**2. Autophagic regulation and dysfunction in atherosclerosis**

and macrophages [1]. Accumulated lipoproteins bound to infiltrated macrophages to form a fibrous cap composed mostly of collagen in vascular smooth muscle cells, and this develops into an atherosclerotic plaque [2]. The developed plaque can cause stenosis, which can lead to ischemic conditions in surrounding tissues. When a plaque ruptures, in the worstcase scenario, thrombogenic materials are exposed, platelets are aggregated, and they form a thrombus. The detached thrombus becomes an embolus capable of blocking blood flow. Thus, atherothrombosis has the capability to cause ischemic stroke and myocardial

Lipoproteins transport cholesterol through the blood. Low-density lipoprotein (LDL) consists of esterified cholesterol and triglycerides that contain phospholipids, free cholesterol, and apolipoprotein B100 (ApoB100) [3]. Cellular LDL uptake is well-regulated. Their feedback mechanisms systemically limit excessive uptake and lipid overload in cells [4]. In contrast, oxidized LDL (oxLDL) mostly bypasses the feedback system, and results in intracellular lipid accumulation as foam cells present in atherosclerotic plaques [5]. Generally, the scavenger receptor-mediated uptake system misses the modified lipoproteins such as oxidized forms of LDL. During oxidation, physico-chemical properties such as lipid charge, size, and content are changed. Because oxidation modifies the LDL particles, oxLDL particles have already undergone physico-chemical changes. The oxLDL is technically different from natural LDL. The components of oxLDL can activate endothelial cells, and also induce the expression of adhesion molecules (E-selectin and VCAM-1) on the endothelial surface of the artery [2]. With endothelial activation by these oxidized lipids, oxLDL help macrophages to infiltrate into tissues and to produce chemokines as well

The role of macrophages is critical in the development of atherosclerosis. Macrophages infiltrate to the arterial intima in response to oxLDL in the vessel. Macrophages engulf various lipids containing oxLDL and show a changed phenotype in comparison to lipid-laden foam cells. Spontaneously, they progress to a pro-inflammatory state. This is an early event in forming atherosclerotic lesion plaques. Macrophages secrete pro-inflammatory cytokines and recruit additional macrophages into the artery, and continuously increase atherosclerotic plaque size and complexity [7]. The early events of atherosclerosis induce additional immune cell infiltration and a progressive dysfunction to initiate a cell death pathway [5]. When atherosclerotic lesions develop, apoptotic as well as necrotic cell death occur. Cell debris and cholesterol form a necrotic core in the lesion covered by a fibrous cap of variable thickness [8]. Atherosclerosis forms under chronic exposure to cellular stressors, which promotes accumulated lipid degrading cascades and consequently dysfunction. It has been revealed that macrophage autophagy is linked to lipid metabolism [7]. In atherosclerotic plaques, there is intracellular accumulation of LDL as well as damaged tissue and misfolded/aggregated proteins. Biologically, these extra-accumulating materials are dealt via autophagy. Through the use of adapter proteins, the cells undergo autophagy. The process involves selective events rather than random bulk cleavage [9]. The selective autophagy can be described as: mitophagy, handling mitochondria; pexophagy, charging on peroxisomes; lipophagy, dealing with lipids; aggrephagy, taking care of aggregated proteins; and xenophagy, treating microorganisms. Among them, lipophagy is

infarction.

96 Atherosclerosis - Yesterday, Today and Tomorrow

as adhesion molecules [6].

Autophagy literally means "to eat oneself" and originated in Greek. It is an evolutionary conserved mechanism, that is, a catabolic process to degrade cytoplasmic contents such as cellular proteins and organelles through lysosomes for recycling and use in downstream metabolism [13]. Biomolecules degrade and generate free fatty acids, amino acids, and nucleotides, which can be reused by the cell to maintain energy production and protein synthesis [13]. Degradation of intracellular molecules occurs through two distinct systems: the ubiquitinproteasome system and the lysosome-autophagy system [14, 15]. In mammals, autophagy is the major pathway used to degrade abnormal products besides the ubiquitin-proteasome system. Autophagy is primarily used for the removal of damaged organelles, abnormal proteins, and protein aggregates [16], and this housekeeping function is particularly essential in the heart and brain. When autophagy-specific genes are lacking, a severe cardiomyopathy or neurodegeneration occurs [17].

There are several types of autophagy according to the method of delivery of the cargo to lysosomes: macroautophagy, microautophagy, and chaperone-mediated autophagy (CMA) [18]. Macroautophagy is the predominant mechanism among these three types. Macroautophagy starts with the formation of double-membrane vesicles, autophagosomes. Autophagosomes fuse with lysosomes and finally progress to autolysosomes. Physiological stress conditions such as starvation upregulates autophagy. Identified genes and molecules involved include around 30 genes and they are called autophagy-related genes (ATGs) required for autophagic pathways [19]. Among them, ATG5 and ATG12 are involved in the first step, controlling autophagy with two ubiquitylation-like reactions. ATG12 links to ATG5 requiring ATG7 (serves as an ubiquitin-activating enzyme, E1) and ATGT10 (serves as an ubiquitin-activating enzyme, E2). Then, the ATG5-ATG12 complex is involved in autophagosome formation. Autophagosomes randomly formed in the cytoplasm are trafficked along microtubules to lysosomes in a way that is dynein-dependent. Autophagosomes, then, are fused with lysosomes. The SNARE proteins of yeast are thought to be involved in this fusion [20, 21].

Microautophagy raises the possibility of direct cytoplasmic engulfment by the lysosome in mammals or the vacuole in plant and fungi [22]. In macroautophagy, a double- or multi-membrane-surrounded autophagosome forms, which fuse with lysosomes in a nonspecific way for degradation [22]. In contrast to macroautophagy, in microautophagy, the lysosomal/vacuolar membrane is randomly engulfed and differentiates into the autophagic tube enclosing the cytosolic portion [23]. Microautophagy starts with making membrane knobs into the surface of the lysosome, and constructs small smooth areas that are able to degrade. The invaginations move laterally and also can shrink, which are specified into particular tubular shape "autophagic tubes" [23]. This characteristic is unique and gives them an autophagic function. After that, a dramatic decrease occurs along with the autophagic tube intramembranous proteins toward the top of the tube. Collectively, microautophagy performs degradation of cargo lipids and proteins in the following order: vesicle formation, vesicle expansion, vesicle scission, and eventual vesicle degradation and recycling [22].

tail in LAMP-2A. When they are exposed on the surface of the lysosomes, substrate proteins

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The general architecture and cellular composition of blood vessels have basic components in the wall of blood vessels: endothelial cells (EC), vascular smooth muscle cells (VSMC), and extracellular matrix (ECM), including elastin, collagen, and glycosoaminoglycans. Each vessel is composed of the three concentric layers intima, media, and adventitia [31]. In normal mature blood vessels, VSMC predominantly exist in a contractile or differentiated phenotype that regulates blood flow and blood vessel diameter with vasodilation and vasoconstriction. The contractile VSMCs are surrounded by their own basement membrane, some macrophages, and fibroblasts. When damaged, VSMC generates intimal vascular lesions. The VSMC layers of the nearest vessel lumen receive oxygen as well as nutrients by direct diffu-

VSMC in atherosclerosis consists of aberrantly proliferated VSMC to promote plaque formation. However, VSMC in advanced plaques is involved in preventing rupture of the fibrous cap [32]. The tensile strength of the protective cap relies on structural properties that are determined by the number of VSMCs and the collagen [33]. This is the reason why loss of VSMC leads to plaque destabilization and rupture. In advanced plaques, disintegrating VSMCs in the fibrous cap undergo programmed cell death, which is not apoptosis but autophagy. This has been shown in electron-microscopy imaging as formation of myelin figures [34], accumulation of ubiquitinated inclusions in the cytosol [13], and severe vacuolization [34]. In this

VSMCs loading a lot of cholesterol activate multiple pro-inflammatory genes and are altered to form macrophage-like cells driven by lipid accumulation in the plaque, and are then induced to perform phagocytic activity [32]. In the fibrous cap of advanced human plaques, VSMCs die showing ubiquitinated inclusions indicating they are undergoing autophagic death [13, 19]. Actually, the fibrous cap is a thick layer of basal lamina. It may be easier to let these "caged" cells undergo autophagy. Also, it has been suggested in *in vitro* studies that caged cells can trigger autophagy in atherosclerotic plaques. It has been reported in human plaques that lipid-laden VSMCs increase the expression of deathassociated protein (DAP) kinase, a pro-apoptotic mediator, and regulate the formation of

Generally, autophagy is well-recognized as a survival mechanism under starvation and not as a death pathway [36]. In atherosclerotic plaques, autophagy most likely plays a safeguarding role for plaque cells against oxidative stress, a hallmark of advanced atherosclerotic lesions. The successful autophagy in the atherosclerotic plaque is anti-apoptotic and eventually contributes to cellular recovery. However, it becomes another story under acute or persistent oxidative stress. In this case, over-produced intracellular ROS can be harmful to the lysosomal membrane. When autophagy does not work in the oxidative stress response in atherosclerotic plaques, or when

oxidative injury overwhelms the cellular defenses, cells might undergo apoptosis.

bind to them [25].

sion from the vessel lumen.

autophagic vesicles [35].

**2.1. Autophagy in VSMC of atherosclerotic plaques**

stage, macrophages actively induce SMC death [13].

Another case of autophagy is chaperone-mediated autophagy (CMA). Cargo recognition in macroautophagy has a non-selective process, because soluble cytosolic proteins cannot be selected as single protein molecules and are targeted for degradation through this pathway [24]. CMA targets only single proteins. In CMA, proteins are identified one by one, and the identified proteins are degraded by using a cytosolic chaperone system that delivers them to the surface of the lysosomes [25]. Selectivity in CMA uses a pentapeptide amino acid sequence motif in the substrate proteins. When the substrate proteins are recognized by a cytosolic chaperone, it results in targeting substrates to lysosomes [26]. CMA proceeds in sequential multi-steps: (i) recognition of substrate proteins; (ii) binding and unfolding of substrates; (iii) translocation of substrates inside the lysosomes; and (iv) degradation of substrates in the lysosomal lumen through its cellular functions [27].

CMA-targeting motifs are generated through posttranslational modifications with KFERQ on the targets. This pentapeptide was first reported to be critical in the degradation of RNase A [28], and it is shared by all identified substrate proteins to date [24]. The proteins carrying the KFERQ motif are targeted by a constitutive chaperone, the heat shock-cognate protein of 70 kDa (Hsc70). Hsc70 is the only chaperone to interact with the substrate via regulated ATP/ADP binding cycles [29]. The chaperone Hsc70 combines the proteins with a KFERQ motif, and binds with the cytosolic tail of the single-span membrane protein lysosome-associated membrane protein type 2A (LAMP-2A19, LAMP-2A), which shuttles the chaperone complex and the targeted protein into the lysosomal lumen [29]. Hsc70 also interacts with protein aggregates, and has mediated their degradation by macroautophagy, which is called a chaperone-assisted selective autophagy (CASA) [30]. This reaction works the same way on a responsible disassembly of clathrin from coated vesicles, and is needed to fold the unfolded cytosolic proteins upon recognition of exposed hydrophobic regions [28]. Once the substrate is translocated into the lysosomal lumen, LAMP-2A is rapidly dissembled from the complex into monomers, which endows LAMP-2A to bind with other substrates again [27]. LAMP-2A is one of the three splice variants of the *lamp2* gene [9], and is a single-span membrane protein. There is a very heavily glycosylated luminal region and a short (12-amino acid) C-terminus tail in LAMP-2A. When they are exposed on the surface of the lysosomes, substrate proteins bind to them [25].

#### **2.1. Autophagy in VSMC of atherosclerotic plaques**

Microautophagy raises the possibility of direct cytoplasmic engulfment by the lysosome in mammals or the vacuole in plant and fungi [22]. In macroautophagy, a double- or multi-membrane-surrounded autophagosome forms, which fuse with lysosomes in a nonspecific way for degradation [22]. In contrast to macroautophagy, in microautophagy, the lysosomal/vacuolar membrane is randomly engulfed and differentiates into the autophagic tube enclosing the cytosolic portion [23]. Microautophagy starts with making membrane knobs into the surface of the lysosome, and constructs small smooth areas that are able to degrade. The invaginations move laterally and also can shrink, which are specified into particular tubular shape "autophagic tubes" [23]. This characteristic is unique and gives them an autophagic function. After that, a dramatic decrease occurs along with the autophagic tube intramembranous proteins toward the top of the tube. Collectively, microautophagy performs degradation of cargo lipids and proteins in the following order: vesicle formation, vesicle expansion, vesicle scission, and eventual vesicle degradation and

Another case of autophagy is chaperone-mediated autophagy (CMA). Cargo recognition in macroautophagy has a non-selective process, because soluble cytosolic proteins cannot be selected as single protein molecules and are targeted for degradation through this pathway [24]. CMA targets only single proteins. In CMA, proteins are identified one by one, and the identified proteins are degraded by using a cytosolic chaperone system that delivers them to the surface of the lysosomes [25]. Selectivity in CMA uses a pentapeptide amino acid sequence motif in the substrate proteins. When the substrate proteins are recognized by a cytosolic chaperone, it results in targeting substrates to lysosomes [26]. CMA proceeds in sequential multi-steps: (i) recognition of substrate proteins; (ii) binding and unfolding of substrates; (iii) translocation of substrates inside the lysosomes; and (iv) degradation of substrates in the

CMA-targeting motifs are generated through posttranslational modifications with KFERQ on the targets. This pentapeptide was first reported to be critical in the degradation of RNase A [28], and it is shared by all identified substrate proteins to date [24]. The proteins carrying the KFERQ motif are targeted by a constitutive chaperone, the heat shock-cognate protein of 70 kDa (Hsc70). Hsc70 is the only chaperone to interact with the substrate via regulated ATP/ADP binding cycles [29]. The chaperone Hsc70 combines the proteins with a KFERQ motif, and binds with the cytosolic tail of the single-span membrane protein lysosome-associated membrane protein type 2A (LAMP-2A19, LAMP-2A), which shuttles the chaperone complex and the targeted protein into the lysosomal lumen [29]. Hsc70 also interacts with protein aggregates, and has mediated their degradation by macroautophagy, which is called a chaperone-assisted selective autophagy (CASA) [30]. This reaction works the same way on a responsible disassembly of clathrin from coated vesicles, and is needed to fold the unfolded cytosolic proteins upon recognition of exposed hydrophobic regions [28]. Once the substrate is translocated into the lysosomal lumen, LAMP-2A is rapidly dissembled from the complex into monomers, which endows LAMP-2A to bind with other substrates again [27]. LAMP-2A is one of the three splice variants of the *lamp2* gene [9], and is a single-span membrane protein. There is a very heavily glycosylated luminal region and a short (12-amino acid) C-terminus

recycling [22].

98 Atherosclerosis - Yesterday, Today and Tomorrow

lysosomal lumen through its cellular functions [27].

The general architecture and cellular composition of blood vessels have basic components in the wall of blood vessels: endothelial cells (EC), vascular smooth muscle cells (VSMC), and extracellular matrix (ECM), including elastin, collagen, and glycosoaminoglycans. Each vessel is composed of the three concentric layers intima, media, and adventitia [31]. In normal mature blood vessels, VSMC predominantly exist in a contractile or differentiated phenotype that regulates blood flow and blood vessel diameter with vasodilation and vasoconstriction. The contractile VSMCs are surrounded by their own basement membrane, some macrophages, and fibroblasts. When damaged, VSMC generates intimal vascular lesions. The VSMC layers of the nearest vessel lumen receive oxygen as well as nutrients by direct diffusion from the vessel lumen.

VSMC in atherosclerosis consists of aberrantly proliferated VSMC to promote plaque formation. However, VSMC in advanced plaques is involved in preventing rupture of the fibrous cap [32]. The tensile strength of the protective cap relies on structural properties that are determined by the number of VSMCs and the collagen [33]. This is the reason why loss of VSMC leads to plaque destabilization and rupture. In advanced plaques, disintegrating VSMCs in the fibrous cap undergo programmed cell death, which is not apoptosis but autophagy. This has been shown in electron-microscopy imaging as formation of myelin figures [34], accumulation of ubiquitinated inclusions in the cytosol [13], and severe vacuolization [34]. In this stage, macrophages actively induce SMC death [13].

VSMCs loading a lot of cholesterol activate multiple pro-inflammatory genes and are altered to form macrophage-like cells driven by lipid accumulation in the plaque, and are then induced to perform phagocytic activity [32]. In the fibrous cap of advanced human plaques, VSMCs die showing ubiquitinated inclusions indicating they are undergoing autophagic death [13, 19]. Actually, the fibrous cap is a thick layer of basal lamina. It may be easier to let these "caged" cells undergo autophagy. Also, it has been suggested in *in vitro* studies that caged cells can trigger autophagy in atherosclerotic plaques. It has been reported in human plaques that lipid-laden VSMCs increase the expression of deathassociated protein (DAP) kinase, a pro-apoptotic mediator, and regulate the formation of autophagic vesicles [35].

Generally, autophagy is well-recognized as a survival mechanism under starvation and not as a death pathway [36]. In atherosclerotic plaques, autophagy most likely plays a safeguarding role for plaque cells against oxidative stress, a hallmark of advanced atherosclerotic lesions. The successful autophagy in the atherosclerotic plaque is anti-apoptotic and eventually contributes to cellular recovery. However, it becomes another story under acute or persistent oxidative stress. In this case, over-produced intracellular ROS can be harmful to the lysosomal membrane. When autophagy does not work in the oxidative stress response in atherosclerotic plaques, or when oxidative injury overwhelms the cellular defenses, cells might undergo apoptosis.

#### **2.2. Autophagy in macrophages of atherosclerotic plaques**

Macrophages are immune cells having a strong phagocytic potential. They migrate into tissues derived from the differentiation of monocyte precursors in blood [7]. They are primarily involved in the phagocytosis against extracellular pathogens. They are also responsible for treating cellular debris, antigen presentation, and activation of the adaptive immune system. Macrophages secrete either pro- or anti-inflammatory cytokines according to their activation state [8]. Monocytes are recruited to the vessel intima, and they are initiated by chemokines secreted from endothelial cells, which are activated by excess lipoprotein accumulation [21]. These events show a profound effect on the reduction of atherosclerotic plaque burden through a lower number of circulating monocytes or to prevent their interactions with the endothelium via chemokine/chemokine receptor blockage [37].

efflux, suppressing inflammasome activation, and improving apoptosis in atherosclerotic

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Endothelial cells are arranged in many layers in large blood vessels in which they form a tough wall as connective tissue. The endothelial cells in mature vessels send signals to the surrounding connective tissue, and take on an important part in regulating the vessel's function and structure [40]. For regulating roles, the endothelial cells mediate fluid filtration, hormone trafficking, neutrophil recruitment, and finally maintaining hemostasis [41]. Endothelial cell dysfunction (ECD) in the artery is the first detectable change of a forming atherosclerotic lesion [42]. The changes in the sub-endothelial area contain: the focal permeation and trapping, and the physiological and chemical modification of circulating lipoprotein particles [43]. The term endothelial dysfunction has already entered the lexicon of modern cardiovascular medicine [40]. However, the concept has not been developed to our present understanding of the cellular and molecular mechanisms of atherosclerosis. Under atherogenesis, the earlier characterization of endothelial dysfunction was focused on whether anatomical integrity of the intima was intact. The simplest definition of endothelial dysfunction is a lack of nitro oxide (NO), which is involved in various disease states: atherosclerosis, diabetes mellitus, coronary artery disease, hypertension, and hypercholesterolemia [41]. Endothelium-derived NO can modulate leukocyte adhesion. Endothelium-derived NO prevents leukocyte recruitment to the vascular wall via the anti-inflammatory effects of NO. Endothelium-derived NO suppresses the expression level of VCAM-1, ICAM-1, and E-selectin, which respond to proinflammatory cytokines. The cellular adhesion molecules mediate activation of the transcription factor NF-κB, and NF-κB inhibited by endothelial NO prevents endothelial cell activation [44]. It has been found that inhibition of basal eNOS activity rapidly induces VCAM-1 and also increases monocyte adhesion [45]. This linkage could induce or enhance endothelial cell activation. The mediators of endothelial dysfunction such as hypercholesterolemia or oxidative stress can lead to increasing vasoconstriction, smooth muscle proliferation, platelet aggregation, leukocyte adhesion, LDL oxidation, and MMP activation. In the vessel wall when there is turbulent flow, endothelial cell activation and atherosclerosis may occur more readily because there is less endothelium-derived NO. With support, atherosclerotic lesions develop more frequently at vascular branching sites when exposed to turbulent flow rather than laminar flow. In animal studies, eNOS deleted mice develop increased atherosclerosis

In contrast to endothelial dysfunction, endothelial cell activation is defined by the endothelial cell surface adhesion molecules, such as VCAM-1, ICAM-1, and endothelial leukocyte adhesion molecule (E-selectin) [41]. Endothelial cell activation is typically induced by pro-inflammatory cytokines, such as TNF-α and IL-6. When endothelial cells are activated, they facilitate the recruitment and attachment of circulating leukocytes to the vessel wall. Progressive structural remodeling of developing lesions starts with the formation of a fibrous cap. The lateral edges of these complicated atherosclerotic plaques contain a rich population of inflammatory cells such as activated macrophages, T-cells, natural killer T-cells (NK T-cell), and dendritic

macrophages.

**2.3. Autophagy in vascular endothelial cells**

and vascular inflammation [46].

In atherosclerotic plaques, macrophages contribute to cytokine production, the maintenance of vessel wall inflammation, and finally atherosclerotic progression [38]. Inflammatory signaling is a general and major event in atherosclerosis. Several other pathways, besides inflammation signals, are triggered by macrophages involved in wreaking havoc on the plaque [7]. They are over-expressed reactive oxygen intermediates containing myeloperoxidase-induced reactive nitrogen species. Under oxidative stress, secreted cathepsins and matrix metalloproteinases (MMPs) worsen the sub-endothelial environment. This toxic environment results in a vicious cycle of lipoprotein oxidation, enhanced lipoprotein uptake, and increased inflammatory signaling [7]. When the membrane cholesterol content of macrophages exceeds their handling capacity, a lipid droplet is formed. Cells with lipid droplets are defined as foam cells in the atherosclerotic lesion. A primary response to such lipid overload is the efflux of excess cholesterol out of macrophages with the help of high-density lipoproteins (HDLs). This process occurs in the cytoplasm where a family of cholesteryl ester hydrolases releases cholesterol from macrophage lipid droplets. This is followed by ATP-binding cassette transporter ABCA1 (ABCA-1), known as the cholesterol efflux regulatory protein (CERP). Finally, exogenously derived cholesteryl esters are hydrolyzed in lysosomes [7]. After that, the free cholesterols are distributed to different cellular membrane compartments. In addition to cholesterol, the focal lipid substrate and other lipid species may affect macrophage lysosomal function [7].

Under physiological states in contrast to pathological states, most cells turn on compensatory mechanisms for handling such insults. Autophagy is one of the responses to toxic intermediates found in the atherosclerotic plaque, and autophagic processes concomitantly increase in macrophages [14]. Lipophagy was first discovered in the liver where a specific mechanism handles lipids [39]. It has also been evaluated in foam cell macrophages. Autophagic uptake of lipid droplets is subsequently subjected to lysosomal acid lipase (LAL)-dependent degradation of cholesteryl esters in lysosomes. This is also an alternative mechanism of generating free cholesterol for ABCA1-mediated efflux to HDL [14]. It can be concluded that autophagy deficiency in macrophages increases macrophages' susceptibility to foam cell formation. It is undoubtedly true that macrophage autophagy has an essential role in the atherosclerotic process. In mice lacking *Atg*5, atherosclerotic plaques are enlarged and overloaded with lipids, there is extensive pro-inflammation, and the atherosclerotic core is filled with apoptotic and necrotic cells [7]. Recently, autophagy has been implicated in regulating cholesterol efflux, suppressing inflammasome activation, and improving apoptosis in atherosclerotic macrophages.

#### **2.3. Autophagy in vascular endothelial cells**

**2.2. Autophagy in macrophages of atherosclerotic plaques**

100 Atherosclerosis - Yesterday, Today and Tomorrow

endothelium via chemokine/chemokine receptor blockage [37].

Macrophages are immune cells having a strong phagocytic potential. They migrate into tissues derived from the differentiation of monocyte precursors in blood [7]. They are primarily involved in the phagocytosis against extracellular pathogens. They are also responsible for treating cellular debris, antigen presentation, and activation of the adaptive immune system. Macrophages secrete either pro- or anti-inflammatory cytokines according to their activation state [8]. Monocytes are recruited to the vessel intima, and they are initiated by chemokines secreted from endothelial cells, which are activated by excess lipoprotein accumulation [21]. These events show a profound effect on the reduction of atherosclerotic plaque burden through a lower number of circulating monocytes or to prevent their interactions with the

In atherosclerotic plaques, macrophages contribute to cytokine production, the maintenance of vessel wall inflammation, and finally atherosclerotic progression [38]. Inflammatory signaling is a general and major event in atherosclerosis. Several other pathways, besides inflammation signals, are triggered by macrophages involved in wreaking havoc on the plaque [7]. They are over-expressed reactive oxygen intermediates containing myeloperoxidase-induced reactive nitrogen species. Under oxidative stress, secreted cathepsins and matrix metalloproteinases (MMPs) worsen the sub-endothelial environment. This toxic environment results in a vicious cycle of lipoprotein oxidation, enhanced lipoprotein uptake, and increased inflammatory signaling [7]. When the membrane cholesterol content of macrophages exceeds their handling capacity, a lipid droplet is formed. Cells with lipid droplets are defined as foam cells in the atherosclerotic lesion. A primary response to such lipid overload is the efflux of excess cholesterol out of macrophages with the help of high-density lipoproteins (HDLs). This process occurs in the cytoplasm where a family of cholesteryl ester hydrolases releases cholesterol from macrophage lipid droplets. This is followed by ATP-binding cassette transporter ABCA1 (ABCA-1), known as the cholesterol efflux regulatory protein (CERP). Finally, exogenously derived cholesteryl esters are hydrolyzed in lysosomes [7]. After that, the free cholesterols are distributed to different cellular membrane compartments. In addition to cholesterol, the focal lipid substrate and other lipid species may affect macrophage lysosomal function [7]. Under physiological states in contrast to pathological states, most cells turn on compensatory mechanisms for handling such insults. Autophagy is one of the responses to toxic intermediates found in the atherosclerotic plaque, and autophagic processes concomitantly increase in macrophages [14]. Lipophagy was first discovered in the liver where a specific mechanism handles lipids [39]. It has also been evaluated in foam cell macrophages. Autophagic uptake of lipid droplets is subsequently subjected to lysosomal acid lipase (LAL)-dependent degradation of cholesteryl esters in lysosomes. This is also an alternative mechanism of generating free cholesterol for ABCA1-mediated efflux to HDL [14]. It can be concluded that autophagy deficiency in macrophages increases macrophages' susceptibility to foam cell formation. It is undoubtedly true that macrophage autophagy has an essential role in the atherosclerotic process. In mice lacking *Atg*5, atherosclerotic plaques are enlarged and overloaded with lipids, there is extensive pro-inflammation, and the atherosclerotic core is filled with apoptotic and necrotic cells [7]. Recently, autophagy has been implicated in regulating cholesterol Endothelial cells are arranged in many layers in large blood vessels in which they form a tough wall as connective tissue. The endothelial cells in mature vessels send signals to the surrounding connective tissue, and take on an important part in regulating the vessel's function and structure [40]. For regulating roles, the endothelial cells mediate fluid filtration, hormone trafficking, neutrophil recruitment, and finally maintaining hemostasis [41]. Endothelial cell dysfunction (ECD) in the artery is the first detectable change of a forming atherosclerotic lesion [42]. The changes in the sub-endothelial area contain: the focal permeation and trapping, and the physiological and chemical modification of circulating lipoprotein particles [43].

The term endothelial dysfunction has already entered the lexicon of modern cardiovascular medicine [40]. However, the concept has not been developed to our present understanding of the cellular and molecular mechanisms of atherosclerosis. Under atherogenesis, the earlier characterization of endothelial dysfunction was focused on whether anatomical integrity of the intima was intact. The simplest definition of endothelial dysfunction is a lack of nitro oxide (NO), which is involved in various disease states: atherosclerosis, diabetes mellitus, coronary artery disease, hypertension, and hypercholesterolemia [41]. Endothelium-derived NO can modulate leukocyte adhesion. Endothelium-derived NO prevents leukocyte recruitment to the vascular wall via the anti-inflammatory effects of NO. Endothelium-derived NO suppresses the expression level of VCAM-1, ICAM-1, and E-selectin, which respond to proinflammatory cytokines. The cellular adhesion molecules mediate activation of the transcription factor NF-κB, and NF-κB inhibited by endothelial NO prevents endothelial cell activation [44]. It has been found that inhibition of basal eNOS activity rapidly induces VCAM-1 and also increases monocyte adhesion [45]. This linkage could induce or enhance endothelial cell activation. The mediators of endothelial dysfunction such as hypercholesterolemia or oxidative stress can lead to increasing vasoconstriction, smooth muscle proliferation, platelet aggregation, leukocyte adhesion, LDL oxidation, and MMP activation. In the vessel wall when there is turbulent flow, endothelial cell activation and atherosclerosis may occur more readily because there is less endothelium-derived NO. With support, atherosclerotic lesions develop more frequently at vascular branching sites when exposed to turbulent flow rather than laminar flow. In animal studies, eNOS deleted mice develop increased atherosclerosis and vascular inflammation [46].

In contrast to endothelial dysfunction, endothelial cell activation is defined by the endothelial cell surface adhesion molecules, such as VCAM-1, ICAM-1, and endothelial leukocyte adhesion molecule (E-selectin) [41]. Endothelial cell activation is typically induced by pro-inflammatory cytokines, such as TNF-α and IL-6. When endothelial cells are activated, they facilitate the recruitment and attachment of circulating leukocytes to the vessel wall. Progressive structural remodeling of developing lesions starts with the formation of a fibrous cap. The lateral edges of these complicated atherosclerotic plaques contain a rich population of inflammatory cells such as activated macrophages, T-cells, natural killer T-cells (NK T-cell), and dendritic cells. These inflammatory cells further modulate the endothelial cells into a pro-inflammatory phenotype, and have the endothelial cells work on structural instability of the plaque by modifying the proteolytic activity of extracellular matrix components [47].

collagen synthesis in reduced and also the fibrous plaque cap gets thinning. Of course, autophagic cell death is triggered in endothelial cells, which is detrimental role in the sustaining structure of the atherosclerotic plaque. It is an acute clinical event promoting thrombosis

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Lipid modification such as LDL oxidation brings about a range of modifications with various physiological and biochemical properties [8]. Modified lipids in macrophage cells are able to induce lysosomal dysfunction which can result in the accumulation of intra-lysosomal cholesteryl esters [56]. A number studies have shown that uptake of modified lipids induces a lysosomal lipid storage disease-like condition [5]. Accumulated lipids in lysosomes cause lysosomal dysfunction and affects the intracellular transport machinery. When macrophages are exposed to oxLDL and cholesterol, so-called atherogenic or modified lipids, lysosomal dysfunction occurs [16]. The oxLDL-derived cholesteryl esters form cholesterol crystal when oxLDLderived cholesteryl esters are inefficiently hydrolyzed and transported in lysosomes [57]. Through CD36-dependent mechanisms, oxLDL is moved to macrophage lysosomes; cholesterol crystals accumulate in the lysosomes. Cholesterol crystals beyond the dealing range initiate lysosomal damage and result in leaking lysosomes [57]. As an example, phagocytosis of apoptotic cells (efferocytosis) is detected in plaque progression and is regarded as a critical feature of increasing plaque complexity [5]. PRPs, cell surface receptors and also scavenger receptors, recognize modified lipids (oxLDL) and pathogens. Plasma levels of soluble CD36, one of scavenger receptors, are higher in the context of risk factors for the development of atherosclerosis such as diabetes [58]. The altered "eat-me" signals can also affect efferocytosis and the targets of apoptotic cells. For example, mice lacking complement factor C1q exhibited efferocytosis dysfunction and atherosclerotic plaque burden [59]. In human atherosclerotic plaques, efferocytosis is impaired and also shaded phagocytic receptors, which impedes phagocytic capacity of macrophages and involves activation of the inflammatory response [60]. The LDLR-related protein 1 (LRP1) is one of the important receptors interacting with

Prolonged oxidative damage induces protein misfolding and the accumulation of dysfunctional proteins to be degraded [61]. Large protein aggregates are ubiquitinated, and the poly-ubiquitinated protein aggregates are shuttled to the autophagosome. This is generally performed via chaperone proteins such as p62/SQSTM1 [11]. The reason for inflammasome activation in the plaque is not currently unclear, but two mechanisms have been suggested. One is that inefficient mitophagy clearing of damaged mitochondria results in increasing reactive oxygen species (ROS), which induces inflammasome activation. However, the level of protein oxidation and superoxides are augmented in autophagy-deficient macrophages and atherosclerotic plaques [12]. The other mechanism is that overloaded oxLDLs and cholesterol crystals destabilize the lysosomal membrane, resulting in inflammasome activation by producing IL-1β [7]. In the atherosclerotic context, it has been shown that aggregated proteins activate inflammasomes and aggravate atherosclerosis in autophagy-deficient systems [12]. Atherosclerosis progression presents the features of impaired autophagy. Autophagy is sequential events called as autophagic flux (autophagosome formation, cargo sequestration, and autolysosomal fusion), and unfortunately, hard to assess the flux in vivo. When p62/SQSTM1, a

on the atherosclerotic lesion.

C1q for opsonizing.

Because of the above-mentioned characteristics, exogenous NO has been implicated as a therapeutic target. NO has benefits in vascular inflammatory diseases, and some researchers have tried to ameliorate atherosclerosis and other vascular diseases with NO donor therapy [41]. The therapeutic use of NO therapy has been reported to ameliorate atherosclerosis [48]. It is an important aspect of therapy whether atherogenesis initiates the formation of endothelial dysfunction or activation. Although it is unclear how endothelial cells recruit inflammatory cells, it is clear that inflammatory cytokines secretion of endothelial cells is tightly linked to eNOS expression. This relationship gives us hints for therapy. Also, vascular endotheliumderived NO has a protective role extending to endothelial-leukocyte interactions, leukocyte trafficking to hinder platelet activation, and smooth muscle contraction and proliferation. Statins (HMG-CoA reductase inhibitors) restore endothelial function, and protect vessels by boosting endothelium-derived NO. Endostatin has been reported to induce autophagic cell death in human endothelial cells (EA.hy926) [49]. When human endothelial cells are exposed to oxLDL, autophagy in the cells is increased to deal with plaque components. It is accepted that endostatin induces damaged endothelial cells by overloaded lipid through autophagic cell death pathways [50].
