4.1 Autophagy in number of macrophages

The disturbed count of macrophages is one of the characteristics of CVDs.¹⁵ Although there are two sources of macrophages, monocyte-derived and tissue-resident, the former is the primary source and is mainly responsible for uncontrolled inflammation in CVDs. In addition to monocyte recruitment, macrophages proliferation is required to maintain continuous replenishment of macrophages in lesions. Hyperlipidemia enhances macrophage proliferation, which aids in the formation of atherosclerotic plaques. Cell death also influences the number of macrophages, which triggers an immunological cascade via the release of inflammatory factors, exacerbating pathogenic alterations in CVDs. In this section, we explore the role of autophagy in regulating macrophage numbers through various steps and the underlying mechanisms based on published research (Table 1).

4.2 Autophagy in macrophage polarization

Differently polarized types of macrophages play a role in CVDs by influencing cardiac inflammation. M1 macrophages aggravate injury by releasing pro-inflammatory factors, whereas M2 macrophages are involved in wound repair and ECM reconstruction. Wang et al.⁸² demonstrated that the autophagy-related gene, HSPB8, is involved in macrophage polarization in AS. M2 macrophages are the predominant subtype in the low HSPB8 group. Furthermore, Tanshinone IIA has a propensity to M2 polarization by reestablishing the interaction between KLF4 and MCPIP, which facilitates the ERS-dependent autophagy and activation of signal transducer and activator of transcription (STAT)-6.⁸³ Altogether, these findings indicate a potential correlation between autophagy and macrophage polarization.

SIRT1 and mTOR may be crucial factors in autophagy and macrophage polarization. Gypenoside (GP) and Laminaria japonica polysaccharide (LJP61A) restore ox-LDL-induced autophagy impairment to attenuate M1 polarization and AS by the SIRT1-FOXO1-mediated pathway.^{84, 85} Araloside C, a natural saponin, exerts an anti-inflammatory effect in AS with tendentious M2 polarization by boosting autophagic flux in a SIRT1-dependent pathway.⁸⁶ In addition, 5-aminolaevulinic acid-mediated non-lethal sonodynamic therapy provokes a shift in M1/M2 equilibrium toward M2 via prompting the ROS-AMPK-mTOR-autophagy signalling pathway.⁸⁷ Atheroma lesions treated with rosuvastatin (RVS) have a lower lipid profile and necrotic core. Due to the suppression of PI3K-Akt-mTOR, RVS can improve autophagy initiation, enhancing M2 polarization conversion, and cholesterol efflux.⁸⁸

miRNAs also participate in macrophage polarization by altering autophagy. miR-29a can increase autophagic activity and promote M2 differentiation to ameliorate AS.⁸⁹ Forced expression of miR-520a-3p dampens the expression of LC3 and degradation of p62 to retard IL-4-triggered M2 differentiation through the interaction with 3′ untranslated region of UVRAG to accelerate the progression of AS (Figure 4).⁹⁰

Notably, the cell composition of the plaques is intricate at different stages. Macrophages predominate during early atherosclerotic plaques; however, foam cells take over in the late stages and transition from macrophages by phagocytizing lipids. Sun et al.⁹¹ found that RAP, an mTOR inhibitor, induces autophagic flux with upregulated LC3 in both peritoneal macrophages and foam cells. However, it augments the expression of IL-6 and COX2 (M1 markers) and reduces lipid droplet accumulation in peritoneal macrophages only. Conversely, fingolimod permits foam cells to undergo autophagy-mediated M2 polarization to ameliorate advanced AS. As a result, the atheroprotective benefits vary depending on the autophagy modulators used for each cell type.

4.3 Autophagy in inflammasome activation

Inflammasomes comprise a cytosolic sensor protein (either a nucleotide-binding domain and leucine-rich-repeat-containing [NLR] protein or an AIM2-like receptor [ALR] protein), an adaptor protein (apoptosis-associated speck-like protein containing a CARD [ACS]), and an effector pro-caspase-1.⁹² Assembly of inflammasome leads to autocatalytic cleavage of an inactive pro-caspase-1 into an active caspase-1, which regulates the proteolytic maturation of IL-1β and IL-18 in turn.⁹³ Activated caspase-1 cleaves GSDMD to cause a pyrogenic, inflammatory form of programmed cell death called pyroptosis.⁹²

Dysregulation of inflammasome activation accelerates the pathogenesis of CVDs, attributed to the undesirable inflammatory response.⁹⁴ Increased autophagic flux can subside NLRP3 inflammasome activation and subsequent macrophage pyroptosis.⁹⁵ TFEB evokes atheroprotection via autophagy-lysosome system, suppressing the inflammasome and cytotoxic protein aggregates.⁹⁶ Additionally, an evident reduction in IL-1β, IL-18, and NLRP3 inflammasome is observed in arglabin-treated macrophages concomitantly with increasing clustering of LC3 at the autophagosomal membrane.⁹⁷

Autophagy may hinder inflammasome activity by degrading inflammasome constituents. This depends on the increased amount of the autophagy adaptor p62 that co-immunoprecipitates with ASC.⁹⁸ Remarkably, chaperone-mediated autophagy is also involved in the progression of AS. Its impairment promotes NLRP3 inflammasome activation, predominantly in macrophages, among the cell types in the plaques. The NLRP3 protein, but not ASC or pro-caspase-1, interacts with heat shock cognate 71 kDa protein (HSC70) and lysosome-associated membrane protein type-2A (LAMP-2A) to be degraded by chaperone-mediated autophagy.⁹⁹ Besides, precision autophagy can selectively mar inflammasome components. Previous reports have stated that NLRP3 is explicitly degraded by the tripartite motif-containing (TRIM)-20 and E3 ubiquitin ligase MARCH7.^{100, 101}

Mitochondrial dysfunction directly activates NLRP3 inflammasome by inducing K⁺ efflux and releasing damage-associated molecular patterns (DAMPs), such as mitochondrial DNA (mtDNA) and mitochondrial ROS (mtROS).¹⁰² Shen et al.¹⁰³ reported that peritoneal macrophages from LDLR^−/− mice decrease IL-1β production and caspase-1 cleavage to reduce AS when fed dietary polyunsaturated fatty acids (PUFA). The mechanistic study discovered that PUFA-enriched diets potentiate autophagic activation and mitigate dysfunctional mitochondria. Mitophagy phagocytizes the damaged and dysfunctional mitochondria to control inflammasome activity.¹⁰⁴ Melatonin-mediated mitophagy suppresses NLRP3 inflammasome activation in atherosclerotic lesions via the SIRT3-FOXO3a-Parkin signalling pathway.¹⁰⁵ Synergizing with DNase II, mitophagy limits mtDNA damage and degrades ROS-producing mitochondria to reduce LPS-induced NLRP3 inflammasome expression in THP-1 and primary macrophages.¹⁰⁶ In contrast to these findings, Liu et al.¹⁰⁷ indicated that autophagy could activate NLRP3 inflammasome in MI. An autophagy specific-inhibitor, 3-MA attenuates IL-1β production and NLRP3 inflammasome formation to improve cardiac function and fibrosis. However, further studies are required to elucidate the underlying mechanisms by which autophagy upregulates NLRP3 activation in MI.

Inflammasomes also participate in the regulation of autophagy function. The activation of AIM2 and NLRP3 inflammasomes can induce autophagy, which does not require ASC, caspase-1, IL-1β, or complete assembly of inflammasome; however, it depends on the presence of the cytosolic sensor protein.⁹⁸ NLRP3 inflammasome antagonizes TLR4-TRIF-mediated autophagy in microglia. It generates active caspase-1 to cleave the signalling protein TRIF.¹⁰⁸ Notably, the TLR4-TRIF pathway also stimulates autophagy in macrophages.³⁹ As a result, the interaction between autophagy and inflammasomes is closely related but complex, requiring more studies to better understand the distinct mechanisms (Figure 5).

4.4 Autophagy in cytokine secretion

Cytokine production by macrophages, such as IL-1, IL-6, IL-10, IL-18, and TGF-β, orchestrates various immune regulations in CVDs. The conventional and unconventional secretion pathways are two types of cytokine secretion pathways. In the conventional secretion pathway, cytokines with specific signal peptides facilitate endoplasmic reticulum (ER) targeting. They are then translocated into the Golgi and delivered to the cell plasma membrane for extracellular secretion through secretory vesicles.¹⁰⁹ Previous studies have found that autophagy processes affect the conventional secretion of IL-6 and IL-8 through the TOR-autophagy spatial coupling compartment (TASCC), a specialized compartment and lysosomal-like organelle juxtaposed to the Golgi.¹¹⁰ Autophagosomes wrap and deliver cargo to the TASCC for degradation. It simultaneously produces amino acids and other metabolites to promote the activation of TASCC-resident mTOR and provides essential building blocks for the extracellular translation and secretion of IL-6 and IL-8.¹¹¹ HL60 human promyelocytic leukaemia cells differentiate into macrophage-like cells after stimulation with 12-O-tetradecanoylphorbol-13-acetate, accompanied by transient production of IL-8 and activation of autophagy. A substantial population of attached cells exerts local enrichment for mTOR, lysosomes, and autophagy vacuoles in the perinuclear regions.¹¹²

Cytokines deficient in leader peptides cannot reach the ER and are secreted by an unconventional pathway where autophagy takes part.¹¹³ Stimulation of autophagy by starvation contributes to the unconventional secretion of IL-1β in macrophages, which depends on ATG5, Golgi reassembly stacking protein 55 (GRASP55/GORASP2), and Ras-related protein (Rab)-8a.¹¹⁴ According to mechanical research, specific TRIM proteins and soluble N-ethylmaleimide sensitive factor attachment protein receptor (SNARE) proteins are responsible for the unconventional secretion route in bone marrow monocytes. IL-1β is recognized by TRIM16 and translocated to the LC3-II packing carrier when TRIM16 combines with Sec22b (a protein involved in vesicle trafficking). In conjunction with synaptosomal-associated protein 23/29 (SNAP23/29) and syntaxin3/4 (STX3/4), Sec22b molecules in autophagosomes fuse with the plasma membrane to deliver IL-1β for extracellular secretion.¹¹⁵ However, none of these experimental findings have been confirmed in animal studies, and it is debatable whether they are involved in regulating macrophage cytokine secretion in CVDs. Conversely, autophagy can suppress IL-1β expression by removing inflammasomes and pro-IL-1β.¹¹⁶ Targeted delivery of RAP perturbs IL-1β production to reverse plaque inflammation and burden via mTOR-SIRT1-mediated restriction of NF-κB.¹¹⁷ It suggests that autophagy may act as a double-switch mechanism that promotes IL-1β secretion for defence during the early phase of stress conditions while inhibiting IL-1β synthesis to avoid over-inflammation during the late stages.

Additionally, the role of autophagy in cytokine secretion extends to the unconventional pathways of TGF-β1, IL-18, and the DAMP HMGB1.^{114, 118} TGF-β1 interacts with GRASP55/GORASP2 in the Golgi and is secreted by Golgi-derived microtubule-associated protein 1 light chain 3 (MAP1LC3)-positive autophagosomal intermediates.¹¹⁸ TGF-β1 is the first cytokine that contains a signal peptide to enter the Golgi apparatus for maturation and proper folding, but still needs an autophagic pathway for secretion. This innovative study could be utilized for the development of anti-myocardial fibrosis strategies.

Apart from IL-1β and IL-18, IL-1α is also delivered to the cellular membrane by autophagosome-mediated unconventional secretion when caspase-1 is activated.¹¹⁰ A calpain-dependent process stimulated by mitochondrial uncoupling also involves the activation and production of IL-1α.¹¹⁹ Therefore, mitophagy can downregulate both the initiation of inflammasomes and calpain pathway, leading to the alleviative secretion of IL-1α. Furthermore, the explicit block of PI3K-Akt-mTOR signalling pathway encourages macrophage autophagy. It reduces atherosclerotic plaque susceptibility and lipid load with decreased IL-10 and increased IFN.¹²⁰ These findings broaden the functional manifestations of autophagy beyond its autodigestive role as a more widespread process in cytosolic protein extracellular delivery.

4.5 Autophagy in metabolism

Emerging reports emphasize the predominant role of metabolic reprogramming in the diversity and plasticity of macrophages, also known as immunometabolism.¹²¹ Macrophages can alter their metabolic programmes in response to stimuli to meet the specialized needs of host defence, tissue repair, and homeostasis. Aerobic glycolysis and the pentose phosphate pathway are integral to M1 polarization and function. Upregulation of glycolysis can maintain ATP production, acidize the environment, and provide precursors for lipid biosynthesis.¹²² Whereas M2 macrophages use the fatty acid oxidation (FAO) pathway and enhance mitochondrial biogenesis to produce ATP.¹²³ Fatty acid synthesis (FAS) modifies macrophage adhesion, recruitment, and inflammatory initiation by organizing plasma membranes.¹²² Aberrant accumulation of intercellular lipid causes macrophages to activate ERS and NLRP3 inflammasome activation.¹²⁴ This reveals the close relationship between metabolic reprogramming and divergent macrophage activities.

mTOR and AMPK, two principal autophagy regulators, have been used to probe the relationship between metabolic status and the autophagy machinery.^{122, 123} mTOR can induce protein synthesis, cap-dependent translation, and ribosomal biogenesis under amino acid abundance. Macrophages isolated from the patients with IL-10 receptor mutations display a pro-inflammatory response. Mechanistic study suggested that IL-10 inhibits the activation of mTOR by STAT3-DDIT4 pathway, which promotes mitophagy and oxidative phosphorylation and hinders aerobic glycolysis.¹²⁵ mTOR signalling pathway supports the transition toward glycolysis required by M1 macrophages via elevating HIF-1 expression. It results in the upregulation of glycolytic enzymes and pro-inflammatory genes.¹²⁶ mTOR catalyses fatty acids and triacylglycerol synthesis, while reducing lipolysis and FAO. It also exacerbates intracellular lipid accumulation via sterol regulatory element-binding protein, which codes for the expression of lipogenesis-associated enzymes.^{127, 128} AMPK, as a primary sensor of cellular energy status, enhances FAO and suppresses FAS, protein biogenesis, and other anabolic programmes to meet the demands of ATP.¹²³ AMPK encourages FAO to mitigate lipid intermediates and inflammation via antagonizing acetyl-coA carboxylase1, 3-Hydroxy-3-methylglutaryl-CoA reductase, and augmenting ULK1-dependent mitophagy.^{124, 129}

In cooperation with neutral lipase, autophagy hastens the hydrolysis of lipids to FC and move out of macrophages to ameliorate plaque stability.¹³⁰ Ginsenoside Rb1 increases lipolysis to mitigate lipid accumulation in foam cells by inducing AMPK phosphorylation and autophagy activation.¹³¹ miR-33, a post-transcriptional regulator of genes involved in cholesterol homeostasis, targets essential autophagy effector genes (ATG5, TFEB, ATG12, and ATG4b) and AMPK-activated transcription factors (FOXO3 and Tcfeb) to reduce lipid droplet catabolism. Additionally, miR-33 inhibits the clearance of apoptotic cells and the digestion of their cargo by an autophagy-dependent mechanism, which aggravates the pathological aspects of AS (Figure 6).¹³² In addition, cordycepin and ursolic acid have been demonstrated to promote macrophage autophagy and cholesterol efflux, exerting anti-atherosclerotic effects.^{133, 134} A recent study found that Ca²⁺-calpain signalling is a pivotal upstream of autophagy. LJP61A markedly reduces intracellular Ca²⁺ to provoke macrophage autophagy, resulting in the suppression of lipid accumulation and AS.¹³⁵

Apart from increasing lipophagy, autophagy can also regulate the ABCA1/ABCG1 transport pathway to alleviate AS. Liang et al.¹³⁶ reported that activation of autophagy enhances the cholesterol trafficking out of foam cells by promoting the expression of ABCA1, ABCG1, and their upstream transcription factor LXRα. However, the impairment of autophagy inhibits FC delivery extracellularly by p62-mTOR-LXRα pathway. A reduction in ox-LDL ingestion abrogates the accumulation of cholesterol ester to relieve AS. ox-LDL is identified and engulfed into macrophages by scavenger receptors, such as LOX-1, class A scavenger receptor (SRA), and CD36.¹³⁰ According to the study, enhanced autophagy significantly retards ox-LDL uptake, foam cell formation, and migration by autolysosome-dependent CD36 degradation at the post-transcriptional level.¹³⁷

4.6 Autophagy in phagocytosis

Phagocytosis is defined as the pathway for the recognition and internalization of particles (>0.5 μm). Through endocytosis of soluble ligands, targets are engulfed within the plasma membrane envelope of phagocytes.¹³⁸ Engulfment of exogenous particles results in the maturation of single-membrane phagosome vacuoles. They subsequently fuse with lysosomes and promote acidification, culminating in the formation of phagolysosomes for digestion.¹³⁸ Apart from microbial particles, phagocytosis can remove cellular corpses and debris, predominantly by macrophages, in a process known as efferocytosis. They can recycle cellular components and mitigate inflammation in the local tissue milieu. These processes are essential for the anti-inflammation and removal of apoptotic cells in plaques and dead cardiomyocytes during MI.¹³⁹ Advanced atherosclerotic lesions are characterized by impaired efferocytosis.¹⁴⁰

Autophagy plays a favourable role in AS and abates its progression by promoting the efferocytosis of apoptotic cells. Dynamin-related GTPase1 (Drp1)-deficient macrophages subside mitophagy and render efferocytosis defective, contributing to atherogenesis and aortic aneurysms.¹⁴¹ Macrophage-targeted photoactivation, a promising therapeutic strategy for CVDs, promotes macrophage autophagy activation with the specific affinity binding to the scavenger receptor. Mechanistic results show that imaging-guided DS-Ce6 light activation dampens plaque burden and inflammation owing to the elevation in apoptotic cell engulfment via increasing autophagy and phagocytic receptor Mer tyrosine kinase (MerTK) expression in foam cells (Figure 7).¹⁴² Moreover, resveratrol enhances SIRT1-mediated autophagy and improves the defective efferocytosis of ox-LDL-induced apoptotic cells.¹⁴³ PUFA-mediated atheroprotection is associated with the arrest of caspase-1 cleavage, apoptosis, necrosis, and amelioration of efferocytosis, whereas myeloid ATG5 deletion abolishes these protective effects.¹⁴⁴ Additionally, TEFB, an essential autophagy molecule, facilitates phagocytosis-related genes and impedes lesion complexity.¹⁴⁵ These findings indicate that the activation of macrophage autophagy-mediated efferocytosis may serve as a cutting-edge approach for treating AS.

In 2007, Sanjuan et al.¹⁴⁶ first reported that activation of the TLR pathway promotes LC3 conjugation with the phagosome membrane in macrophages requiring LC3, ATG7, and Beclin1 activity, defined as LC3-associated phagocytosis (LAP). Distinct from canonical autophagy, LAP is independent of the ULK1 complex for phagophore formation under nutrient deprivation or stress stimulation; however, it utilizes a portion of key canonical autophagy proteins. The PI3K complex is necessary for both autophagy and LAP. The core components of the PI3K complex are Beclin1, VPS15, VPS34, UVRAG, and Rubicon during LAP without ATG14 and Ambral, which are the two indispensable subunits of autophagy. Furthermore, ubiquitin-like systems, including ATG3, ATG5, ATG7, ATG10, ATG12, and ATG16L, are functional in the processing and ligation of LC3 to phagosomes.¹⁴⁷ Therefore, LAP may represent a significant additional mechanism of phagocytosis and autophagy interaction.

Increasing evidence shows that the restraint of autophagy activation damages standard macrophage LAP. miR-33 enhances lipid droplet accumulation in macrophages and inhibits apoptotic cell efferocytosis via LAP pathway. Recovery of autophagy and lysosomal biogenesis treatment improves efferocytosis and plaque necrotic areas using anti-miR-33.¹³² Besides, Liao et al.¹⁴⁸ investigated that ATG5 ablation in macrophages exacerbate nicotinamide adenine dinucleotide phosphate oxidase expression and apoptosis in response to oxidative stress and ERS. Simultaneously, it makes apoptotic cells less visible to efferocytosis, which synergistically worsens plaque necrosis in advanced AS. However, whether LAP participates in the impairment of efferocytosis needs to be determined because AGT5 is a crucial component of LAP.

5.1 Atherosclerosis

AS is the primary cause of CVDs and is characterized by foam cell formation and necrotic cores in plaques. Macrophages are pivotal effector cells that migrate to the inflamed sites of arteries to become cholesterol-loaded foam cells and release various pro-inflammatory factors. As a defence mechanism in AS, macrophage autophagy digests damaged proteins and accumulated lipids, and modulates inflammation in response to ROS, ox-LDL, and ERS.

Plaque rupture and erosion eventually leads to atherothrombosis. Mechanical research has shown that SIRT6 diminishes contact with ECs in a macrophage autophagy-dependent manner. It inhibits macrophage infiltration, inflammatory response, and occlusive thrombosis.¹⁴⁹ Tissue factor (TF) is a coagulation and hemostatic modulator secreted by macrophages when the fibrous cap ruptures, which activates the clotting system and causes thrombosis. Knockdown of mTOR suppresses the expression of TF, MMPs, and monocyte chemoattractant protein (MCP)-1 and promotes macrophage autophagy.¹⁵⁰ Moreover, impaired monocyte autophagy jeopardizes the stability of atherosclerotic plaques, which may be related to the attenuated phosphorylation of AMPK.^{151, 152} ATG16L1 is reportedly expressed around the necrotic core of human carotid atherosclerotic plaques and co-localizes with CD68 (a macrophage marker). ATG16L1 facilitates the production of pro-inflammatory cytokines and MMPs, exacerbating plaque vulnerability.¹⁵³

To stabilize plaques, VSMCs produce collagen to build fibrous caps. Canagliflozin increases VSMCs and exerts atheroprotective effects by enhancing macrophage autophagy.¹⁵⁴ Apoptotic macrophages are exacerbated by MST1-mediated autophagy inhibition, which secrete inflammatory factors to deteriorate the death of VSMCs, altogether contributing to plaque vulnerability.¹⁵⁵ Furthermore, FURIN increases VSMC proliferation and lipid droplet translocation to arrest AS by the AMPK-ULK-PI3K pathway.¹⁵⁶ Notably, chaperone-mediated autophagy also takes part in AS development. This process is initiated early in atherogenesis but gradually decreases with disease progression, leading to a pro-inflammatory state in macrophages and de-differentiation of VSMCs.^{157, 158}

Macrophage autophagy is a potential therapeutic approach for AS. SR-BI regulates TFEB-mediated macrophage autophagy to defend against atherogenesis by abrogating lipid accumulation, apoptosis, and inflammation.⁴⁰ LJP61A reduces the M1/M2 macrophage phenotype ratio and suppresses lesion burden in AS, which provokes autophagic flux by upregulating the expression of SIRT1 and FOXO1.⁸⁴ Research has demonstrated that calycosin, a flavonoid from Radix Astragali, helps resist macrophage inflammation and apoptosis. It enhances lipid hydrolysis and cholesterol efflux via KLF2-MLKL-mediated autophagy activation.¹⁵⁹ Melatonin exacerbates macrophage autophagy and limits the inflammatory response to inhibit AS progression by galectin-3 downregulation.¹⁶⁰

mTOR is a potential target for the regulation of macrophage autophagy in AS. A high-protein diet is a popular method of weight loss; however, it has been shown to raise the risk of AS. According to Zhang et al.,⁷⁶ dietary proteins prevent the degradation of dysfunctional mitochondria via amino acid-mTOR-mitophagy-mediated autophagy inhibition, which contributes to a rise in plaque complexity and caspase-9-mediated intrinsic apoptosis. Moreover, a recent investigation demonstrated that rivaroxaban administrates atheroprotection through abrogating PAR2-mTOR-mediated suppression of macrophage autophagy and obstructing inflammasome activation.¹⁶¹

According to the atheroprotective effect of autophagy activation, targeting macrophage autophagy in plaques has attracted increasing attention in recent studies. A type of NO-driven carrier-free Tr-Arg-PS (TAP) nanomotor, based on the reaction between trehalose (Tr, an mTOR-independent autophagy inducer), L-arginine (Arg), and PS, has been reported to prolong the circulatory half-life and improve the drug utilization of Tr. To achieve the first-phase targeting of the AS site, high expression of ROS and iNOS in the AS microenvironment is used as a chemoattractant, which interacts with Arg to generate NO as the nanomotor driving force to facilitate the chemotactic behaviour of the nanomotor. In the second-phase targeting macrophages, the ‘eat me’ signal sent by PS is exploited to target macrophages to promote cell uptake in the plaques precisely, thus delivering Tr to macrophages using the step-by-step strategy. Subsequently, intercellular Tr can alleviate impaired autophagy in macrophages to constrict AS.¹⁶²

In addition, Guo et al.¹⁶³ constructed a RAP spherical nucleic acid (SNA) structure for plaque-targeted delivery. Multiple hydrophobic RAPs are covalently grafted to the PS segment, designed at the 3′ terminus of the DNA strand, to form amphiphilic drug-grafted DNA (RAP-DNA), and consequently, self-assembles into micellar SNA (RAP-SNA). At the outer shell of the RAP-SNA, the phosphodiester-DNA segment can hybridize with LOX-1 siRNA to form the drug-co-delivered SNA (LOX-1/RAP-SNA). This compound enhances macrophage autophagy by RAP-mediated mTOR inhibition and prohibits the LOX-1-dependent formation of foam cells, thereby reducing the progression of AS and instability of atheromatous plaques. Utilizing the alterations in the endothelial monomolecular layer at the site of plaque formation, RAP-loaded biomimetic nanoparticles (leukosomes) target inhibition of the mTOR-SIRT-NF-κB pathway in macrophages of plaques to reverse vascular inflammation and plaque burden.¹¹⁷ Moreover, two novel macrophage-targeted acidic nanoparticles developed by Zhang et al.¹⁶⁴ have been shown to attenuate lysosomal dysfunction, autophagy retardation, and apoptosis activation in treating AS.

Although macrophage autophagy plays an essential protective role in AS, excessive autophagy activation can induce undesired results, such as apoptosis and sustained inflammation, which intensify pathological changes. Furthermore, regulation of macrophage autophagy may refer to the AS period. The large formation of ceroids is found in foam cell-like macrophages isolated from late plaques. Its continuous degradation via autophagy increases the production of hydrogen peroxide and peroxidation of the lysosomal membrane, resulting in the uncontrolled release of lysosomal enzymes.¹⁶⁵ In conclusion, further studies should discuss and investigate the optimum level of macrophage autophagy in atherogenesis and whether the effect of autophagy varies depending on the stage of AS.

5.2 Myocardial infarction

MI is a type of ACS with sudden blockage or occlusion of a major branch of coronary artery flow secondary to atherothrombosis, contributing to the blockage of cellular supply and ischaemic injury.¹⁶⁶ Cheng et al.¹⁵¹ found that peripheral blood monocytes of patients with non-ST-segment elevation ACS and ST-segment elevation MI have lower Atg7, Beclin1, and p-AMPK levels, while having higher phosphorylated mTOR. An increasing number of studies have demonstrated that improving macrophage autophagy is advantageous for alleviating the prognosis of MI.

Metformin attenuates the release of pro-inflammatory cytokines, infiltration of inflammatory cells, and NLRP3 inflammasome activation in macrophages to diminish the disarrangement of cardiomyocytes and infarct size in ischaemic myocardial injury. Further research found that metformin increases AMPK phosphorylation to upregulate autophagy function.⁴³ Moreover, the alpha7 nicotinic acetylcholine receptor (α7nAChR) alleviates systemic inflammatory reaction and hypoxemic myocardium damage through mTOR-related autophagy signalling in macrophages.¹⁶⁷ miR-144 is a cardioprotective miR, of which hypoxia depletes the endogenous level in the infarct zone. Macrophages, cardiomyocytes, and ECs take up intravenous miR-144 to exacerbate autophagy by targeting mTOR. It reduces the production of TNF-β, SMAD-3, MMP2, and MMP9 to ameliorate fibrosis, left ventricle dilation, and heart failure (HF).¹⁶⁸ Remarkably, Liu et al.¹⁰⁷ found that inhibition of macrophage autophagy by Calhex231 retards myocardial fibrosis and cardiomyocyte necrosis, which is attributed to the constricted NLRP3 inflammasome activation.

Angiogenesis is a self-regulating physiological process of tissue repair after MI accompanied by macrophage infiltration. M2-type macrophages exert an anti-inflammatory role and stimulate wound healing during recovery.¹⁶⁹ They produce various pro-angiogenic growth factors, such as MMP9 and vascular endothelial growth factor (VEGF).¹⁷⁰ Salvianolic acid B boosts the expression of VEGF and subsequent neovascularization to evoke cardioprotective effects against MI by enhancing autophagy activity.¹⁷¹ However, intraplaque angiogenesis and haemorrhages are risk factors in plaque vulnerability and heart attack. SIRT6 abrogates TLR4 and HIF-α secretion from macrophages through an autophagy-mediated method, which abolishes intraplaque angiogenesis and stabilizes the plaques.¹⁴⁹ Similarly, inhibition of mTOR counteracts circulating monocytes and neovascularization in plaques.¹⁷² Tongxinluo, a traditional Chinese medication, depletes inflammatory angiogenesis and macrophage apoptosis to stabilize vulnerable plaque by elevating Beclin-1-induced macrophage autophagy.¹⁷³ Therefore, the role of macrophage autophagy in regulating blood vessel formation is not invariable, and should be applied according to the characteristics of different diseases.

Research has demonstrated that autophagy activation improves ischaemic myocardial growth and regeneration adjacent to ischaemic focal regions because salvaged amino acids are employed to synthesize new proteins.¹⁷⁴ Macrophages facilitate neovascularization and cardiomyocyte proliferation owing to the timely resolution of inflammation.^{175, 176} Interference of NLRP3 by autophagy ameliorates I/R-damaged cardiomyocyte regeneration.¹⁷⁷ Disruption of inflammatory regression causes higher protease activity and jeopardizes recovery after MI.¹⁷⁸ Additionally, it is reported that mammalian sterile 20-like kinase (MST)-1 may exert a crossover between macrophage autophagy and myocardial regeneration. MST1 inhibition elevates macrophage autophagy to restrain excessive inflammation and promote cardiomyocyte regeneration after injury.¹⁷⁹ Thus, the macrophage autophagy-mediated immune micro-environment is crucial for cardiomyocytes with limited regenerative capacity.

5.3 Myocardial I/R injury

Javaheri et al.¹⁸⁰ verified that macrophages subjected to hypoxia/reoxygenation stress exhibit a statistically significant decrease in LC3-II, likely secondary to a relative deficiency in acidified lysosomes. This suggests that I/R injury is sufficient to provoke macrophage autophagy impairment. Although the effects of upregulated macrophage autophagy have not been reported in myocardial I/R injury, it has been confirmed to be protective in other organs, such as the liver and kidney.^{181, 182}

Cardiomyocyte autophagy is controversial and dynamic during I/R. Cardiomyocytes exert defective autophagy and metabolism during ischemia. In contrast, they exhibit overactivated autophagy resulting from the heavy oxidative burden, which leads to excessive cell death during reperfusion.^{183, 184} For instance, aldehyde dehydrogenase 2 (ALDH2), an enzyme that catalyses the oxidation of aldehydes, induces cardioprotection through the paradoxical regulation of autophagy. It exacerbates autophagic flux by activating AMPK and inhibiting mTOR during hypoxia, while abrogating autophagy accompanied by the activation of the Akt–mTOR signalling pathway during reoxygenation.¹⁸⁴ Similarly, ginsenoside Rb1 abolishes reperfusion-induced over-activation of cardiomyocyte autophagy by the PI3K-Akt–mTOR pathway to alleviate myocardial I/R injury.¹⁸⁵

Macrophages are the primary sources of oxidative stress during myocardial reperfusion. As a result, the ability of autophagy to reduce the oxidative burden and inflammation can contribute to the amelioration of myocardial injury during I/R. However, when regulating macrophage autophagy to defend against myocardial I/R injury, the dynamic changes in cardiomyocyte autophagy should be considered and undesirable effects on cardiomyocytes should be avoided. Macrophage autophagy-targeted treatment has become a hotspot and more applicable approach for I/R injury.

5.4 Heart failure and cardiac remodelling

In addition to acute myocardial injury, elevated serum pro-inflammation cytokines are detected in patients with chronic HF.¹⁸⁶ Macrophages increase abundantly in the myocardium to promote the pathogenesis of HF via cytokine secretion. Moreover, the imbalance between primary cardiomyocyte death and macrophage-mediated efferocytosis is a crucial trigger for the ongoing clinical deterioration of HF development. It results in chronic inflammation and collateral tissue injury.¹⁸⁷ Macrophage autophagy can provide a method for HF anesis owing to the regulation of inflammation. The investigation shows that Heat shock transcription factor 1 (HSF1) alleviates cardiac malfunction and fibrosis in pressure overload-induced HF. The autophagic flux of macrophages is activated with increased expression of LC3-II, Beclin-1, and autophagosome formation, associated with upregulated HIF-1.¹⁸⁸

Cardiac remodelling is a decisive factor in impaired ventricular function and the clinical course of HF. Macrophage accumulation has been implicated in cardiac remodelling and contributes to fibrosis through TGF-β production.¹⁸⁹ In ATG5 haplodeficient mice, macrophage-mediated ROS production and inflammatory response elevate cardiac fibrosis and inflammation.¹⁹⁰ Thus, activation of macrophage autophagy also plays a protective role in ameliorating cardiac remodelling. Adiponectin, a 30-kDa adipokine secreted by adipocytes, exerts anti-inflammatory effects and deters fibrosis against hypertensive cardiac remodelling. Adiponectin can mitigate the expression of pro-inflammatory cytokines in an autophagy-dependent manner. By phosphorylating AMPK, adiponectin induces macrophage autophagy and inhibits NF-κB activation.¹⁹¹ In addition, Cathepsin S-deficient mice show robust production of pro-inflammatory cytokines and severe cardiac fibrosis, attributed to the abnormal accumulation of autophagosomes in macrophages. Impaired autophagy blocks the elimination of damaged mitochondria, thus leading to ROS production and NF-κB activation to exacerbate hypertension-mediated cardiac remodelling.¹⁹²

5.5 Metabolic cardiomyopathy

With the clinical features of cardiac hypertrophy and diastolic dysfunction, metabolic cardiomyopathy develops in the context of chronic pathological metabolic dysfunction.¹⁹³ Excessive nutrients trigger enormous inflammatory signalling pathways. The characteristics of early-stage cardiomyopathy include metabolic disturbance-induced subcellular low-grade inflammation and subsequent component abnormalities, such as ERS and mitochondrial dysfunction. While cardiomyocyte injury and fibrosis with impaired myocardial structure and function contribute to a vicious cycle of inflammatory cell infiltration and neurohumoral activation in advanced stage.⁴²

Macrophages migrate into the heart by intramyocardial chemokines and secret pro-inflammatory cytokines, such as IL-1β, TNF-α, and MCP-1.¹⁹⁴ Free fatty acids are capable of activating macrophages to potentiate inflammatory response and insulin resistance via IL-1β-mediated islet β-cell dysfunction.¹⁹⁵ Upregulated expression of M1 polarization in the myocardium leads to the inflammatory micro-environment and activation of metabolic cardiomyopathy-related pathways.¹⁹⁶ These findings indicate that macrophage-induced inflammation takes part in the pathogenesis of metabolic cardiomyopathy.

Notably, metabolic diseases impede autophagy and mitophagy with a reduction in AMPK activity, resulting in oxidative stress and NLRP3 inflammasome activation in macrophages.¹⁹⁷ Furthermore, the generation of toxic lipid intermediates and dyslipidemia are accompanied by the onset of cardiomyopathy.¹⁹³ Considering that autophagy is insufficient in metabolic cardiomyopathy and its function in affecting inflammation and lipid metabolism, we suspect that the upregulation of autophagy activation may play a dominant role in treating cardiometabolic syndrome. SIRT3-mediated autophagy ameliorates diabetic cardiac dysfunction. It can impede NLRP3 inflammasome activation and IL-1β to ameliorate hyperlipidemia-induced endothelial dysfunction. SIRT3 overexpression of macrophage abates the protein levels of α-SMA, collagen-I, VACM-1, and intercellular adhesion molecule 1 in ECs by SIRT3-ATG5-IL-1β axis.¹⁹⁸

5.6 Myocarditis

Myocarditis is an inflammatory disease of the myocardium and is pathologically ruled out by pronounced macrophage infiltration and cardiomyocyte death.¹⁹⁹ On account of its high plasticity and pathophysiological role, the modulation in macrophages provides a new approach to myocarditis therapy.²⁰⁰ For example, miR-30a-5p silencing skews the macrophage subset into M2 and suppresses the inflammatory response to ameliorate viral myocarditis (VMC) injury.²⁰¹ Autophagy, as a regulator of macrophage function, may play a protective role in myocarditis. Androgen receptor (AR) inhibition exerts anti-inflammatory effects to promote tissue repair in an experimental autoimmune myocarditis (EAM) mouse model. Further investigation showed that AR suppression alleviates the secretion of pro-inflammatory cytokines and the M1/M2 polarization rate via enhancing autophagy in macrophages. This is corroborated by the increased LC3-II/I ratio and autophagosome formation, thus diminishing apoptotic injury and tissue fibrosis in EAM.²⁰²

It is worth noting that coxsackievirus B3 (CVB3), one of the major pathogens of VMC, can increase cellular autophagosome formation to support its replication in the host and release it from cells depending on apoptosis.²⁰³ Autophagy is not enhanced because there are no apparent changes in autophagic adaptor p62 expression after CVB3 infection. However, activation of autophagosome–lysosome fusion can abrogate viral proliferation. It suggests that suggesting that lysosomal fusion is commendatory and autophagosome formation should be avoided when treating CVB3-infected viral myocarditis.²⁰⁴

5.7 Abdominal aortic aneurysm

The histopathological features of abdominal aortic aneurysm (AAA) are chronic inflammation and production of oxidative stress.²⁰⁵ In particular, macrophage infiltration of the aortic wall leads to the sustained local and systemic inflammatory response in AAA.¹⁵ Secretion of IL-1β, especially by macrophages, has been documented to be elevated in aneurysmal tissue.²⁰⁶ Studies have found that macrophage autophagy is activated by AAA-related factors, thus postponing the AAA process.

Angiotensin-II, a principal mediator peptide of the renin-angiotensin system, can mediate adverse vascular remodelling and dysfunction in AAA through ROS generation and inflammation. These processes can stimulate macrophage autophagy and are mitigated by protective mitophagy.²⁰⁷ As a result, upregulation of macrophage autophagy may be a candidate method for suppressing and treating AAA. Liu et al.²⁰⁸ demonstrated that spermidine markedly limits aortic inflammatory cell infiltration, angiogenesis, and neutrophil circulation in an AAA mouse model by accumulating autophagosomes in medial VSMCs and adventitial macrophages. Moreover, naringenin has been shown to effectively reverse the progression of established AAA by targeting the macrophage TFEB-14-3-3 epsilon interface. Naringenin can directly bind to 14-3-3 epsilon to inhibit TFEB-14-3-3 epsilon interactions, and subsequently promote the induction of TFEB and autolysosome formation in macrophages. TFEB-dependent inhibition of NLRP3 inflammasome and encouragement of GATA3-IRF4-STAT6-mediated reparative M2 phenotype synergistically exert beneficial roles in abolishing AAA.²⁰⁹ Notably, TEFB is a vital regulator of autophagy-related genes. Further experiments are needed to clarify whether TEFB-dependent modulation of inflammasomes and polarization of macrophages are associated with autophagy to hinder inflammation in aortic tissue.