**2. Background**

### **2.1 Pathogenesis of atherosclerosis**

### *Vascular anatomy*

Arteries have three tissue layers: the intima, media, and adventitia. The intima is lined with a monolayer of ECs in direct contact with blood. The ECs act as a protective membrane, allowing diffusion from the blood stream into the artery. ECs are capable of expressing specific genes in response to physical stresses which cause the vessel to remodel leading to the development of atherosclerosis. In addition, the intima can contain other cells (smooth muscle cells, fibroblasts and inflammatory cells), an extracellular matrix (ECM), and is only a few cell layers thick in healthy tissue. The internal elastic membrane, consisting of a layer of elastic connective tissues, separates the intima and media. The media layer is mainly comprised of smooth muscle cells (SMCs) and ECM. Although the media is involved in atherosclerosis, remodelling is less evident. The adventitia is relatively unaffected by atherosclerosis. It is separated from the media by the external elastic membrane comprised mainly of collagen, providing structural support yet allowing for artery expansion when required (Waller et al., 1992).

Over time, an atherosclerotic plaque grows by the accumulation of lipids, inflammatory cells, vascular cells and matrix material in the intima. It often produces a fibrous cap, over a necrotic lipid core, which can weather and rupture over time. The artery is able to compensate for some intimal thickening by expanding outwards, instead of allowing for the plaque to impede blood flow. Eventually the vessel can no longer expand outwards, and negative remodelling can occur. Blood flow is therefore disturbed through the formation of a stenosis (Shah, 2006). This may lead to ischemia and angina pectoris (Libby, 2002).

Atherosclerosis can occur in any size of artery. However, clinical manifestations frequently occur in medium and large arteries when the EC layer is breached by erosion or disruption of the fibrous cap. Disruption may occur from a thinning of the fibrous cap as there is increased lipid accumulation, inflammatory cell recruitment and matrix metalloproteinase (MMP) expression, as well as the expression of cytokines inhibiting collagen synthesis. When the plaque is opened up to the blood stream, platelets cause blood coagulation and thrombus formation. There are two possible outcomes after thrombus formation. First, the thrombus may be broken down and reabsorbed. A second outcome is that the thrombus is disrupted by the blood flow and detached from the site of injury. This embolism may then travel through the vasculature to small arteries, where it causes ischemia and potential heart attack or stroke (Libby, 2002).

### *The role of inflammation*

Over the past two decades, it has been recognized that inflammation plays a critical role in the development and progression of atherosclerosis (Libby, 2002). Indeed, the localization of plaques to regions of disturbed blood flow (curvature, bifurcations, and branches) has been linked to an inflammatory response of ECs due to hemodynamic forces (Libby, 2002; Shah, 2006). In these areas, ECs become inflamed, causing an influx of leukocytes (Shah, 2006). It has been found that nuclear factor κB (NF-κB), a transcription factor responsible for expressing genes involved in the inflammatory cascade, is activated at sites of disturbed blood flow (Van der Heiden et al., 2010).

Additionally, monocytes, part of the family of leukocytes, are attracted into the intima through the EC layer due to the existence of a chemical gradient. During inflammation, a chemokine called monocyte chemoattractant protein-1 (MCP-1) is expressed within the intima layer (Libby, 2002). MCP-1 is expressed constitutively, by both the EC layer, and the SMCs within the intima (Schwartz et al., 1991). The receptor for MCP-1 on the monocyte (the CCR2 receptor) is attracted to the MCP-1 within the intima, and monocytes migrate into the intima through diapedesis (Libby, 2002).

Also flowing in the blood stream are low-density lipoproteins (LDL), including cholesterol. LDLs are brought across the EC layer and into the intima. Reactive oxygen species within the intima, including ·OH and ·O2, oxidize the LDLs, turning them into oxidized lowdensity lipoproteins (Ox-LDL). These Ox-LDL molecules are also responsible for stimulating ECs and SMCs to secrete additional MCP-1 (Schwartz et al., 1991).

Once inside the intima, monocytes begin to express characteristics of macrophages, activated by the presence of macrophage colony stimulating factor (M-CSF) (Libby, 2002). M-CSF also activates Ox-LDL receptors on the macrophages, turning them into scavengers for Ox-LDL (Libby et al., 2002). Macrophages begin to take up Ox-LDL, filling themselves with lipids and transforming into foam cells (Ross, 1993; Shah, 2006). The accumulation of macrophage foam cells, as well as collagen, elastin, and proteoglycans, within the intimal layer is known as the fatty streak, and is an early indication of a complex atherosclerotic lesion (Ross, 1993; Schwartz et al., 1991). It has been shown that the progression of a foam cell to a more advanced lesion may be halted or reversed, possibly through a decrease in blood LDL levels (Schwartz et al., 1991).

compensate for some intimal thickening by expanding outwards, instead of allowing for the plaque to impede blood flow. Eventually the vessel can no longer expand outwards, and negative remodelling can occur. Blood flow is therefore disturbed through the formation of

Atherosclerosis can occur in any size of artery. However, clinical manifestations frequently occur in medium and large arteries when the EC layer is breached by erosion or disruption of the fibrous cap. Disruption may occur from a thinning of the fibrous cap as there is increased lipid accumulation, inflammatory cell recruitment and matrix metalloproteinase (MMP) expression, as well as the expression of cytokines inhibiting collagen synthesis. When the plaque is opened up to the blood stream, platelets cause blood coagulation and thrombus formation. There are two possible outcomes after thrombus formation. First, the thrombus may be broken down and reabsorbed. A second outcome is that the thrombus is disrupted by the blood flow and detached from the site of injury. This embolism may then travel through the vasculature to small arteries, where it causes ischemia and potential heart

Over the past two decades, it has been recognized that inflammation plays a critical role in the development and progression of atherosclerosis (Libby, 2002). Indeed, the localization of plaques to regions of disturbed blood flow (curvature, bifurcations, and branches) has been linked to an inflammatory response of ECs due to hemodynamic forces (Libby, 2002; Shah, 2006). In these areas, ECs become inflamed, causing an influx of leukocytes (Shah, 2006). It has been found that nuclear factor κB (NF-κB), a transcription factor responsible for expressing genes involved in the inflammatory cascade, is activated at sites of disturbed

Additionally, monocytes, part of the family of leukocytes, are attracted into the intima through the EC layer due to the existence of a chemical gradient. During inflammation, a chemokine called monocyte chemoattractant protein-1 (MCP-1) is expressed within the intima layer (Libby, 2002). MCP-1 is expressed constitutively, by both the EC layer, and the SMCs within the intima (Schwartz et al., 1991). The receptor for MCP-1 on the monocyte (the CCR2 receptor) is attracted to the MCP-1 within the intima, and monocytes migrate into the

Also flowing in the blood stream are low-density lipoproteins (LDL), including cholesterol. LDLs are brought across the EC layer and into the intima. Reactive oxygen species within the intima, including ·OH and ·O2, oxidize the LDLs, turning them into oxidized lowdensity lipoproteins (Ox-LDL). These Ox-LDL molecules are also responsible for stimulating

Once inside the intima, monocytes begin to express characteristics of macrophages, activated by the presence of macrophage colony stimulating factor (M-CSF) (Libby, 2002). M-CSF also activates Ox-LDL receptors on the macrophages, turning them into scavengers for Ox-LDL (Libby et al., 2002). Macrophages begin to take up Ox-LDL, filling themselves with lipids and transforming into foam cells (Ross, 1993; Shah, 2006). The accumulation of macrophage foam cells, as well as collagen, elastin, and proteoglycans, within the intimal layer is known as the fatty streak, and is an early indication of a complex atherosclerotic lesion (Ross, 1993; Schwartz et al., 1991). It has been shown that the progression of a foam cell to a more advanced lesion may be halted or reversed, possibly through a decrease in

ECs and SMCs to secrete additional MCP-1 (Schwartz et al., 1991).

a stenosis (Shah, 2006). This may lead to ischemia and angina pectoris (Libby, 2002).

attack or stroke (Libby, 2002).

blood flow (Van der Heiden et al., 2010).

intima through diapedesis (Libby, 2002).

blood LDL levels (Schwartz et al., 1991).

*The role of inflammation* 

In advanced lesions, macrophages are unable to take up any additional Ox-LDL, and these lipid molecules begin to accumulate within the intima instead (Schwartz et al., 1991). Ox-LDLs are toxic, and begin to injure and kill ECs, SMCs, and macrophages. When the macrophage is injured, its lipid contents are released into the intimal layer, forming a lipid core, also comprised of enzymes, cytokines, and growth factors that have accumulated within the intima (Schwartz et al., 1991; Shah, 2006).

In order to protect the body from the growing necrotic lipid core within the intima, a fibrous plaque is formed over the lesion. This prevents direct contact between the accumulation of cells within the intima and blood flow. The fibrous plaque is composed primarily of collagen, elastin, and proteoglycans that were found in the original fatty streak (Ross, 1999). T lymphocytes and macrophages release MMPs, which break down the extracellular matrix within the intima, allowing for these components to be used within the fibrous plaque (Libby, 2002).

### **2.2 Mechanisms in leukocyte-endothelium adhesion 2.2.1 Intercellular adhesion at the arterial surface**

The initiation of atherosclerosis is hypothesized to start with endothelial injury, which triggers inflammatory pathways integral to the progression of the disease (Ross et al., 1977). Leukocytes, including neutrophils and monocytes, preferentially adhere to sites of inflammation. The events that take place during leukocyte recruitment are shown in the representative drawing, Figure 1.

Fig. 1. Leukocyte recruitment to a site of endothelial injury. When injured, the endothelium expresses an increase in cell adhesion molecules (CAMs). (A) Leukocytes circulate within the blood stream. (B) Ligands on the leukocytes attach to selectins on the endothelium, effectively tethering the leukocyte. (C) The leukocyte begins to roll across the endothelium, reducing its velocity by forming and breaking selectin-ligand bonds. (D) Leukocytes begin to make bonds between CAMs and integrins. This firmly attaches the leukocyte to the endothelium. (E) Leukocytes migrate towards a cellular junction through CAM-integrin interactions. (F) CAM-CAM interactions allow the leukocyte to migrate through the endothelium by diapedesis.

## *Leukocyte tethering and rolling*

Leukocytes circulating within the bloodstream must make their way to the site of injury, located on the endothelium. Although leukocytes flow in close contact with the endothelial layer, they do not stick until the inflamed endothelium starts to express adhesive molecules (Kelly et al., 2007). Tethering and rolling of leukocytes along the endothelial wall is due to a class of cell adhesion molecules (CAMs) known as selectins. There are three selectins involved: E-selectin, P-selectin, and L-selectin. Both E- and P-selectins are expressed on the endothelium; E-selectin is synthesized and expressed after endothelial stimulation, whereas P-selectin is expressed constitutively and stored, then quickly released upon stimulation (Kelly et al., 2007). L-selectin differs in that it is not expressed on the endothelium, but instead is constitutively expressed on the leukocyte surface. Both E- and P-selectin recognize carbohydrate ligands on the surface of leukocytes, while L-selectin recognizes a series of ligands expressed on the endothelium. When the selectins come in contact with their ligands they will bind, tethering the leukocyte to the endothelium (Miyasaka et al., 1997). Tethering facilitates leukocyte rolling along the EC surface. The velocity of the travelling leukocyte will be reduced as more selectin-ligand bonds form, allowing leukocyte adhesion (Kelly et al., 2007; Kubes & Kerfoot, 2001).

### *Leukocyte adhesion*

Firm adhesion begins when integrins, another class of CAMs, are activated on the surface of leukocytes by chemoattractant cytokines, termed chemokines. Chemokines are secreted by circulating leukocytes and ECs (Kelly et al., 2007). One class of integrin responsible for neutrophil adhesion is the β2-integrin. When activated by cytokines, certain β2-integrins bind to intracellular adhesion molecules 1 and 2 (ICAM-1 and ICAM-2, respectively) on the EC surface, effectively adhering the leukocyte to the EC. Also activated by cytokines, α4β1 integrin will bind with vascular cell adhesion molecule 1 (VCAM-1) (Kelly et al., 2007; Rao et al., 2007). The bound integrin-CAM complex results in firm adhesion of the leukocyte to the endothelial surface (Miyasaka et al., 1997). Although under transcriptional regulation, both adhesion molecules ICAM-1 and VCAM-1 have been shown to be upregulated at sites in the vasculature prone to developing atherosclerotic lesions (Iiyama et al., 1999).

### *Leukocyte migration*

Once the leukocytes have firmly adhered to the endothelium, they may migrate from the lumen of the blood vessel into the subintimal space. Initially, leukocytes must make their way to the closest EC junction through a process termed locomotion. The movement of the leukocyte is made possible through interactions with leukocyte integrins and both ICAM-1 and -2 located on the endothelial surface (Schenkel et al., 2004). At the junction, the leukocytes will encounter another cell adhesion molecule, called platelet endothelial CAM 1 (PECAM-1). PECAM-1 is expressed both on the leukocyte and the endothelium. An interaction between the complementary PECAM-1 molecules allows leukocytes to migrate through the gap junction by diapedesis (Rao et al., 2007; Schenkel et al., 2004), a process also called transmigration.

### **2.2.2 Idealized arterial hemodynamics**

Hemodynamics, the mechanics of blood flow, influence many of the physiological processes of the vascular system (Glagov et al., 1988). From an engineering perspective, blood flow through medium and small arteries (such as the right and left coronary arteries) is often

Leukocytes circulating within the bloodstream must make their way to the site of injury, located on the endothelium. Although leukocytes flow in close contact with the endothelial layer, they do not stick until the inflamed endothelium starts to express adhesive molecules (Kelly et al., 2007). Tethering and rolling of leukocytes along the endothelial wall is due to a class of cell adhesion molecules (CAMs) known as selectins. There are three selectins involved: E-selectin, P-selectin, and L-selectin. Both E- and P-selectins are expressed on the endothelium; E-selectin is synthesized and expressed after endothelial stimulation, whereas P-selectin is expressed constitutively and stored, then quickly released upon stimulation (Kelly et al., 2007). L-selectin differs in that it is not expressed on the endothelium, but instead is constitutively expressed on the leukocyte surface. Both E- and P-selectin recognize carbohydrate ligands on the surface of leukocytes, while L-selectin recognizes a series of ligands expressed on the endothelium. When the selectins come in contact with their ligands they will bind, tethering the leukocyte to the endothelium (Miyasaka et al., 1997). Tethering facilitates leukocyte rolling along the EC surface. The velocity of the travelling leukocyte will be reduced as more selectin-ligand bonds form, allowing leukocyte adhesion (Kelly et

Firm adhesion begins when integrins, another class of CAMs, are activated on the surface of leukocytes by chemoattractant cytokines, termed chemokines. Chemokines are secreted by circulating leukocytes and ECs (Kelly et al., 2007). One class of integrin responsible for neutrophil adhesion is the β2-integrin. When activated by cytokines, certain β2-integrins bind to intracellular adhesion molecules 1 and 2 (ICAM-1 and ICAM-2, respectively) on the EC surface, effectively adhering the leukocyte to the EC. Also activated by cytokines, α4β1 integrin will bind with vascular cell adhesion molecule 1 (VCAM-1) (Kelly et al., 2007; Rao et al., 2007). The bound integrin-CAM complex results in firm adhesion of the leukocyte to the endothelial surface (Miyasaka et al., 1997). Although under transcriptional regulation, both adhesion molecules ICAM-1 and VCAM-1 have been shown to be upregulated at sites

in the vasculature prone to developing atherosclerotic lesions (Iiyama et al., 1999).

Once the leukocytes have firmly adhered to the endothelium, they may migrate from the lumen of the blood vessel into the subintimal space. Initially, leukocytes must make their way to the closest EC junction through a process termed locomotion. The movement of the leukocyte is made possible through interactions with leukocyte integrins and both ICAM-1 and -2 located on the endothelial surface (Schenkel et al., 2004). At the junction, the leukocytes will encounter another cell adhesion molecule, called platelet endothelial CAM 1 (PECAM-1). PECAM-1 is expressed both on the leukocyte and the endothelium. An interaction between the complementary PECAM-1 molecules allows leukocytes to migrate through the gap junction by diapedesis (Rao et al., 2007; Schenkel et al., 2004), a process also

Hemodynamics, the mechanics of blood flow, influence many of the physiological processes of the vascular system (Glagov et al., 1988). From an engineering perspective, blood flow through medium and small arteries (such as the right and left coronary arteries) is often

*Leukocyte tethering and rolling* 

al., 2007; Kubes & Kerfoot, 2001).

*Leukocyte adhesion* 

*Leukocyte migration* 

called transmigration.

**2.2.2 Idealized arterial hemodynamics** 

simplified by assuming steady laminar flow in a straight, rigid vessel (Ku, 1997; Nichols & O'Rourke, 1990). Additionally, blood is assumed to be a Newtonian fluid to simplify the flow dynamics to Hagen-Poiseuille flow (Nichols & O'Rourke, 1990). Such assumptions allow us to reduce the governing equations describing pressure-driven flow into a onedimensional velocity profile in the form of Hagen-Poiseuille flow (Nichols & O'Rourke, 1990). For a cylindrical vessel model of arterial perfusion, the velocity profile as a function of a radial dimension is described by:

$$w(r) = 2\left(\frac{q}{\pi R^2}\right)\left[1 - \left(\frac{r}{R}\right)^2\right] \tag{1}$$

where *Q* is the volumetric flow rate of blood and *R* is the hydraulic radius of the vessel. Wall shear stress (WSS) is a tangential force per unit area of a fluid-wall interface that results from flow parallel to the vessel wall. For fluids with constant dynamic viscosity *μ* (Newtonian fluid), the WSS is the product of the viscosity and the shear rate *γ*, evaluated at the vessel wall:

$$\tau\_{\mathcal{W}} = \|\mu\mathcal{Y}\|\_{r=R} \tag{2}$$

The wall shear rate of a fluid is the velocity gradient evaluated at the fluid-wall interface. For Hagen-Poiseuille flow through a cylindrical vessel (Equation 1), the wall shear rate is expressed as:

$$\left. \mathcal{Y} \right|\_{r=R} = \left. \frac{dv(r)}{dr} \right|\_{r=R} = \frac{4Q}{\pi R^3} \tag{3}$$

Combining Equations (2) and (3) produces an expression for WSS that is dependent on both the vessel geometry and the volumetric flow rate:

$$
\pi\_{\mathcal{W}} = \mu \frac{{}^{4Q}\!}{{}^{\pi R^3}} \tag{4}
$$

This equation is accepted as a reasonable model of the average WSS for arteries that are absent from serious geometric disturbances (Ku, 1997). Arterial WSS values range from 5 to 70 dyne/cm2 with average WSS values of approximately 15 dyne/cm2 being observed in coronary arteries (Glagov et al., 1988; Malek et al., 1999). Moderate levels of steady, laminar shear stress (> 10-15 dyne/cm2) are believed to induce an atheroprotective EC phenotype while low shear stresses (< 4 dyne/cm2) are believed to induce an atheroprone EC phenotype (Malek et al., 1999). An atheroprone EC phenotype describes one which facilitates the disease pathway marked by an increase in adhesion molecules and a decrease in vasodilators (as described in Sections 2.1 & 2.2) (Libby, 2002; Malek et al., 1999).

The fluid flow regime is determined by the dimensionless Reynolds number (Re), which represents the ratio of inertial to viscous forces. For flow in a cylindrical channel, the Reynolds Number is described by:

$$Re = \frac{\rho DU}{\mu} \tag{5}$$

where *ρ* is the fluid density, *D* is the hydraulic diameter, and *U* is the average fluid velocity. A Reynolds Number below 2300 indicates laminar flow that will behave predictably while a value above this threshold suggests the presence of flow disturbances. The average arterial conditions are within a laminar flow regime (Nichols & O'Rourke, 1990) and vary depending on the artery and metabolic demand (Myers et al., 2001; Nichols & O'Rourke, 1990).

### *Localized hemodynamics of leukocyte adhesion*

The progression of leukocyte adhesion is strongly influenced by local hemodynamic forces. As the cell is passing along the wall, the torque imparted on the cell by the blood stream causes the cell to spin. As a result, the state of loose attachment with selectin-ligand bonds constantly forming and breaking has become known as cell rolling. The blood stream imposes not only torque but also shear stress on the slow moving cell. In turn, the membrane of the cell will try to distribute this stress by elongating in the direction of flow, allowing for increased binding with the vessel wall. Firrell and Lipowsky found that leukocytes rolling along rat arteriolar walls would elongate by around 140%, allowing their contact area to jump from approximately 14 µm2 to 50 µm2 (Firrell & Lipowsky, 1989).

Modelling of bond forces and leukocyte attachment is well documented in the literature (Cozens-Roberts et al., 1990; Evans et al., 2004; Lawrence et al., 1997; Tees & Goetz, 2003). For successful adhesion, a fine balance between the adhesive force and the hemodynamic force must be met. This adhesive force is dependent on several factors, including: the receptor density of both cells, the rate of reaction with respect to both bond formation and dissociation and the strength of the bonds and their response to strain. For instance, the bonds between E-, P-, and L-selectins and their respective ligands behave as catch-slip bonds (Lawrence et al., 1997; Marshall et al., 2003; Sarangapani et al., 2004). Though receptor-ligand bonds spontaneously dissociate (Tees & Goetz, 2003), slip bond behaviour describes an increasing probability of dissociation with increasing tensile force. Catch-slip bond behaviour, on the other hand, strengthens with increasing tensile force until some optimal force has been met. Furthermore, the rate at which this force is increased, known as the force gradient, also affects the strength of the selectin-ligand bonds (Evans et al., 2004). This is of particular interest to the study of stenotic arteries as plaque formation leads to distinct regions of varying force gradients.

### **2.3 Leukocyte adhesion in Parallel-Plate Flow Chambers (PPFCs)**

The flow between two parallel plates has often been used to investigate the effects of blood flow on ECs and their interactions with blood components. Traditionally, parallel-plate flow chambers (PPFCs) have been used to provide an environment suited for tissue and suspension culture experiments under laminar flow. In classical PPFCs, fluid is driven through a channel formed by two narrowly separated plates in parallel. ECs are cultured on the bottom surface of the upper plate (often, a glass coverslip) while suspension cultures of leukocytes (neutrophils or monocytes) are prepared in the perfusion medium and their movement visualized within the chamber (Lawrence et al., 1987). This allows for regional or complete surface quantification of cells that are either adherent or, if observed in real-time, leukocytes that are undergoing rolling adhesion. A schematic of a PPFC is presented in Figure 2.

In a well defined PPFC, the WSS can be accurately characterised for steady, laminar flow of a Newtonian fluid as a function of a constant measurable volumetric flow rate *Q*:

$$
\tau\_{\rm w} = \frac{{}^{6\mu Q}}{{}^{\nu h^2}} \tag{6}
$$

where *w* is the width of the plate perpendicular to flow and *h* is the height of the interstitial gap between the two plates. For a constant volumetric flow rate, the velocity profile is parabolic and the WSS is uniform across the upper and lower plates save for the boundaries defined by the gasket (where the flow field approaches zero) and in the region of

The progression of leukocyte adhesion is strongly influenced by local hemodynamic forces. As the cell is passing along the wall, the torque imparted on the cell by the blood stream causes the cell to spin. As a result, the state of loose attachment with selectin-ligand bonds constantly forming and breaking has become known as cell rolling. The blood stream imposes not only torque but also shear stress on the slow moving cell. In turn, the membrane of the cell will try to distribute this stress by elongating in the direction of flow, allowing for increased binding with the vessel wall. Firrell and Lipowsky found that leukocytes rolling along rat arteriolar walls would elongate by around 140%, allowing their contact area to jump from approximately 14 µm2 to 50 µm2 (Firrell & Lipowsky, 1989). Modelling of bond forces and leukocyte attachment is well documented in the literature (Cozens-Roberts et al., 1990; Evans et al., 2004; Lawrence et al., 1997; Tees & Goetz, 2003). For successful adhesion, a fine balance between the adhesive force and the hemodynamic force must be met. This adhesive force is dependent on several factors, including: the receptor density of both cells, the rate of reaction with respect to both bond formation and dissociation and the strength of the bonds and their response to strain. For instance, the bonds between E-, P-, and L-selectins and their respective ligands behave as catch-slip bonds (Lawrence et al., 1997; Marshall et al., 2003; Sarangapani et al., 2004). Though receptor-ligand bonds spontaneously dissociate (Tees & Goetz, 2003), slip bond behaviour describes an increasing probability of dissociation with increasing tensile force. Catch-slip bond behaviour, on the other hand, strengthens with increasing tensile force until some optimal force has been met. Furthermore, the rate at which this force is increased, known as the force gradient, also affects the strength of the selectin-ligand bonds (Evans et al., 2004). This is of particular interest to the study of stenotic arteries as plaque formation leads to

*Localized hemodynamics of leukocyte adhesion* 

distinct regions of varying force gradients.

Figure 2.

**2.3 Leukocyte adhesion in Parallel-Plate Flow Chambers (PPFCs)** 

The flow between two parallel plates has often been used to investigate the effects of blood flow on ECs and their interactions with blood components. Traditionally, parallel-plate flow chambers (PPFCs) have been used to provide an environment suited for tissue and suspension culture experiments under laminar flow. In classical PPFCs, fluid is driven through a channel formed by two narrowly separated plates in parallel. ECs are cultured on the bottom surface of the upper plate (often, a glass coverslip) while suspension cultures of leukocytes (neutrophils or monocytes) are prepared in the perfusion medium and their movement visualized within the chamber (Lawrence et al., 1987). This allows for regional or complete surface quantification of cells that are either adherent or, if observed in real-time, leukocytes that are undergoing rolling adhesion. A schematic of a PPFC is presented in

In a well defined PPFC, the WSS can be accurately characterised for steady, laminar flow of

߬௪ ൌ ఓொ

where *w* is the width of the plate perpendicular to flow and *h* is the height of the interstitial gap between the two plates. For a constant volumetric flow rate, the velocity profile is parabolic and the WSS is uniform across the upper and lower plates save for the boundaries defined by the gasket (where the flow field approaches zero) and in the region of

௪<sup>మ</sup> (6)

a Newtonian fluid as a function of a constant measurable volumetric flow rate *Q*:

developing flow at the inlet of the chamber (Bacabac et al., 2005; Lawrence et al., 1987). In practice, PPFCs are designed with a large *w/h* ratio allowing most of the flow field to be homogenous over the surface of the cells (Bacabac et al., 2005).

Fig. 2. Schematic of a Parallel-Plate Flow Chamber.

Studies of leukocyte adhesion using PPFCs have become the benchmark for revealing the role of shear in the adhesion pathway. Early results revealed a discreet shear dependence on both non-specific (Forrester & Lackie, 1984) and adhesion molecule-mediated adhesion (Alon et al., 1995; Finger et al., 1996; Lawrence et al., 1997). Further studies highlighted the role of endothelial dysfunction and inflammation as a precursor to adhesion when conditioned with flow (Alcaide et al., 2009; Sheikh et al., 2003; Sheikh et al., 2005). These findings complement the paradigm of leukocyte adhesion at sites of vascular inflammation; however, they do not address focal adhesion in non-uniform shear fields. This was considered by performing flow experiments using a step disturbance across the plate of the flow chamber (Burns & DePaola, 2005; Chen et al., 2006). The flow fields created by the step introduce regions of flow reversal and spatial WSS gradients to represent physiological hemodynamics (Burns & DePaola, 2005; Chen et al., 2006). Despite disturbed flow, leukocyte adhesion is increased in areas of high WSS gradients, with the highest incidence in reattachment zones (Chen et al., 2006).
