**3. The neutrophil involvement in hypercholesterolemia-related vascular dysfunction**

Neutrophils make up the majority of the nucleated leukocytes in human blood with the remaining being monocytes and lymphocytes. As the principal gatekeepers of the acute inflammatory response of the body's immune system, neutrophils are extremely sensitive to inflammatory stimuli allowing them to rapidly (i.e., on the order of milliseconds) transition from an inactivated to an activated state. Upon activation, the upregulation of cell-cell adhesion molecules (e.g., selectins, integrins) enables neutrophils to roll and adhere onto the endothelium prior to their transmigration (via diapedesis) across the vascular wall and to the target tissues (i.e., sites of infection and tissue injury) where they release a potent array of biochemicals including proinflammatory mediators, ROS, and proteases to fight infection and orchestrate tissue repair. During this process, neutrophils also undergo changes in their physical attributes such as their size, deformability, and adhesiveness. It is these features that cause neutrophils to be major players in the pathobiology of hypercholesterolemiarelated cardiovascular diseases for both the macro- and micro- circulations.

#### **3.1. Potential roles of neutrophils in atherosclerosis**

While it has long been appreciated that monocytes and their descendants, along with T lymphocytes, mast cells, and platelets, contribute to the development and destabilization of atherosclerotic lesions, only recently has the neutrophil been seriously considered as a contributing factor for disease onset and/or progression. Direct evidence comes from the identification of neutrophils in different locations of atherosclerotic lesions present in hypercholesterolemic mice and humans using antibodies to neutrophil-specific antigens including Ly6G, CD177, and CD66b[18-21]. Neutrophils, in fact, have been reported to accumulate in atheroprone arteries preceding plaque formation in hypercholesterolemic murine models of atherosclerosis[18, 20]. Further evidence of a neutrophilic component in the early stages of atherosclerosis is the positive correlation between the number of circulating neutrophils and lesion sizes[18]. Experimental data also point to neutrophil infiltration into the highly inflamed areas of atherosclerotic arteries during late disease stages[19] with contribution to lesion destabilization and thromboembolus formation[21, 22].

One way chronically activated neutrophils may enter atheroprone regions in the macrocirculation are at sites of disturbed flow and recirculation[23] where their enhanced residence times promote capture at the vascular wall[24]. Alternatively, activated neutrophils may disrupt vascular (i.e., adventitial or medial) wall perfusion in the vasa vasorum (or microcirculation) of large vessels (e.g., aorta) leading to vessel tissue injury followed by atherogenesis (from within the vessel wall to the luminal surface)[25-27]. In these ways, neutrophils may initiate or exacerbate atherosclerosis at different stages via their capability to release vast amounts of ROS and proteins stored in their cytosolic granules[28]. For example, while MPO released by activated neutrophils can reduce the bioavailability of NO[29, 30] and contribute to the onset of endothelial dysfunction, a number of granule proteins, such as LL-37, azurocidin, cathepsin G, and -defensins, exert direct chemotactic activity for monocytes[31, 32]. Moreover, neutrophil secretory products, e.g., -defensins, also promote macrophage maturation and activation, contributing to the uptake of oxLDL and the formation of foam cells[28]. Finally, neutrophil-derived proteolytic enzymes, particularly matrix metalloproteinase (MMP) -2 and -9[28], play critical roles in plaque destabilization and eventual rupture.

#### **3.2. Effects of neutrophils on microvascular dysfunction**

226 Lipoproteins – Role in Health and Diseases

inflammatory stimuli allowing them to rapidly (i.e., on the order of milliseconds) transition from an inactivated to an activated state. Upon activation, the upregulation of cell-cell adhesion molecules (e.g., selectins, integrins) enables neutrophils to roll and adhere onto the endothelium prior to their transmigration (via diapedesis) across the vascular wall and to the target tissues (i.e., sites of infection and tissue injury) where they release a potent array of biochemicals including proinflammatory mediators, ROS, and proteases to fight infection and orchestrate tissue repair. During this process, neutrophils also undergo changes in their physical attributes such as their size, deformability, and adhesiveness. It is these features that cause neutrophils to be major players in the pathobiology of hypercholesterolemia-

While it has long been appreciated that monocytes and their descendants, along with T lymphocytes, mast cells, and platelets, contribute to the development and destabilization of atherosclerotic lesions, only recently has the neutrophil been seriously considered as a contributing factor for disease onset and/or progression. Direct evidence comes from the identification of neutrophils in different locations of atherosclerotic lesions present in hypercholesterolemic mice and humans using antibodies to neutrophil-specific antigens including Ly6G, CD177, and CD66b[18-21]. Neutrophils, in fact, have been reported to accumulate in atheroprone arteries preceding plaque formation in hypercholesterolemic murine models of atherosclerosis[18, 20]. Further evidence of a neutrophilic component in the early stages of atherosclerosis is the positive correlation between the number of circulating neutrophils and lesion sizes[18]. Experimental data also point to neutrophil infiltration into the highly inflamed areas of atherosclerotic arteries during late disease stages[19] with

One way chronically activated neutrophils may enter atheroprone regions in the macrocirculation are at sites of disturbed flow and recirculation[23] where their enhanced residence times promote capture at the vascular wall[24]. Alternatively, activated neutrophils may disrupt vascular (i.e., adventitial or medial) wall perfusion in the vasa vasorum (or microcirculation) of large vessels (e.g., aorta) leading to vessel tissue injury followed by atherogenesis (from within the vessel wall to the luminal surface)[25-27]. In these ways, neutrophils may initiate or exacerbate atherosclerosis at different stages via their capability to release vast amounts of ROS and proteins stored in their cytosolic granules[28]. For example, while MPO released by activated neutrophils can reduce the bioavailability of NO[29, 30] and contribute to the onset of endothelial dysfunction, a number of granule proteins, such as LL-37, azurocidin, cathepsin G, and -defensins, exert direct chemotactic activity for monocytes[31, 32]. Moreover, neutrophil secretory products, e.g., -defensins, also promote macrophage maturation and activation, contributing to the uptake of oxLDL and the formation of foam cells[28]. Finally, neutrophil-derived proteolytic enzymes, particularly matrix metalloproteinase (MMP) -2 and -9[28], play critical roles in

related cardiovascular diseases for both the macro- and micro- circulations.

contribution to lesion destabilization and thromboembolus formation[21, 22].

plaque destabilization and eventual rupture.

**3.1. Potential roles of neutrophils in atherosclerosis** 

Similar to situations in large arteries, activated neutrophils promote microvascular dysfunction through the sustained release of proinflammatory, cytotoxic, and degradative agents. However, while leukocytes have no significant effect on the macrovascular flow properties of blood (which is dominated by the substantially greater numbers of erythrocytes), these cells, particularly the neutrophils, influence blood flow in the microcirculation (Figure 1). This is because vessel diameters in the microcirculation are in the range of 6 – 100 m, which are comparable in size to the diameters of leukocytes.

**Figure 1. Potential rheological effects of leukocyte activation on the blood flow in the microcirculation.** Sustained activation, e.g., due to proinflammatory stimuli, hinders leukocyte passage through the small vessels either by promoting pseudopod projections or through enabling cell adhesion to the vascular wall. Ultimately, these may elevate peripheral resistance and contribute to microvascular dysfunctions (adapted from Shin, H.Y., et al., 2011[79]).

The relatively comparable size scales of the neutrophil and vessel diameters are important to note since the quiescent neutrophil under physiological (i.e., non-inflamed) conditions is capable of efficient transit through the microvessels due to their inherently round, deformable, and non-adhesive state. On the other hand, cell activation physically hinders the passage of neutrophils through the small vessels of the microcirculation[1]. Pseudopods projected by activated neutrophils, while enabling cells to attach to other cells (e.g., endothelial cells, other blood cells) or phagocytose particles, also contribute to a reduction in cell deformability due to their enriched content of F-actin, and increases in geometric size and irregularity[1, 33], all of which serve to increase leukocyte transit time or enhance leukocyte retention in the microvasculature[34, 35]. In turn, activated leukocytes disrupt the motion of erythrocytes, leading to increases in the apparent viscosity of blood and microvascular resistance[1, 34, 36, 37]. Moreover, activated neutrophils are hyperadhesive and exhibit extensive interactions with other leukocytes or platelets, e.g. during hypercholesterolemia[7], which may also enhance the apparent viscosity of blood. Finally, once neutrophils adhere to endothelium, they further increase flow resistance by reducing microvessel diameters (resistance ∝ 1/[diameter]4)[34, 37]. The state of neutrophil activation is, thus, a critical determinant of tissue blood flow and perfusion.

In summary, as a result of their arsenal of noxious agents and their effects on microvascular blood flow, it is evident that tight regulation of neutrophil activity is an essential requirement for a healthy circulation. A failure to either prevent or "turn-off" cell activity, e.g., due to hypercholesterolemia, leads to sustained neutrophil activation which has potential impacts not only in terms of the initiation and progression of atherosclerosis in large arteries but also as it relates to microvascular blood flow and downstream tissue perfusion.
