Autophagy is a self-degenerative process that participates in organelle turnover, and the recycling of cytoplasmic components as well as protein degradation in response to extracellular stresses. Currently, autophagy can be categorized into three classes (6). Microautophagy is a non-selective lysosomal process describing the direct engulfment of cytoplasmic cargos by lysosomes. This process is accomplished by the inward invagination of cargos into the lysosomal membrane. In contrast, chaperone-mediated autophagy enables the selective degradation of cytoplasmic proteins by recognizing chaperone proteins. During this process, lysosomal-associated membrane protein type 2A on the lysosomal membrane recognizes the target proteins via their chaperone (e.g., heat shock cognate protein of 70 kDa), allowing internalization of the target protein into lysosomes. Macroautophagy is a process whereby cytoplasmic components are degraded by lysosomes, and it is accompanied by the formation of autophagosomes. These autophagosomes are cytosolic double-layered membrane vesicles, in which cytoplasmic components are separated from the cytoplasmic environment, and which finally fuse with a lysosome where lysosomal digestion occurs. One of the key regulators of autophagy is the kinase mammalian target of rapamycin (mTOR), which negatively regulates autophagy (6). Autophagosome formation is mediated by Atg genes, which is regulated by Atg12–Atg5–Atg16L1 and LC3–PE (Atg8 homolog) complexes (6). It was reported that nutrient supply by macroautophagic protein catabolism sustained anabolic reactions to generate macromolecules, such as nucleic acids, proteins, and organelles. As a result, the nutrient supply can exert a prosurvival mechanism, particularly under starving conditions (15). In contrast, the overactivation of autophagic systems elicits cell death, which may be mediated by a process morphologically distinct from apoptosis (16). The contribution of macroautophagy to tumorigenesis, infection, diabetes, and cardiovascular diseases was described previously (6, 7, 15). In vascular systems, macroautophagy is detectable in macrophages in atherosclerotic lesions as well as in vascular-resident cells, including ECs and vascular smooth muscle cells (VSMCs), where it participates in physiologic and pathogenic responses (6).

Razani et al. reported that macroautophagy was associated with the pathogenesis of atherosclerosis (17). The expression of p62 protein, but not of p62 mRNA, was potentiated in lesional macrophages suggesting insufficient autophagy in these cells. Macrophage-specific deficiency of Atg5 accelerated the development of atherosclerosis in Apoe^−/− mice by insufficient autophagy in cells. Furthermore, Liao et al. also reported abnormal expression patterns of LC3 and the presence of p62-positive cells in atheromas in atherogenic mice, indicating impaired autophagy in the lesions (18). Accordingly, macrophage-specific Atg5 deficiency augmented the expansion of lesional necrotic cores and progression of atherosclerosis. Therefore, insufficient autophagy might decrease the stability of atherosclerotic plaques as well as its pathogenic implications.

The ubiquitin proteasomal systems are part of the central protein catabolism machinery in cells and are involved in the degradation of misfolded proteins, as well as normal protein degradation. These systems are also implicated in cellular events, including antigen presentation in immune cells (19) and the cell cycle (20). The polyubiquitination of target molecules triggers proteasomal degradation, which is driven by E1 ubiquitin-activating enzymes, E2 ubiquitin-conjugating enzymes, and E3 ubiquitin-protein ligases (21). Following the ubiquitination process, ubiquitinated target proteins are recognized by proteasomal complexes and are proteolytically degraded into peptides. In this process, the 26S proteasome comprising regulatory particle 19S and core particle 20S, has a crucial role in the proteasomal systems (9). Similar to autophagic systems, dysfunctional proteasomes can cause degenerative diseases, such as Alzheimer’s disease (22), Parkinson’s disease (22), Huntington’s disease (22), and atherosclerosis (23, 24). An atherosclerosis study in high fat diet-fed pigs identified that proteasomal activity was elevated in coronary arteries during the progression of atherosclerosis (25). However, in a human atherosclerosis study, the carotid atherosclerotic lesions in subjects complicated with transient ischemic attack or stroke exhibited lower proteasomal activity compared with that in asymptomatic subjects (26). These studies suggest that proteasomal activity declines in advanced atherosclerotic plaques, whereas it is sustained in mild lesions. In addition to these observations, animal experiments with pharmacological proteasomal intervention have been performed. Herrmann et al. examined the long-term administration of the proteasome inhibitor MLN-273 in high cholesterol diet-fed pigs and identified the elevation of oxidative stress and exacerbation of atherosclerosis (25). Conversely, Wilck et al. reported that a moderate dose of bortezomib (also known as PS-341) led to partial proteasomal inhibition in high cholesterol diet-fed Ldlr^−/− mice, which reduced oxidative stress and NADPH oxidase 4 expression levels in the aortae, thereby suppressing atherosclerosis (27). Therefore, it hardly defines pathophysiologic roles of proteasomal systems in atherosclerosis. It is well known that macrophage cytotoxicity is prominent in advanced atheromas, while vascular inflammation is sustained throughout moderate-to-advanced atherosclerotic lesions (1). Thus, it is suspected that the contribution of proteasomal systems to the pathogenesis of atherosclerosis is substantially dependent upon the stage of the disease.

The calpain family comprises Ca²⁺-dependent intracellular proteases (28–30). These proteolytic systems are unique because calpains modulate their substrates through limited proteolytic cleavage in addition to proteolytic degradation. The calpain system comprises the endogenous inhibitor calpastatin as well as 15 homologs of the catalytic subunits, and 2 regulatory subunit homologs in mammals (28–30). Calpain catalytic subunits are categorized into conventional and unconventional subtypes (28–30). The conventional subtype consists of two isozymes, calpain-1 and calpain-2, which are ubiquitously expressed in vertebrates (28–30). These are heterodimers of the catalytic subunits, CAPN1 and CAPN2, which form calpain-1 and calpain-2 isozymes, respectively, together with their common regulatory subunit, CAPNS1 (28–30). The substrate selectivity of those conventional calpains is currently unclear, but they exhibit enzymatically distinct properties. Indeed, calpain-1 and calpain-2 require micromolar and millimolar levels of Ca²⁺ for half-maximal activation, respectively. It was reported that a variety of biophysical and biochemical stimuli elevate intracellular Ca²⁺ levels (31–33); therefore, the activity of these protease systems has been the focus of numerous pathophysiologic studies (13, 14). Our previous investigation indicated that oxidized or enzymatically modified LDL or its component, lysophosphatidylcholine, induced calpain-2; accordingly, this molecule is enriched in ECs in human and mouse atheromas (34). Calpain-2 exerts pro-atherogenic roles because the calpain inhibitors calpeptin and ALLM counteracted aortic atherosclerotic development in high cholesterol diet-fed Ldlr^−/− mice. It was also reported that the calpain inhibitor BDA-410 inhibited atherosclerotic development in angiotensin II-infused Ldlr^−/− mice (35). Furthermore, angiotensin II- or hypercholesterolemia-induced atherosclerosis in Ldlr^−/− mice was suppressed by the transgenic overexpression of calpastatin; in contrast, abdominal aneurysmal formation, a complication of angiotensin II-infused mice, was unaltered (36). A study using angiotensin II-infused mice showed that calpain-1 in myeloids and calpain-2 in leukocytes were responsible for atherogenesis (36). Collectively, conventional calpains participate in pathophysiologic regulation in ECs and leukocytes, thereby facilitating atherosclerosis.

Our previous data identified that unconventional calpains also contribute to atherogenesis (37). Expression analysis in Ldlr^−/− mice showed that Capn2, Capn6, and Capn9 were upregulated in atheroprone aortae. Capn6 deficiency decelerated the progression of atherosclerosis in Ldlr^−/− mice, while Capn9 deficiency did not have any effect. Calpain-6 is a non-proteolytic calpain because the cysteine residue in its CysPc domain is substituted with a lysine (28–30). This molecule is expressed preferentially in macrophages in advanced atheromas in humans and mice (37). Interestingly, calpain-6 was originally identified in the skeletal muscle, cartilage, and heart of a murine fetus (38); however, its expression is absent in adult tissues with the exception of the placenta under non-disease conditions (39). Therefore, calpain-6 might be potentiated under developmental and pathogenic conditions. Regarding atherosclerosis, it is likely that calpain-6 is upregulated in macrophages after cells infiltrate into lesions, whereas it is absent in bone marrow cells (37). Furthermore, calpain-6 induction can be reproduced in cultured bone marrow-derived macrophages by cytokine stimulation. In particular, tumor necrosis factor-α markedly induced calpain-6. Atherogenesis appears to be dependent upon myeloid Capn6 but not upon other cell lineages in mice. Therefore, the proatherogenic transformation of macrophages by calpain-6 is responsible for atherosclerotic disease.

Endothelial cells cover the luminal surface of blood vessels and act as a physical barrier to limit the infiltration of active plasma factors through the vascular wall. It is well known that the endothelial barrier is disorganized in atheroprone arteries because of inflammatory insults (1), which accelerate the recruitment of immune cells that subsequently promote proinflammatory responses in the vessels. It is noteworthy that immune cells can also infiltrate atheroprone vascular walls through the vasa vasorum (40). Previous studies identified three major types of interendothelial junctions: tight junctions, gap junctions, and adherence junctions (41). In human umbilical vein ECs, tight junctions are present in up to 20% of the total junctional complexes, while adherence junctions account for approximately 90% (42). Vascular endothelial (VE)-cadherin forms adherence junctions in a Ca²⁺-dependent homophilic manner (43). Furthermore, vascular permeability in mice was elevated by the administration of anti-VE-cadherin neutralizing antibody, whereas this treatment had no effect on other types of junctions in ECs (44); thus, adherence junctions have a pivotal role in barrier maintenance in ECs. Our previous investigations identified that administration of the calpain inhibitors calpeptin and N-acetyl-Leu-Leu-methional (calpain inhibitor II) recovered the dysfunction of the EC barrier in dyslipidemic mice (34). Mechanistically, a juxtamembrane domain of VE-cadherin is proteolyzed by calpain-2, thereby dissociating β-catenin from the VE-cadherin complex, leading to the destabilization of adherence junctions. As a result, macrophage infiltration and the subsequent development of atherosclerosis in the dyslipidemic mice were suppressed by pharmacological inhibition of conventional calpains. Calpain proteolyzes E-cadherin and N-cadherin; thus, calpain systems regulate the posttranslational processing of cadherin family proteins (45, 46). In contrast to the calpain family, the contribution of autophagy and proteasomal systems to atherosclerotic endothelial barrier dysfunctions has not been reported to date, although these pathways were reported to participate in the disorganization of tight junctions in septic pulmonary ECs (47) and ischemic cerebral ECs (48).

Endothelial nitric oxide (NO) production is crucial for ensuring vascular integrity. NO reacts with superoxide to neutralize its toxicity; therefore, a reduction of NO bioavailability facilitates ROS-dependent vascular inflammation. Furthermore, uncoupled endothelial nitric oxide synthase (eNOS), which generates superoxide radicals instead of NO, is largely responsible for ROS production even in atheroprone vessels (49, 50), in addition to xanthine oxidase and NADPH oxidase. It was reported that conventional calpains were responsible for physiological NO production in ECs. Indeed, calpain inhibitors N-acetyl-leucyl-leucyl-norleucinal (calpain inhibitor I) or calpeptin prevented vascular endothelial growth factor (VEGF)-driven NO production in cultured ECs (51). Furthermore, VEGF accelerated the translocation of calpain to the plasma membrane to form an ezrin-containing molecular complex. Accordingly, AKT, AMP-dependent kinase (AMPK), and eNOSs1179 were phosphorylated through the molecular complex, which is indispensable for NO production in ECs. In addition to VEGF-driven NO synthesis, disorders of NO production under pathophysiologic conditions might be caused by the dysregulation of calpain systems. Yu et al. documented that the administration of calpain inhibitor I increased the activity and protein expression of aortic eNOS in high fat diet-fed rats along with the elevation of NO (52) indicating excessive calpain activity may disturb NO synthesis in ECs. Herrmann et al. reported that in contrast to calpain inhibitors, the chronic administration of the proteasome inhibitor MLN-273 increased oxidative stress and exacerbated the development of atherosclerosis in pigs fed a high cholesterol diet (25). In vitro experiments further showed that inhibition of the proteasome prevented EC-dependent vasodilation in isolated coronary arteries. Collectively, protein catabolism pathways are critical for endothelial NO production. It appears that proteasomal systems ensure NO synthesis, while calpain systems act as a negative regulator for NO production in atheroprone vessels.

It is widely known that fluid shear stress, the blood flow-generated dragging force tangential to the EC surface, is a critical determinant for atherosclerosis susceptibility in blood vessels. This is because shear stress-dependent pathways in ECs are involved in barrier regulation and NO production (53). Interestingly, aortic arch, coronary artery, and aortic bifurcation, where blood flow is disturbed, are proatherogenic; thus, atherosclerosis susceptibility in blood vessels is largely dependent upon the vascular architecture because of the limited EC mechanotransduction in these regions (54). Yang et al. reported that physiological fluid shear stress sustained substantial autophagic activity in cultured ECs, but was reduced in cells subjected to low shear stress (55). Shear stress-induced eNOS phosphorylation and NO production were markedly blunted by autophagic deficiency (56). Similar to impaired NO production, the loss of autophagic activity potentiates ROS and cytokine production in ECs, suggesting that autophagy may play a suppressive role in eNOS uncoupling. Consistent with these results, autophagic inhibition by 3-methyladenine downregulated eNOS expression in sheared carotid arteries in rabbit, while autophagic activation by rapamycin potentiated eNOS expression (57). In addition to shear stress-dependent autophagic regulation, we previously showed that endothelial calpain-2 was activated in response to physiologic shear stress in cultured ECs (58). This activity was mediated through Ca²⁺ influx and phosphatidylinositol 3-kinase and was responsible for EC alignment in the direction of the blood flow. Interestingly, calpain-2 is enriched in specific vascular regions, such as the lesser curvature of the aortic arch and origin of vascular branch, in normal physiological murine aortae, where the blood flow is structurally disturbed (59). Thus, the autophagic and calpain systems orchestrate endothelial mechanotransduction.

It was reported that cardiovascular defects such as hypertension and atherosclerosis are strongly related to age-associated alterations in arterial structure and function (60, 61). The hallmark of such disorders is the stiffening of large arteries caused by ECM remodeling and vascular calcification and is triggered by the phenotypic conversion of contractile VSMCs to a synthetic phenotype. Mechanistically, synthetic VSMCs orchestrate elastin fragmentation as well as the synthesis and degradation of collagen through ROS-dependent inflammatory cascades. It was reported that RhoA, TGF-β, Notch, and integrin–matrix pathways modulated SMC differentiation (62) and that platelet-derived growth factor (PDGF) orchestrated the conversion of VSMCs to a contractile state along with autophagic activation (63). The pharmacological intervention of autophagy appears to inhibit the PDGF-induced phenotypic conversion in VSMCs, indicating the contribution of autophagic signals to the conversion. Interestingly, VSMC-specific deficiency of autophagy exacerbated high fat diet-induced atherosclerosis in Apoe^−/− mice, which was accompanied by a thickening of the fibrous cap (64). Furthermore, the autophagic flux resulted in an increase in collagen content, TGF-β expression, and matrix metalloproteinase-9 in VSMCs. Thus, autophagy in VSMCs is protective in atherogenesis, but augments plaque vulnerability.

In addition to autophagy, calpain systems were reportedly associated with the phenotypic regulation in VSMCs. The transgenic overexpression of calpastatin antagonized arterial fibrosis and hypertrophy in mice infused with angiotensin II (65) by reduced MMP levels and the downregulation of NF-κB signals in arterial media. Similarly, the age-associated overactivation of calpain-1 in VSMCs participated in the upregulation of MMP2 and VSMC motility (66). Furthermore, osteopontin and osteonectin levels in VSMCs were reduced by the overexpression of calpain-1 in rats, inducing the calcification and fibrosis in the arteries of aged rats (67). Furthermore, VSMC proliferation and collagen synthesis were accelerated during ligation-induced carotid restenosis in mice, which was opposed by the transgenic overexpression of calpastatin (68). Therefore, calpain systems have a pivotal role in inflammation-related fibrogenic responses in VSMCs.

Macrophages exert an essential role in the pathogenesis and progression of atherosclerosis (69). Bone marrow-derived circulating monocytes can be divided into two subsets, LY6C^hi and LY6C^low, in mice (70–72). The increased number of circulating monocytes in hypercholesterolemic mice is responsible for the expansion of the LY6C^hi subset (70), which is a major source of recruited macrophages in atheromas. In the early stage of atherosclerosis, circulating monocytes adhere to luminal ECs and migrate into the intimal layer according to the gradient of cytokines/chemokines (1). Subsequently, monocytes are differentiated into classically (M1) and alternatively (M2) activated macrophage subsets. M1 subset is mainly associated with inflammatory responses in atherosclerotic lesions and is derived from LY6C^hi monocytes (70–72). The M2 subset can be further divided into at least 4 subgroups, M2a, M2b, M2c, and M2d, which participate in tissue repair and inflammatory resolution in some instances (73). Foam cells in atherosclerotic lesions are mostly derived from the M1 subset, which is mediated through receptor-dependent and receptor-independent uptake of LDL cholesterol. In contrast, M2c macrophages incorporate cholesterol by engulfing dead cells efferocytically (74). The balance of M1 and M2 polarization can be disrupted in the presence of inflammatory burden in atherosclerotic lesions at least in part by disturbed poly (ADP-ribose) polymerase- or Dll4/Notch-related signals (73). Because cytosolic-free cholesterol exhibits robust cytotoxicity, the excessive incorporation of cholesterol results in cell death in macrophages. Accordingly, the acceleration of cholesterol uptake in macrophages in atheroprone vascular walls increases the number of dying macrophages, thereby forming cholesterol-enriched vulnerable atherosclerotic lesions (75). In addition to their defective cholesterol handling, atheroprone macrophages play central roles in innate immunity, including toll-like receptor and NOD-like receptor-mediated inflammasome signaling, in lesions (69). Although the protein catabolism pathway critically influences both cholesterol handling and inflammatory cascades in cells, cholesterol handling is not discussed in this issue. Please refer to our previous review articles for information on proatherogenic cholesterol handling in macrophages by defective cholesterol catabolism (12).

It was reported that macrophage-specific deficiency of autophagy by the deletion of Atg5 facilitated atherogenesis in Apoe^−/− mice (17). Interestingly, Atg5-deficient macrophages had increased oxidative stress and enhanced inflammasome secretion of IL-1β. The inflammasome systems in Atg5-null macrophages were potentiated by the addition of cholesterol crystals to the culture systems. It is likely that the inflammasome secretion of IL-1β by Atg5-null macrophages in atheromas may be potentiated by cholesterol crystals, because they are enriched in atherosclerotic lesions in Atg5-deficient mice. Furthermore, necrotic cores and oxidative stress in lesions of Ldlr^−/− mice were enhanced by the macrophage-specific deletion of Atg5 (18). Autophagic activity in isolated macrophages was potentiated by proatherogenic stressors, such as 7-ketocholesterol or 1-(palmitoyl)-2-(5-keto-6-octene-dioyl) phosphatidylcholine. Such autophagic activity suppressed apoptotic cell death by the reduction of endoplasmic reticulum stress and oxidative stress by NADPH oxidase 2. Furthermore, atherogenicity in macrophages was also mediated by proteasomal defects. For example, Brand et al. reported that the pharmacological inhibition of proteasomes prevented the proteolytic degradation of inhibitor κBα, an endogenous inhibitor of NF-κB transcriptional systems, by oxidized LDL in monocytes (76). This indicates that proteasomal inhibitors possess anti-inflammatory effects in atherosclerosis. In contrast, it was reported that oxidized LDL-stimulated macrophages showed increased ubiquitination activity, which suppressed apoptosis (10). Similarly, the complex mode of cell death in VSMCs was abrogated by 7-ketocholesterol-indced proteasomal activity (11). Thus, proteasomal activity may be involved in cell survival under stressed conditions, while it participates in cytotoxic inflammatory burden. In addition to the autophagic and proteasomal flux, the participation of calpain systems in inflammatory cascades of macrophages was proposed. It appears that calpain can proteolytically degrade inhibitor κBα, thereby accelerating NF-κB signaling (77); thus, the overactivation of calpain systems potentiates inflammatory responses. The transduction of CAST prevented the production of inflammatory cytokines in peritoneal macrophages isolated from high fat diet-fed Apoe^−/− mice (36). Collectively, macrophages can be susceptible to proatherogenic burden by the overactivation of calpain systems. Whether defective protein catabolism influences macrophage polarity is currently unclear and, therefore, should be studied in the future.

Vulnerable atherosclerotic lesions harbor vast number of inflammatory cells, a necrotic core, and a thin fibrous cap (78). In particular, expansion of the cholesterol-enriched necrotic core by the accumulation of dead cells weakens the physical stability of plaques. It was reported that macrophages engulf and remove cellular corpses in lesions through efferocytic activity, thereby reducing the necrotic core (78). Furthermore, the efferocytic actions of macrophages participate in the reprogramming of cells toward anti-inflammatory phenotypes, leading to the resolution of inflammation. Importantly, the clearance of dead cells by efferocytosis is limited in advanced atheromas, because “eat-me/find-me” signals in apoptotic cells and phagocytosis pathways in macrophages are prevented in the lesions (78). Thus, the coordination of integrated cell death signals in dying cells and phagocytic signals in phagocytes are necessary for efferocytosis. In the case of mammary gland involution in pregnancy, efferocytic activity was accelerated in mammary epithelial cells, resulting in remodeling of the tissues and the production of milk (79). Mammary epithelial-specific Atg7 deficiency markedly reduced efferocytic activity, Rac1 activity, and MertK expression; thus, autophagic signaling is associated with efferocytosis-associated tissue remodeling. In the case of atherosclerosis, autophagic defects caused by Atg5 deficiency reduced the efficiency of the efferocytic clearance of dead cells, leading to an expansion of the necrotic core (18). It is likely that Atg5 deficiency in dying cells, but not phagocytic macrophages, prevented the efficiency of efferocytosis. Therefore, the autophagic disorder in dying cells or phagocytic macrophages induced defects in efferocytosis, although this needs to be elucidated in the future. While the participation of conventional calpains in efferocytosis has not been reported to date, calpain-6 seems to be involved in this event. Indeed, the efferocytic clearance of dead cells by cultured macrophages was modestly inhibited by Capn6 deficiency (37). Accordingly, the deletion of Capn6 resulted in a reduction of necrotic cores in atheromas in Ldlr^−/− mice, which was accompanied by the upregulation of Rac1 protein in the cells. Rac1 prevention by a pharmacological inhibitor suppressed efferocytic activity in macrophages (80); therefore, it is interpreted that efferocytic activity in lesional macrophages was prevented by calpain-6, probably through Rac1 downregulation. Because conventional calpains are associated with cellular dynamic processes, such as migration (81) and trafficking of intracellular vesicles (82), it will be interesting to investigate the participation of these calpains in efferocytosis.