*5.3.3. Membrane cholesterol versus membrane fluidity in hypercholesterolemic impairment of neutrophil mechanosensitivity*

Hypercholesterolemia is associated with chronic neutrophil activation and elevated blood cholesterol as well as cholesterol enrichment in the plasma membranes of blood cells. Based on the intimate relationship between protein dynamics (e.g., surface expression, conformational activity) and the cell membrane (as described in the previous section), the chemical and mechanical properties of the lipid bilayer may be critical determinants of the ability of neutrophils to sense fluid shear stress. Along this line, hypercholesterolemiarelated membrane perturbations may reduce the neutrophil responsiveness to shear stress by interfering with critical mechanotransduction events, e.g. GPCR and CD18 conformational activity, protease release, and/or production of ROS, that must bidirectionally transmit biological activity across the cell membrane (Figure 4).

**Figure 4. Effects of cholesterol abundance on neutrophil mechanotransduction.** Elevations in extracellular cholesterol lead to membrane cholesterol enrichment which may alter cell mechanosensitivity either by influencing shear-induced structural changes of surface sensors, or by interfering with shear-induced release of lysosomal proteases. The cell membrane may also influence contributions from ROS/RNS (not shown).

*of neutrophil mechanosensitivity* 

mechanosensitivity. This is the topic of the next section.

shear, come to the forefront in terms of how hypercholesterolemia modifies neutrophil

*5.3.3. Membrane cholesterol versus membrane fluidity in hypercholesterolemic impairment* 

Hypercholesterolemia is associated with chronic neutrophil activation and elevated blood cholesterol as well as cholesterol enrichment in the plasma membranes of blood cells. Based on the intimate relationship between protein dynamics (e.g., surface expression, conformational activity) and the cell membrane (as described in the previous section), the chemical and mechanical properties of the lipid bilayer may be critical determinants of the ability of neutrophils to sense fluid shear stress. Along this line, hypercholesterolemiarelated membrane perturbations may reduce the neutrophil responsiveness to shear stress by interfering with critical mechanotransduction events, e.g. GPCR and CD18 conformational activity, protease release, and/or production of ROS, that must

bidirectionally transmit biological activity across the cell membrane (Figure 4).

**Figure 4. Effects of cholesterol abundance on neutrophil mechanotransduction.** Elevations in extracellular cholesterol lead to membrane cholesterol enrichment which may alter cell

contributions from ROS/RNS (not shown).

mechanosensitivity either by influencing shear-induced structural changes of surface sensors, or by interfering with shear-induced release of lysosomal proteases. The cell membrane may also influence

**Figure 5. Relationship between membrane cholesterol-dependent fluidity and neutrophil shear responses.** A: Recovery effects of benzyl alcohol (BnOH; a membrane fluidizer) on the shear response by neutrophils treated with cholesterol-enhancing agents (CH). B: Does-dependent effects of cholesterol enrichment on neutrophil shear response and membrane fluidity. Cone-plate shear: 5 dyn/cm2 for 10 min. Bars are mean percentage of reductions in activated cells by shear ± SEM. \*, #p < 0.001 compared to untreated cells using Student's t-test with Bonferrroni's adjustment.

Recently, we reported that neutrophil deactivation by shear stress depends on the cholesterol-dependent physicochemical properties (i.e., fluidity) of the cell membrane[40]. Fundamentally, we showed that the deactivating actions of fluid shear require a cell membrane containing an optimal level of cholesterol. Shear stress mechanotransduction is impaired if there is too much or too little cholesterol. Moreover, the membrane must be capable of supporting the formation of lipid rafts. But, the critical evidence from this work are our observations[40] that membrane fluidizer, benzyl alcohol, was capable of counteracting the rigidifying effects of membrane cholesterol enhancement (with

cyclodextrin-cholesterol conjugates) and that the concentration of benzyl alcohol to achieve this depended on the amount of cholesterol loaded into the neutrophil membranes (Figure 5A). Thus, there is also an optimal membrane fluidity level permissive for shear-induced neutrophil deactivation. This was confirmed by regression analysis[40] which revealed a linear relationship (Figure 5B) between membrane cholesterol-related fluidity and the degree to which neutrophils within a population are inactivated by fluid flow. Membrane cholesterol enrichment therefore impairs neutrophil mechanosensitivity, at least in part, through its impact on membrane fluidity.

Interestingly, neutrophils from LDLr-/- mice fed a HFD exhibit a reduced and even reversed shear stress response relative to cells from similar mice maintained on a regular chow (i.e., normal) diet (ND)[40]. These observations were consistent with our in vitro data correlating membrane cholesterol levels with neutrophil mechanosensitivity[40]. In fact, the shear sensitivity of neutrophils from hypercholesterolemic mice tracks negatively with timedependent increases in blood levels of cholesterol, particularly of the free form (Figure 6). Presumably, the gradual loading of cholesterol into the neutrophil membrane resulting from the progressive increases in the cholesterol concentration gradient across the outer leaflet of the cell membrane is responsible for the time-dependent decrease in shear mechanosensitivity. Impairment of neutrophil shear responses by membrane cholesterol enrichment may thus underlie the pathogenesis of hypercholesterolemic disorders via an effect on cell membrane fluidity which governs the ability of protein sensors to initiate a sufficient degree of mechanotransduction at the cell surface. As such, a chronic inflammatory state may develop.

**Figure 6. Correlation between neutrophil shear responses and serum levels of free cholesterol.** A: LDLr-/- mice on normal diet (ND); B: LDLr-/- mice on high fat diet (HFD). Cone-plate shear: 5 dyn/cm2 for 10 min. Bars and square dots are mean ± SEM. \*, #p < 0.02 compared to 2-week using Student's t-test with Bonferrroni's adjustment.

#### **6. Future directions**

242 Lipoproteins – Role in Health and Diseases

through its impact on membrane fluidity.

inflammatory state may develop.

with Bonferrroni's adjustment.

cyclodextrin-cholesterol conjugates) and that the concentration of benzyl alcohol to achieve this depended on the amount of cholesterol loaded into the neutrophil membranes (Figure 5A). Thus, there is also an optimal membrane fluidity level permissive for shear-induced neutrophil deactivation. This was confirmed by regression analysis[40] which revealed a linear relationship (Figure 5B) between membrane cholesterol-related fluidity and the degree to which neutrophils within a population are inactivated by fluid flow. Membrane cholesterol enrichment therefore impairs neutrophil mechanosensitivity, at least in part,

Interestingly, neutrophils from LDLr-/- mice fed a HFD exhibit a reduced and even reversed shear stress response relative to cells from similar mice maintained on a regular chow (i.e., normal) diet (ND)[40]. These observations were consistent with our in vitro data correlating membrane cholesterol levels with neutrophil mechanosensitivity[40]. In fact, the shear sensitivity of neutrophils from hypercholesterolemic mice tracks negatively with timedependent increases in blood levels of cholesterol, particularly of the free form (Figure 6). Presumably, the gradual loading of cholesterol into the neutrophil membrane resulting from the progressive increases in the cholesterol concentration gradient across the outer leaflet of the cell membrane is responsible for the time-dependent decrease in shear mechanosensitivity. Impairment of neutrophil shear responses by membrane cholesterol enrichment may thus underlie the pathogenesis of hypercholesterolemic disorders via an effect on cell membrane fluidity which governs the ability of protein sensors to initiate a sufficient degree of mechanotransduction at the cell surface. As such, a chronic

**Figure 6. Correlation between neutrophil shear responses and serum levels of free cholesterol.** A: LDLr-/- mice on normal diet (ND); B: LDLr-/- mice on high fat diet (HFD). Cone-plate shear: 5 dyn/cm2 for 10 min. Bars and square dots are mean ± SEM. \*, #p < 0.02 compared to 2-week using Student's t-test To date, the accumulated evidence strongly points to shear stress mechanotransduction as an important negative control mechanism for neutrophils flowing in blood under noninflamed conditions and, thus, an important mediator of circulatory homeostasis. For the most part, the pathobiology of hypercholesterolemia is a process that takes decades to develop into a serious, life-threatening condition and tracks with gradual elevations in blood cholesterol levels. In addition, hypercholesterolemia is characterized by a chronic inflammatory phenotype associated with elevated levels of neutrophil activity in the blood. The question is how these two factors may be related or linked?

Based on the evidence presented in this chapter, the possibility that elevations in blood cholesterol levels impair the neutrophil-deactivating effects of fluid shear stress further suggests that vascular mechanotransduction is an important aspect of cardiovascular physiology and that the pathobiology of hypercholesterolemia may result, at least in part, from a putative disruption of this mechanotransducing function. This statement applies not only to neutrophils, but also to other cells in the circulation including the other white cells and the endothelium. Moreover, the presented evidence hints at the need to shift focus on the study of vascular mechanobiology from characterizing mechanotransduction (i.e., identifying mechanobiological signaling) in disease to actively investigating the influence of mechanosensitivity (i.e. the degree to which cells transduce fluid stresses) on vascular pathogenesis. In our case, we linked altered neutrophil mechanosensitivity with the gradual changes in blood cholesterol levels and leukocyte membranes during the development of hypercholesterolemia in LDLr-/- mice fed a fat-enriched diet. In light of our own evidence and those of others[3, 4, 8] showing that shear stress is anti-inflammatory for neutrophils, it is possible that a putative source of vascular dysfunction causal for hypercholesterolemic pathobiology is the aberrant neutrophil mechanosensitivity.

Despite recognition that vascular mechanotransduction is critical for circulatory homeostasis, there are no markers currently in use or, to our knowledge, in development that account for mechanosensitivity to predict vascular inflammatory status. Current indicators of inflammation include C-reactive protein (CRP; >3 mg/L is at cardiovascular risk) and serum amyloid protein A (SAA; >10 mg/L is at cardiovascular risk). But even though these two biochemical markers are the gold standard measures of inflammatory activity for blood[138, 139], they are upregulated when leukocyte activity levels are already elevated. It is thus not clear whether these molecules are viable "predictors" or just indicators of chronic inflammatory disorders. As such, understanding, characterizing, and formulating measures of neutrophil mechanosensitivity may prove useful in revealing earlier clues regarding the state of inflammation in blood.

In the end, the likelihood that a cholesterol-dependent loss of neutrophil sensitivity to fluid flow stimuli leads to pathological situations implicates a wide range of cardiovascular (and non-cardiovascular) diseases that correlate with both chronic inflammation and an altered cholesterol environment, e.g. hypercholesterolemia and diabetes[74, 140]. The critical issues are to increase efforts to define the link between chronic inflammation and impaired neutrophil mechanotransduction and to determine if chronic inflammation precedes or results from an impairment of vascular mechanotransduction. Further work is, therefore, needed to determine mechanistic-level connections between the cell surface, the flow sensors, the extracellular flow environment, and the influence of a hypercholesterolemic environment on these. The hope is that by fully defining the role of fluid mechanics in the physiological regulation of leukocytes, particularly the neutrophils, one may gain a better understanding of their role in the pathogenesis of cardiovascular disease.
