associated and surface/extra-cellular associated mediators. In cell associated mediators consist of cytoskeleton (Microfilaments, Microtubules, Intermediate filaments) and nuclei (Ion channels, Nuclear lamina, Chromatin, Gene expression). Surface/extra-cellular mechanosensors include membranes (Ion channels, Caveolae, Surface receptors, Lipid bilayer), surface processes (Primary cilium, Stereocillia), cell-cell adhesions (Cadherins, Gap junctions), ECM (Fibronectin, Collagen, Proteoglycans, Basement membrane) and cell-ECM adhesions (Focal adhesions, Integrins) (Davies, 1995 and Takahashi et al., 1997).
Mediators of mechanotransduction activate signalling molecules, nitric oxide, cGMP, G-proteins and G-protein coupled receptors, ROS, inositol triphosphate, protein kinases, calcium influx and transcription factors, early growth factor-1, NF-ĸβ and activator protein-1 within minutes of surface response. After several hours changes can then be identified through cytoskeleton organisation in the direction of flow and cell elongation (Orr et al., 2006). Signalling also results in growth arrest/proliferation, inflammatory/anti-inflammatory gene activation via the shear stress response elements located on the promoter regions (Chien 2007). According to experimental results, laminar flow is capable of mechanoreceptor activation and transduction however cells adapt to the environment and begin to down regulate pathways. Disturbed flow was able to sustain activation of NF-ĸβ and tissue factor promoting inflammation and thrombosis (Orr et al., 2006). Mechanotransduction in ECs has been studied with both in vivo an in vitro approaches. Many in vitro models have been designed to study this process as they have the advantage of controlling experimental variables. However, in vivo studies are important as follow up studies to verify and determine applicability of in vitro results.