**5. Membrane cholesterol and the neutrophil mechanosensitivity to shear stress**

In addition to the presence of inflammatory stimuli (e.g., oxLDL), elevated neutrophil activity in hypercholesterolemia may result from defects in their mechanotransduction of fluid shear stress, a control mechanism to prevent spontaneous neutrophil activity under physiological conditions[78, 79]. In this regard, the mechanosensitivity of neutrophils may serve as a key regulator of the inflammatory status of the circulation. We will first define the leukocyte mechanosensitivity to shear followed by a brief discussion of cellular mechanisms that link the extracellular flow environment to downstream neutrophil functions. Interestingly, such mechanotransduction processes occur across the plasma membrane that plays a critical role in regulating the activity of membrane proteins as well as the transmembrane movement of bioactive molecules. The direct contact of cell membrane with the extracellular flow environment makes it a likely target of local environmental factors (e.g., enhanced cholesterol abundance) that influence the neutrophil responsiveness to mechanical stimuli.

#### **5.1. Regulation of neutrophil activity by fluid flow-derived shear stress**

Neutrophils, either freely suspended in the bloodstream or adhered to/migrating on vascular endothelium, sense and respond to fluid shear stress[80-82]. Fluid shear stress (ranging from approximately 1 to 10 dyn/cm2) minimizes neutrophil activity levels[78]. The most obvious manifestation of the cell-inactivating effects of shear exposure on cell activity is the retraction of existing pseudopodia by non-cytokine-stimulated human neutrophils adhered to a surface and subjected to a non-uniform flow field imposed by a micropipette with a tip of diameter in the range of 4 – 8 μm[82] (Figure 3A). This situation models brief and spontaneous periods of blood stasis followed by reperfusion, a typical scenario in the microvessels. Under this condition, neutrophils sediment, attach, extend pseudopods, and migrate on the vascular endothelium. Upon reintroduction of fluid flow, these cells retract pseudopods and detach into the flow field in a mechanobiological fashion. Such a scenario has been documented using intravital microscopy of microvascular networks of rodents (e.g., mesentery, spinotrapezius muscle, cremaster muscle)[81-83]. The ability of shear stress to minimize pseudopod activity has been further confirmed for non-adherent heterogeneous leukocyte populations[84] exposed to a constant shear field (5 dyn/cm2) in a cone-plate viscometer (Figure 3B).

**Figure 3. Deactivation of neutrophils under flow stimulation.** A: A migrating/adherent neutrophil exposed to a micropipette flow (~ 2 dyn/cm2) for 2 min. B: Non-adherent neutrophils in suspension exposed to cone-plate shear (5 dyn/cm2) for 10 min. Bars are mean percentage of activated cells with pseudopods (see image insets) in each population tested ± SEM; \*p < 0.05 compared to static condition using paired Student's t-test.

Notably, impairment of shear-induced pseudopod retraction by treating neutrophils with cell agonists above threshold concentrations, e.g. fMLP (>10-8 M), commits these cells to an activated (inflamed) phenotype and leads to their microvascular entrapment due to increases in adhesivity, size, and stiffness[35, 81, 82]. Thus, during inflammation, the biochemical milieu of the neutrophil overrides mechanobiological deactivation. Exposure to shear of magnitudes typically found in the macro- and micro- circulations is also associated with other attributes of neutrophil deactivation such as decreased surface expression of integrin receptors (i.e., CD18), depolymerization of the F-actin cytoskeleton, cell detachment, and attenuated phagocytic activity[81, 82]. Moreover, shear stress exposure enhances caspase 3-dependent apoptosis[85], in line with the relatively short lifespan (18 to 24 hrs) of these cells when they are passively circulating in the physiologic bloodstream. These observations support the key role of fluid flow-related shear stress as a biophysical stimulus that promotes neutrophil inactivation when cell activity is below a threshold level. As such, the mechanical influence of fluid flow serves an anti-inflammatory role.

#### **5.2. Shear stress mechanotransduction at the neutrophil surface**

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viscometer (Figure 3B).

using paired Student's t-test.

and spontaneous periods of blood stasis followed by reperfusion, a typical scenario in the microvessels. Under this condition, neutrophils sediment, attach, extend pseudopods, and migrate on the vascular endothelium. Upon reintroduction of fluid flow, these cells retract pseudopods and detach into the flow field in a mechanobiological fashion. Such a scenario has been documented using intravital microscopy of microvascular networks of rodents (e.g., mesentery, spinotrapezius muscle, cremaster muscle)[81-83]. The ability of shear stress to minimize pseudopod activity has been further confirmed for non-adherent heterogeneous leukocyte populations[84] exposed to a constant shear field (5 dyn/cm2) in a cone-plate

**Figure 3. Deactivation of neutrophils under flow stimulation.** A: A migrating/adherent neutrophil exposed to a micropipette flow (~ 2 dyn/cm2) for 2 min. B: Non-adherent neutrophils in suspension exposed to cone-plate shear (5 dyn/cm2) for 10 min. Bars are mean percentage of activated cells with pseudopods (see image insets) in each population tested ± SEM; \*p < 0.05 compared to static condition

Notably, impairment of shear-induced pseudopod retraction by treating neutrophils with cell agonists above threshold concentrations, e.g. fMLP (>10-8 M), commits these cells to an activated (inflamed) phenotype and leads to their microvascular entrapment due to increases in adhesivity, size, and stiffness[35, 81, 82]. Thus, during inflammation, the biochemical milieu of the neutrophil overrides mechanobiological deactivation. Exposure to shear of magnitudes typically found in the macro- and micro- circulations is also associated with other attributes of neutrophil deactivation such as decreased surface expression of integrin receptors (i.e., CD18), depolymerization of the F-actin cytoskeleton, cell detachment, and attenuated phagocytic activity[81, 82]. Moreover, shear stress exposure enhances caspase 3-dependent apoptosis[85], in line with the relatively short lifespan (18 to 24 hrs) of these cells when they are passively circulating in the physiologic bloodstream. These observations support the key role of fluid flow-related shear stress as a biophysical stimulus that promotes neutrophil inactivation when cell activity is below a threshold level.

As such, the mechanical influence of fluid flow serves an anti-inflammatory role.

An understanding of the fluid flow mechanoregulation of neutrophil activity in the circulation reveals clues regarding how impaired mechanosensitivity to flow may be a mitigating factor for hypercholesterolemic disorders. Membrane detachment during pseudopod retraction by migrating neutrophils in response to fluid shear stress points to two fundamental requirements that must be fulfilled by the cell signaling apparatus: 1) depolymerization of the F-actin cytoskeleton that serves as a structural and a signaling scaffold for neutrophil motility and 2) rapid disengagement of adhesion receptors that anchor the pseudopod to the underlying substrates. For suspended neutrophils, similar events are needed but, in this case, mechanisms must be in place to prevent the expression of adhesive proteins or interfere with engagement of adhesion molecule with substrates (e.g. foreign surfaces, other cells) presenting counter-receptors. These fundamental requirements point to the neutrophil surface components as critical players in mechanotransduction since the cell must sense the extracellular flow environment and remediate its interactions with the cellular microenvironment (e.g., the surrounding matrix and cells).

#### *5.2.1. GPCRs and shear stress control of neutrophil pseudopod activity*

Shear stress-induced pseudopod retraction by neutrophils occurs in parallel with a rapid decrease in F-actin content[86, 87]. Typically, remodeling of the F-actin cytoskeleton in leukocytes is controlled by the Ras superfamily of small guanine triphosphate (GTP) binding proteins, particularly the small GTP-binding phosphatases (GTPases) including Rac1, Rac2, cdc42 and members of the Rho family (as reviewed in the literature[88-90]). Rather than stimulating the activity of molecules that coordinate pseudopod retraction (e.g., RhoA, MLCK), fluid shear stress appears to either inhibit (e.g., possibly through release of an inhibitor) or interfere with the ability of neutrophils to form and sustain pseudopod projections via reducing cytosolic activity of the key small GTPases (e.g., Rac1, Rac2) involved in actin polymerization[83]. These reported effects point to the actions of fluid shear stress on G protein signaling downstream of GPCRs that regulate neutrophil chemotaxis, such as the formyl peptide receptor (FPR).

Notably, fMLP, a ligand for FPR, dose-dependently impairs neutrophil pseudopod retraction responses to shear stimulation[81]**.** Along this line, HL-60-derived neutrophils subjected to shear stress exhibit reduced activity of Gαi downstream of FPR[91]. A critical piece of evidence pointing to FPR as a mechanosensory regulator of pseudopod retraction is the observation that transfection of FPR expression plasmid in undifferentiated HL-60 cells not only confers expression of this receptor but imparts on these cells the ability to form pseudopods that retract under the influence of fluid shear stress[91]. Furthermore, HL-60 promyelocytes differentiated into neutrophils and subsequently transfected with siRNA to silence FPR expression exhibit an attenuated pseudopod retraction response to shear exposure, despite the fact that these cells retain the ability to project pseudopods because of the presence of other cytokine-related GPCRs[91]. Together, these observations point to a role of fluid flow in regulating the activity of membrane-associated receptors by establishing the importance of membrane-bound GPCRs, specifically FPR, in the neutrophil pseudopod retraction response to shear stress. In conjunction with the dependence of GPCR activity on the membrane cholesterol content, it is conceivable that the influence of shear stress on GPCR activity is impacted by perturbations in extracellular cholesterol abundance and their effects on the cell membrane properties.

#### *5.2.2. Cell surface CD18 integrins and shear stress regulation of neutrophil adhesion*

Pseudopod retraction by migrating neutrophils subjected to fluid flow depends on their expression levels of CD18 integrins[92], consistent with the requirement of these receptors for cyclical pseudopod projection and retraction[93]. In addition to modulating CD18 interactions with their ligands (e.g. ICAM-1) during inflammation[94, 95], fluid shear stress appears to regulate integrin dynamics on the neutrophil surface under conditions that mimic low activation states by redistributing these receptors from areas of maximal shear stress to regions where shear is minimal, i.e. at focal adhesions. Moreover, shear exposure reduces CD18 levels on the surfaces of migrating, and also non-adherent, neutrophils even in the presence of inflammatory mediators, e.g. fMLP[81, 96]. Considering the role of CD18 in strengthening neutrophil attachment to the vascular wall, shear-mediated reductions in CD18 likely diminish the ability of cells to maintain adhesive attachments[97]. In this way, shear-mediated reductions in CD18 serve an anti-inflammatory role that ensures neutrophils in a non-inflamed environment remain in a non-adhesive state.

The mechanism underlying shear-induced reductions in CD18 surface levels involves proteolysis that occurs on the surfaces of migrating and suspended neutrophils. Proteolysis modulates the levels of a wide variety of transmembrane receptors on the neutrophil surface including L-selectin (involved in rolling interactions with endothelium)[98] and CD43, an anti-adhesive mucin-like molecule[99]. CD18 integrins also undergo cleavage of the intracellular domain by calpain to promote detachment of the cell uropod during neutrophil migration[100]. But shear-induced truncation of CD18 integrins differs from calpainmediated cleavage in that the former involves lysosomal cysteine proteases (e.g., cathepsin B) that exert extracellular activity[96, 97]. Notably, the cell membrane is critically positioned between the intracellular levels, and the extracellular actions, of these proteases.

Additionally, cleavage of CD18 integrins under fluid flow also requires conformational changes in their extracellular domains[96]. Conformational activity of CD18 integrins involves shifts in the protein tertiary structure from a closed-bent to an open-extended configuration[96]. In the case of cytokine stimulation, this conformational change exposes ligand binding sites[101] that promote cell capture onto the vessel wall[95, 102]. Another consequence of CD18 conformational changes, which occur upon shear stress exposure, is to expose proteolytic cleavage sites[96]. With this evidence in mind, it is apparent that the physicochemical state of the cell membrane is a key factor in neutrophil mechanosensitivity that directly or indirectly affects the ability of shear stress to unfold the CD18 ectodomain.

#### *5.2.3. RNS and ROS in shear mechanotransduction*

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effects on the cell membrane properties.

role of fluid flow in regulating the activity of membrane-associated receptors by establishing the importance of membrane-bound GPCRs, specifically FPR, in the neutrophil pseudopod retraction response to shear stress. In conjunction with the dependence of GPCR activity on the membrane cholesterol content, it is conceivable that the influence of shear stress on GPCR activity is impacted by perturbations in extracellular cholesterol abundance and their

*5.2.2. Cell surface CD18 integrins and shear stress regulation of neutrophil adhesion* 

in a non-inflamed environment remain in a non-adhesive state.

Pseudopod retraction by migrating neutrophils subjected to fluid flow depends on their expression levels of CD18 integrins[92], consistent with the requirement of these receptors for cyclical pseudopod projection and retraction[93]. In addition to modulating CD18 interactions with their ligands (e.g. ICAM-1) during inflammation[94, 95], fluid shear stress appears to regulate integrin dynamics on the neutrophil surface under conditions that mimic low activation states by redistributing these receptors from areas of maximal shear stress to regions where shear is minimal, i.e. at focal adhesions. Moreover, shear exposure reduces CD18 levels on the surfaces of migrating, and also non-adherent, neutrophils even in the presence of inflammatory mediators, e.g. fMLP[81, 96]. Considering the role of CD18 in strengthening neutrophil attachment to the vascular wall, shear-mediated reductions in CD18 likely diminish the ability of cells to maintain adhesive attachments[97]. In this way, shear-mediated reductions in CD18 serve an anti-inflammatory role that ensures neutrophils

The mechanism underlying shear-induced reductions in CD18 surface levels involves proteolysis that occurs on the surfaces of migrating and suspended neutrophils. Proteolysis modulates the levels of a wide variety of transmembrane receptors on the neutrophil surface including L-selectin (involved in rolling interactions with endothelium)[98] and CD43, an anti-adhesive mucin-like molecule[99]. CD18 integrins also undergo cleavage of the intracellular domain by calpain to promote detachment of the cell uropod during neutrophil migration[100]. But shear-induced truncation of CD18 integrins differs from calpainmediated cleavage in that the former involves lysosomal cysteine proteases (e.g., cathepsin B) that exert extracellular activity[96, 97]. Notably, the cell membrane is critically positioned

Additionally, cleavage of CD18 integrins under fluid flow also requires conformational changes in their extracellular domains[96]. Conformational activity of CD18 integrins involves shifts in the protein tertiary structure from a closed-bent to an open-extended configuration[96]. In the case of cytokine stimulation, this conformational change exposes ligand binding sites[101] that promote cell capture onto the vessel wall[95, 102]. Another consequence of CD18 conformational changes, which occur upon shear stress exposure, is to expose proteolytic cleavage sites[96]. With this evidence in mind, it is apparent that the physicochemical state of the cell membrane is a key factor in neutrophil mechanosensitivity that directly or indirectly affects the ability of shear stress to unfold the CD18 ectodomain.

between the intracellular levels, and the extracellular actions, of these proteases.

Reactive nitrogen species (RNS; e.g., NO) and ROS are multi-functional free radical mediators of acute inflammation serving not only as anti-microbial agents but also as biological second messengers that influence leukocyte functions (e.g., chemotaxis, phagocytosis, etc.)[103, 104]. NO from exogenous and endogenous sources (such as membrane-associated NO synthase) inhibits neutrophil recruitment out of the microvasculature during acute inflammation[105, 106]. Interestingly, NO also enhances neutrophil pseudopod retraction in response to shear stress and counteracts the blocking effects of cell agonists (e.g. fMLP and platelet-activating factor)[81]. In contrast, ROS, particularly O2-, interferes with the neutrophil shear response and is thought to contribute to the blocking effects of cell agonist, e.g. fMLP, on flow-induced pseudopod retraction[84].

Notably, the fact that inhibition of NO synthase activity in neutrophils has no effect on shear-induced pseudopod retraction[81] points to an exogenous source and an extracellular role for NO. This finding leaves open the possibility that the facultative effects of NO on the neutrophil shear response (i.e., pseudopod retraction) result from its ability to scavenge O2- [103] and, in this way, mediate cell pseudopod activity[107, 108]. In support of this, SOD (an O2- scavenger) also enhances the shear responses of fMLP-stimulated neutrophils[84]. Thus, O2- is a critical mediator for neutrophil shear response. Since the cell membrane, particularly cholesterol-enriched lipid rafts, plays an important role in regulating the production/release of O2-[109], its state may indirectly influence neutrophil mechanosensitivity to shear stress.

#### **5.3. Neutrophil mechanosensitivity and cardiovascular disease**

The accumulated evidence reported in the vascular mechanotransduction literature (see reviews[23, 78]) points to the following general paradigm. Exposure of vascular cells to physiological flows under normal (i.e., non-diseased, non-inflamed) conditions correlates with quiescence (i.e., baseline activity). This paradigm resulted from a multitude of studies that selectively examined the activity of various signaling pathways and putative force sensors in response to applied mechanical stresses. They, however, overlooked a subtle, but equally important, factor: mechanosensitivity or the degree to which cells respond to mechanical stresses. Just as biochemical perturbations (e.g. pathogens, inflammatory agonists) temporally and dose-dependently alter vascular cell activity leading to pathogenesis, so must changes in cell mechanosensitivity impact circulatory health.

Neutrophils experience wide variations in fluid stresses as they pass through the circulation and, thus function "normally" under a diverse array of mechanical stress distributions and magnitudes. In other words, aberrant mechanical stresses are unlikely to be a cause of cell dysfunction. What may change and contribute to "abnormal" behavior is their sensitivity to the surrounding fluid flow mechanoenvironment with a negative impact on the ability of fluid shear stress to deactivate the neutrophils. Along this line, the work of Geert Schmid-Schönbein at the University of California, San Diego has demonstrated that attenuated neutrophil shear responses contribute to the microvascular pathobiology observed in spontaneously hypertensive rats (SHRs)[110] and, in doing so, illustrated the potential impact of impaired shear stress mechanotransduction on cardiovascular health.

#### *5.3.1. Impaired fluid shear responses and downstream effects on vascular pathophysiology*

Significant features of the blood from SHRs are elevated numbers of circulating neutrophils, suppressed expression of adhesion molecules (e.g., selectins, CD18), and an activated phenotype[111-113]. Although the increased activity of neutrophils is not associated with increased adhesion to microvascular endothelium[78], their increased numbers raise peripheral vascular resistance[110]. One possible explanation is that circulating activated neutrophils in SHRs release vasoactive substances that constrict the small arteries and arterioles; this has been documented for atherosclerosis[114-116]. Extensive evidence, however, points to a hemorheological effect of leukocyte activation on microvascular resistance[1, 34, 36]. Specifically, the disturbed motion of white blood cells, due to pseudopod projection, significantly reduces erythrocyte velocities in the microcirculation increasing hemodynamic resistance and upstream blood pressures[36, 110] (see Figure 1).

The key evidence for the involvement of fluid flow mechanotransduction in microvascular abnormalities due to hypertension is that neutrophils from SHRs lack the ability to retract pseudopods in response to shear stress; in some cases, cells extend cellular projections under flow stimulation[110]. The underlying mechanism associated with the blockade and possible reversal of the pseudopod retraction response to shear stress reportedly involves the dependence of blood pressure in SHRs on the plasma level of glucocorticoid-related steroid hormones and the density of glucocorticoid receptors on the neutrophil surface[117, 118]. In line with this, glucocorticoid-treated[119] rats, like SHRs, exhibit elevated peripheral resistance in parallel with elevated numbers of neutrophils that lack a pseudopod retraction response to shear stress. Taken together, leukocyte shear mechanotransduction appears to be critical for the maintenance of a healthy circulation, particularly the microcirculation. Failure of this regulatory mechanism, e.g., due to impaired cell mechanosensitivity resulting from a pathological blood environment, may not only lead to sustained neutrophil activation but also result in disturbed blood flow. In this way, aberrant neutrophil mechanotransduction may contribute to microvascular damage that exacerbates ischemia-reperfusion injury or leads to peripheral vascular disease and downstream organ/tissue injury.

Studies on spontaneous hypertension also reveal a key point. Factors that drive phenotypic changes in neutrophils (e.g., from an inactivated to an activated state) dramatically alter their ability to sense the surrounding flow environment (i.e., mechanosensitivity) leading to the development of pathological behavior, including immune suppression. Intuitively, cell mechanosensitivity depends on the number and activity of proteins "moonlighting" as putative mechanosensors embedded in the cell membrane positioned at the interface between the intra- and extra- cellular milieu. These studies further strengthen the argument that the plasma membrane is a critical determinant of neutrophil mechanosensitivity.

#### *5.3.2. The plasma membrane and shear stress mechanosensitivity*

The fact that shear stress-induced neutrophil deactivation (e.g., FPR deactivation, G protein signaling, CD18 cleavage, pseudopod retraction, etc.) occurs in the absence of any passive cell deformation due to flow[120] substantiates the presence of a cell surface component(s) that transduces flow stimulation. Interestingly, neutrophils retract pseudopods independently of the fluid shear stress distribution imposed on the cell surface[82]. Thus, membrane properties appear to outweigh the location of mechanosensors on the cell surface. Moreover, non-adherent neutrophils respond to shear stress further emphasizing the importance of cell membrane-mediated over cell deformation-based (e.g., cytoskeletonrelated, cell adhesion-dependent) neutrophil mechanotransduction.

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*5.3.1. Impaired fluid shear responses and downstream effects on vascular pathophysiology* 

Significant features of the blood from SHRs are elevated numbers of circulating neutrophils, suppressed expression of adhesion molecules (e.g., selectins, CD18), and an activated phenotype[111-113]. Although the increased activity of neutrophils is not associated with increased adhesion to microvascular endothelium[78], their increased numbers raise peripheral vascular resistance[110]. One possible explanation is that circulating activated neutrophils in SHRs release vasoactive substances that constrict the small arteries and arterioles; this has been documented for atherosclerosis[114-116]. Extensive evidence, however, points to a hemorheological effect of leukocyte activation on microvascular resistance[1, 34, 36]. Specifically, the disturbed motion of white blood cells, due to pseudopod projection, significantly reduces erythrocyte velocities in the microcirculation increasing hemodynamic resistance and upstream blood pressures[36, 110] (see Figure 1).

The key evidence for the involvement of fluid flow mechanotransduction in microvascular abnormalities due to hypertension is that neutrophils from SHRs lack the ability to retract pseudopods in response to shear stress; in some cases, cells extend cellular projections under flow stimulation[110]. The underlying mechanism associated with the blockade and possible reversal of the pseudopod retraction response to shear stress reportedly involves the dependence of blood pressure in SHRs on the plasma level of glucocorticoid-related steroid hormones and the density of glucocorticoid receptors on the neutrophil surface[117, 118]. In line with this, glucocorticoid-treated[119] rats, like SHRs, exhibit elevated peripheral resistance in parallel with elevated numbers of neutrophils that lack a pseudopod retraction response to shear stress. Taken together, leukocyte shear mechanotransduction appears to be critical for the maintenance of a healthy circulation, particularly the microcirculation. Failure of this regulatory mechanism, e.g., due to impaired cell mechanosensitivity resulting from a pathological blood environment, may not only lead to sustained neutrophil activation but also result in disturbed blood flow. In this way, aberrant neutrophil mechanotransduction may contribute to microvascular damage that exacerbates ischemia-reperfusion injury or leads to

Studies on spontaneous hypertension also reveal a key point. Factors that drive phenotypic changes in neutrophils (e.g., from an inactivated to an activated state) dramatically alter their ability to sense the surrounding flow environment (i.e., mechanosensitivity) leading to the development of pathological behavior, including immune suppression. Intuitively, cell mechanosensitivity depends on the number and activity of proteins "moonlighting" as putative mechanosensors embedded in the cell membrane positioned at the interface between the intra- and extra- cellular milieu. These studies further strengthen the argument

that the plasma membrane is a critical determinant of neutrophil mechanosensitivity.

The fact that shear stress-induced neutrophil deactivation (e.g., FPR deactivation, G protein signaling, CD18 cleavage, pseudopod retraction, etc.) occurs in the absence of any passive

peripheral vascular disease and downstream organ/tissue injury.

*5.3.2. The plasma membrane and shear stress mechanosensitivity* 

The membrane itself may act as a mechanotransducer either via stress-induced changes in its fluidity[121-123] or through lipid rafts[62, 63, 124]. However, the concept that the membrane serves as a fluid stress sensor lacks the specificity that explains the diversity of cell type-specific responses to shear. An alternative, more plausible, viewpoint is that the cell membrane serves as mechanotransduction center for the cell. Along this line, the specificity associated with mechanotransduction depends on the specific mechanoreceptor(s) expressed by the cell. In this regard, a multitude of cell transmembrane proteins including various GPCRs[58, 91, 125], tyrosine kinase receptors[126-130], ion channels[131], NO synthases, and integrin-associated focal adhesions[132, 133] have been implicated as fluid shear stress transducers for a variety of cells (e.g., endothelial cells, osteoblasts, neutrophils) and microorganisms (e.g. dino-flagella)[134].

One potential action of fluid shear stress on transmembrane mechanosensors (e.g., FPR) is to alter their surface levels. In the case of GPCRs, exposing migrating neutrophil-like cells to parallel plate flow redistributes surface-associated FPRs to a perinuclear compartment in the cytosol[135]. These results suggest that internalization of FPRs under fluid shear stimulation leads to pseudopod retraction by counteracting their constitutive activity which drives pseudopod extension. It should be noted, however, that intact FPR must be present since cleavage of FPR is linked to an impaired ability of fluid shear stress to promote retraction of neutrophil pseudopods[136]. Since receptor internalization occurs across the lipid bilayer, shear-induced changes in mechanoreceptor surface levels may thus be a mechanosensitive neutrophil response influenced by properties of the cell membrane.

It is also feasible that the ability of shear stress to alter protein tertiary structure is a function of membrane properties. In addition to evidence regarding the influence of shear stress on the conformation of FPR and CD18 integrins, fluid flow also alters the structure of other membrane-bound GPCRs in other cell types including the bradykinin B2 receptor for endothelial cells and the type I parathyroid hormone receptor for osteoblasts[58, 125]. Interestingly, physiologically relevant magnitudes of mechanical stresses are capable of physically altering the conformation of proteins[132, 133, 137]. Since these proteins are embedded in the cell membrane, it is possible that membrane properties influence flowrelated perturbations of protein structure.

In the end, the physicochemical properties (e.g., fluidity, lipid rafts) of the cell membrane, with their influence on the ability of surface mechanosensors to adopt structural shifts under shear, come to the forefront in terms of how hypercholesterolemia modifies neutrophil mechanosensitivity. This is the topic of the next section.
