Role of pyroptosis in cardiovascular disease

Abstract Cardiac function is determined by the dynamic equilibrium of various cell types and the extracellular matrix that composes the heart. Cardiovascular diseases (CVDs), especially atherosclerosis and myocardial infarction, are often accompanied by cell death and acute/chronic inflammatory reactions. Caspase‐dependent pyroptosis is characterized by the activation of pathways leading to the activation of NOD‐like receptors, especially the NLRP3 inflammasome and its downstream effector inflammatory factors interleukin (IL)‐1β and IL‐18. Many studies in the past decade have investigated the role of pyroptosis in CVDs. The findings of these studies have led to the development of therapeutic approaches based on the regulation of pyroptosis, and some of these approaches are in clinical trials. This review summarizes the molecular mechanisms, regulation and cellular effects of pyroptosis briefly and then discusses the current pyroptosis studies in CVD research.


| INTRODUC TI ON
Cell death (CD) is critical to maintaining tissue homeostasis and basic biological functions, and its changes have significant implications in disease pathology. Since CD was first described in the1960s, many types of CD have been defined based on differences in morphological and biochemical characteristics. 1 In the past, CD in the cardiovascular system was considered passive and negative. In addition, CD was previously believed to occur due to the loss of function of living cells and subsequent inflammation. This situation has changed with the description of apoptosis in the 1970s. In general, apoptosis is essential to maintain cardiovascular homeostasis. Both reduced and increased apoptosis can result in pathology. 2 The maintenance of the normal structure and function of the cardiovascular system requires a balance between cell formation and death in the tissues and organs of the cardiovascular system (including cardiomyocytes [CMs], endothelial cells [ECs], vascular smooth muscle cells [VSMCs] and cardiac fibroblasts [CFs]). Excessive CD (including pyroptosis) often leads to dysfunction of tissues and organs. 3 Pyroptosis was first identified in the macrophage in 1992, which presented rapid lysis after infection with Shigella flexneri, 4 and the name was coined in 2001. 5 Pyroptosis plays a pivotal role in the pathogenesis of various CVDs and involves ECs, 6 VSMCs 7 and so on. This process occurs in patients suffering from myocardial infarction (MI), 8,9 hypertension 10,11 and cardiomyopathy, 12 as well as in animal models of ischaemia-reperfusion injury (IRI), 13 atherosclerosis (As), 6 heart failure (HF) 14 and cardiomyopathy. 15,16 Pyroptosis is a highly regulated cell death process, and inhibition of this process by pharmacological or genetic intervention is cardioprotective under many conditions. 6,17 Therefore, this process is a potential target for therapeutic intervention to prevent CVDs. In summary, the discovery of pyroptosis has broadened our understanding of CD in CVDs, and targeting this manner of CD provides new avenues for the treatment and management of CVDs. This review provides a current overview of the evidence and functional role of pyroptosis in CVDs and discusses the molecular pathways involved in the cardiovascular system.

| OVERVIE W OF PYROP TOS IS
Pyroptosis is a form of programmed cell death (PCD), accompanied by an inflammatory response. 18 PCD refers to the autonomous, ordered death of cells controlled by genes to maintain homeostasis. By contrast, non-PCD (NPCD) mainly refers to cell necrosis, which involves the passive death of cells upon exposure to physical or chemical stimuli in the environment. 19 PCD can be blocked by inhibitors of cellular signal transduction, whereas NPCD cannot. 20 Pyroptosis is triggered by various pathological stimuli, such as oxidative stress, hyperglycaemia (HG), inflammation, and is crucial for controlling microbial infections. At present, pyroptosis can be observed in monocytes, macrophages, dendritic cells, VSMCs, vascular endothelial cells (VECs), CMs, CFs and many other cell types. 21 Pyroptosis is distinct from other forms of CD, such as apoptosis and autophagy, in morphology and mechanism ( Table 1). The main difference between apoptosis and pyroptosis lies in the caspase involved.
Apoptotic caspases mainly include caspase-2,8,9,10 (apoptosis initiation) and caspase-3,6,7 (apoptosis execution). 22 Apoptosis does not form a cell membrane pore mediated by Gasdermin D-N (GSDMD-N) and releases inflammatory factors. 23 Kerr et al 24  Neither the cytoplasmic contents will be released outside the cell nor any inflammatory reactions triggered during this process. 26 Autophagy is a conserved intracellular degradation pathway that delivers cytosolic contents to lysosomes via double-membrane autophagosomes to degrade longevity proteins, misfolded proteins and excess or defective organelles. 27 Autophagy in most tissues occurs at the basal level and contributes to the renewal of cytoplasmic components. It plays a role in the development, differentiation and tissue remodelling of various organisms. Autophagy is highly induced in pathological conditions and increased by more than 10 times. Cell necrosis is a passive process of acute CD mainly caused by physical and chemical stimulation, often leading to cell swelling, rupture and uncontrolled release of inflammatory contents. 28 Necroptosis is also a pro-inflammatory CD that occurs under caspase-8 inhibition, allowing the activation of the receptor interacting protein kinase 1-receptor interacting protein kinase 3-mixed lineage kinase domain-like axis. 29 Necroptosis is a regulated form of cell death morphologically characterized by cell and organelle swelling, which ultimately culminates in the loss of plasma membrane integrity and mild chromatin condensation but intact nuclei. 30 Pyroptosis is characterized by rapid plasma membrane disruption, followed by release of cellular contents and pro-inflammatory mediators, including IL-1β and IL-18. 31 Unlike most cytokines, IL-1β and IL-18 are not secreted by the classical endoplasmic reticulum-Golgi pathway but are produced as biologically inactive precursor proteins that are cleaved prior to their secretion as bioactive cytokines. 32 IL-1β is synthesized initially as an inactive precursor molecule (pro-IL-1β p35), which must be cleaved by caspase-1 at amino acid position 116 to produce the actively mature IL-1β (p17). 33 Mature IL-1β is a pro-inflammatory mediator that recruits innate immune cells to infection sites and modulates adaptive immune cells the production of interferon-γ and potentiation of cytolytic activity of natural killer cells and T cells (Th17 cells) and may polarize T cells towards Th1 or Th2 profiles in combination with other cytokines. 34 Pyroptosis mainly includes the canonical pathway of caspase-1 dependence and the non-canonical pathway involving caspase-4,5 (human) and caspase-11 (mouse; Figure 1). 35 The cells activate their respective inflammasomes, including NLRP3, absent in melanoma 2 (AIM2), or pyrin through the action of pathogen-associated molecular patterns (PAMPs) and danger-associated molecular patterns (DAMPs) under the stimulation of hyperlipidaemia, HG and inflammation ( Figure 2). After the activation of NLRP3, the N-terminal pyrin domain (PYD) of NLRP3 serves as a scaffold to nucleate apoptosis-associated speck-like protein containing a caspase activation and recruitment domain (ASC), which contains a pyrin domain and a caspase activation and recruitment domain (CARD).
Through its pyrin domain, ASC interacts with sensor molecules, and the CARD domain interacts with pro-caspase-1 (p45) and initiates pro-caspase-1 self-cleavage to form a caspase-1 mature body (p10/p20 tetramer). On the one hand, activated caspase-1 recognizes inactive IL-β and IL-18 precursors and converts them into mature inflammatory cytokines. On the other hand, caspase-1 cleaves GSDMD (a member of the Gasdermin protein family consisting of more than 500 amino acids) and oligomerizes 31 kDa amino-terminal products (GSDMD-N) that mediate the formation of membrane pores. The formation of membrane pores promotes the release of inflammatory factors, cell swelling and, finally, pyroptosis. 36,37 In the non-canonical pathway (caspase-1-independent pathway), then activates the IκB kinase complex to release NF-κB from IκBαmediated inhibition and then activates NLRP3 inflammasome to initiate pyroptosis. 15,40 F I G U R E 1 Caspase-1-dependent and independent pyroptotic pathway. In caspase-1-dependent pyroptosis pathway, the cells activate their respective inflammasome (including NLRP3, AIM2 or pyrin) through the action of pathogen-associated molecular patterns (PAMPs) and danger-associated molecular patterns (DAMPs), under the stimulation of hyperlipidaemia, hyperglycaemia and inflammation; NLRP3 oligomerizes and recruits ASC and pro-caspase-1, triggering the activation of caspase-1 and the maturation and secretion of proinflammatory cytokines such as IL-1β and IL-18. GSDMD-N formed by inflammatory caspase cleavage then mediates cell membrane pore formation, and promotes inflammatory factor release, cell swelling and pyroptosis. In caspase-1-independent pyroptosis pathway, Gramnegative bacterial cell wall component LPS activates caspase-4/5/11 pathway to mediate cell pyroptosis Nek7 is an essential component of NLRP3 inflammasome activation. Nek7 has been primarily characterized as a factor regulating microtubule network nucleation and spindle formation during mitosis. 41 In the pyroptosis system, Nek7 could be involved in the formation or provision of a common signal that functions upstream of NLRP3. Nek7 as a regulator of microtubule dynamics could facilitate the interaction between NLRP3 and ASC. 42 Morphologically, pyroptosis appears to be a combination of apoptosis and necrosis and involves the loss of plasma membrane integrity and the release of cellular contents. The GSDMD-N domain mediates the formation of small pores with a diameter of about 10-14 nm in the plasma membrane. Of note, these pores are wide enough for the passage of mature IL-1β (4.5 nm) and caspase-1 (7.5 nm). The cells appear to be osmotically swollen and form spherical vesicles around the nucleus. 43 As the cell expands, the nucleus becomes spherical and condensed, and the DNA fragments. As with apoptosis, the pyroptosis TUNEL assay is also positive. Pyroptosis can also be observed by electron microscopy, lactate dehydrogenase (LDH) release and Hoechst 33342/PI double-staining, etc. 44

| NLRP3 inflammasome
The primary function of the innate immune system is to maintain homeostasis, which is partly achieved through immunological sur-  6) The adipokine visfatin activates the NLRP3 inflammasome to trigger inflammasome activation directly or indirectly through an uncharacterized pathway, especially in obesity-related diseases RIG-I-like receptor (RLR), the AIM2-like receptor (ALR) and the NLR proteins. 46 Inflammasomes are a group of intracellular protein complexes which include NLRs, ASC, caspase-1 and sometimes caspase-11. 47 NLRP family members, including NLRP1, NLRP3, NLRC4, AIM2 and the adaptor ASC, are the critical components of the inflammasome, linking microbial and endogenous "danger" signals to caspase-1 activation. 37,48 The best-characterized inflammasome is the NLRP3 inflammasome multiprotein complex. It comprises the NLR protein NLRP3, the adapter ASC and the pro-caspase-1. Once activated, the inflammasomes act as platforms to trigger caspase-1, cytokine release and pyroptosis. 49 The mechanism underlying the activation of the NLRP3 inflammasome remains controversial. At present, at least three models are widely accepted. The first model assumes that pore formation allows extracellular NLRP3 agonists to enter the cytosol and directly activate NLRP3. The second mode is mediated by lysosomal rupture.
Phagocytosis of particles (such as silica and asbestos) or live pathogens leads to lysosome rupture, releasing cathepsin B (CSTB) or a protein modified by CTSB. 50 Oxidative stresses are also associated with CTSB activity; excessive ROS generation triggers CTSB release to activate the NLRP3 inflammasome. 51

Cathepsin inhibition abolishes the interaction between NLRP3 and
ASC. Furthermore, CTSB has been described in some settings to directly cleave caspase-1 and caspase-11, mediating both canonical and non-canonical pyroptosis. 55,56 In the third mode, the NLRP3 agonist (eg, HG, hyperlipidaemia) triggers the production of reactive oxygen species (ROS) and further activates the NLRP3 inflammasome. 58 In addition to the above three modes, the adipokine visfatin (a major injurious adipokine during obesity) is also thought to activate the NLRP3 inflammasome to induce pyroptosis. 59 This mode plays an important role in vascular endothelial oxidative stress injury and participates in various obesity-related diseases, such as diabetes 60 and coronary atherosclerosis. 61 Moreover, potassium release is associated with all NLRP3 activators, and low-potassium medium alone is sufficient to trigger NLRP3 activation. 62 However, whether NLRP3 directly senses this low level of potassium remains to be determined.
Among the three mechanisms, the third one is the most closely related to CVDs. The source of ROS is currently not clearly understood.
In general, the main source of intracellular ROS is mitochondria and mitochondria produce ROS including electron transport chains and non-electron transfer chains. 63 The electron transport on the mitochondrial electron transport chain is carried out in a step-by-step manner, which dramatically increases the chance of ROS production.
Complexes I and III on the electron transport chain are considered the primary sites for ROS production. In addition to the site of ROS generation in the electron transport chain, other ROS production sites, such as cytochrome b reductase and monoamine oxidase, exist in the mitochondria. 64 In addition to the ROS generation system, the body also has a ROS clearance system, which includes ROS scaveng-

| PYROP TOS IS IN C ARD I OVA SCUL AR DISE A SE
Adult CMs are post-mitotic cells with insufficient ability to respond to injury. In general, acute injury often leads to various types of CD, whereas chronic stress mainly leads to hypertrophy and myocardial remodelling. Increasing evidence shows a slow turnover in normal myocardium maintained by stem cells. However, under pathological conditions, death beyond the mitosis of CMs leads to heart dysfunction. 2 Pyroptosis is involved in various CVDs (eg, As, MI and cardiomyopathy) by mediating CD and inflammation. Intervention in pyroptosis-related molecules (eg, caspase-1, NLRP3, GSDMD and ASC) can significantly affect CVD progression and outcomes. Therefore, an in-depth understanding of the role and molecular mechanisms of pyroptosis in CVDs can provide new potential targets for treatment.

| Atherosclerosis
As is a chronic progressive disease characterized by abnormal lipid deposition in the aorta, obstructing blood flow and subsequent plaque rupture that causes coronary heart disease (CHD) and stroke.
As remains the primary cause of morbidity and mortality worldwide. 74 Both innate and adaptive immune responses, which mainly involve monocytes, macrophages, neutrophils, T lymphocytes and B lymphocytes, are essential for the initiation and progression of As. 75 In summary, As may be considered a chronic inflammatory disease caused by interactions among modified lipoproteins, monocyte-derived macrophages, T cells and ECs. CD can be observed in As and plays a vital role in the development and progression of As lesions.
Pyroptosis is involved in the formation and progression of As by promoting the release of inflammatory factors and is closely related to the stability of the plaque. 44 The most well known of many inflammasomes is NLRP3, which is thought to bridge the gap between lipid metabolism and inflammation because of cholesterol crystals, and oxidized low-density lipoprotein (oxLDL) can activate the NLRP3 inflammasome to induce pyroptosis. Despite this, there are also reports that the NLRP3 inflammasome does not play an important role in As because the absence of NLRP3 does not affect the progression of As lesions and plaque stability in high-fat diet (HFD)-fed ApoE −/− mice (a commonly used animal model of As). 76

| Vascular endothelial cell pyroptosis in atherosclerosis
Vascular endothelial cells are barriers between blood and vascular wall. VEC damage is considered the starting point of As lesions.
Endothelial injury is often accompanied by different types of CD, such as autophagy, apoptosis, pyroptosis and necrosis. Vascular wall integrity is destroyed after pyroptosis, causing local lipid deposition, As formation and plaque instability, and even acute coronary occlusion and sudden death. 77 Caspase-1 is abundantly expressed in human As plaques; in fact, the caspase-1 content in vulnerable plaques and ruptured lesions in patients who died of acute coronary events is significantly increased. 78 Thus, pyroptosis is involved in As formation and plaque hardening.  of target mRNAs to either facilitate their degradation or repress their translation. 85 In the present study, we find that miR-125a-5p mediates oxLDL-induced pyroptosis in VECs by down-regulating tet methylcytosine dioxygenase 2 (TET2), increasing NF-κB activation, activating NLRP3 and caspase-1 p20, and ultimately causing pyroptosis of VECs. After TET2 down-regulation, abnormal DNA methylation occurs, and mitochondrial dysfunction subsequently induces ROS production, which activates the NLRP3 inflammasome, leading to the activation of caspase-1. Activated caspase-1 promotes GSDMD oligomerization, which triggers pore formation of the membrane, DNA fragmentation and release of mature IL-1β and IL-18 from cells, causing a sterile inflammatory response and further contributing to pyroptotic cell death and subsequently promoting As. 86

| Monocyte/macrophage pyroptosis in atherosclerosis
The role of innate and adaptive immune factors in As is gradually being The necrotic core is covered by a fibrous cap, and its "shoulder" region is infiltrated by activated T cells, macrophages and mast cells, which produce pro-inflammatory mediators that can render plaques unstable and can cause rupture of the fibrous cap, leading to vascular embolization and tissue infarction. 87 Although the death of macrophages in early As lesions is beneficial, the reduction in the number of these cells in the plaque can attenuate the inflammatory response and reduce the synthesis of matrix metalloproteinases. However, death of macrophages in advanced lesions promotes the formation of necrotic cores and the instability of As plaques. Macrophage death in As lesions causes the release of growth factors, cytokines, proteases and intracellular lipids to the inflammatory response; promotes plaque rupture and thrombosis; and causes acute cardiovascular events. 88,89 Serum total cholesterol and low-density lipoprotein cholesterol (LDL-C) are risk factors for CHD, and oxLDL has a stronger effect to As. 90 OxLDL-induced macrophage pyroptosis plays an important role in As formation and plaque stability. OxLDL and cholesterol crystals in the plaque necrosis area can activate NLRP3 and caspase-1 to induce cell pyroptosis. This phenomenon causes the release of IL-18 and IL-1β in mouse macrophages, which exacerbates inflammation and As 91 ( Figure 4). Triglycerides are also another As risk factor, which can trigger pyroptosis and aggravate the disease. 92 IL-1β of the IL-1 family is an important pro-inflammatory cytokine that is mainly produced by activated monocytes/macrophages. 93

| Ischaemic heart disease
Many CVDs accompany CD, and As and MI may be the most closely related to pyroptosis because they are often accompanied by CD A recent study has revealed an important endogenous inhibitor of inflammation, namely activated protein C (aPC), which can reduce infarct size in mice with MI. In vitro, aPC inhibits NLRP3 inflammasome activation in macrophages, CMs and CFs via proteinase-activated receptor 1 (PAR-1) and mammalian target of rapamycin complex 1 signalling. The mTOR pathway is related to energy metabolism, and mTOR activation can inhibit autophagy. 108 PARs are members of the G protein-coupled family, and four members of the PAR family have been discovered so far: PAR1-4. PARs represent a component of the innate inflammatory response, being involved in neutrophil recruitment, increased perfusion, pain and swelling. They reportedly serve as the first "alert system" for bacterial invasion. PAR1 is expressed by platelets, osteoblast, ECs, epithelial cells, fibroblasts, myocytes, neurons and astrocytes, and it plays an important role in injured tissues. 109 Diabetes is one of the risk factors for CVDs. Accompanied by mitochondrial swelling and sarcoplasmic reticulum expansion, left ventricular ultrastructure abnormalities and myocardial fibrosis are more severe in diabetic MI rats compared with non-diabetic rats. The creatine kinase isozyme CK-MB and LDH release are significantly higher in diabetic rats than in non-diabetic MI rats under the same conditions. HG promotes NLRP3 inflammasome-mediated pyroptosis and aggravates IRI by causing mitochondrial dysfunction, leading to enhanced ROS production. ROS induces the release of inflammationrelated signalling factors, such as NF-κB, and the subsequent NLRP3 inflammasome activation triggers sterile inflammation and pyroptosis. 110 Activation of inflammasomes can be inhibited by antioxidants, such as SIRT-1. Inhibition of the NLRP3 inflammasome or reduction of ROS production can significantly reduce myocardial IRI. 13,111 In addition to CMs, CFs also play an important role in the maintenance of cardiac physiological functions. CFs are the most abundant cell type in the adult human heart, and they considerably affect the structure and function of the heart. 112 Øystein et al reported that the NLRP3 inflammasome is up-regulated in CFs and mediates myocardial IRI. 113 However, the role of CFs in CVDs remains to be further studied.

| Diabetic cardiomyopathy
Diabetic cardiomyopathy (DCM) is one of the major complications of diabetes and is also the leading cause of death in diabetic patients. DCM is characterized by structural and functional impairments, including cardiomyocyte death, cardiac fibroblast activation, left ventricular dysfunction and metabolic disorders. Among them, the death of CMs and CFs is considered a fundamental change in DCM, which initiates cardiac remodelling and leads to left ventricular dysfunction. 114

| Cardiomyocyte pyroptosis in DCM
Hyperglycaemia-induced ROS overproduction promotes the activation of the NLRP3 inflammasome by NF-kB and thioredoxin-interacting protein (TXNIP). 16 TXNIP, also known as thioredoxin binding protein-2, is a ubiquitously expressed protein that interacts and negatively regulates the expression and function of thioredoxin (TXN).
TXNIP is closely related to energy metabolism. TXNIP influences glucose metabolism by affecting hepatic glucose production and peripheral glucose uptake and regulating beta cell function. In addition, overexpression of TXNIP induces the apoptosis of pancreatic β-cells, reduces the sensitivity of peripheral tissues, such as skeletal muscle and fat, to insulin, and minimizes energy expenditure. 15,115 The inflammatory response is involved in the development of DCM. Studies have shown that IL-1β is an essential pro-inflammatory cytokine in the development of DCM. The NLRP3 inflammasome also plays a crucial role in the inflammatory process in diabetic nephropathy and retinopathy. 116 In mammals, the Hu/ELAV RNA binding protein family consists of four highly conserved members, including HuR/HuA/Elavl1 and the neuronal-specific Hel-N1/HuB/Elavl2, HuC/Elavl3 and HuD/ Elavl4. All family members contain an RNA recognition motif with high affinity for U-and AU-rich sequences (AREs). 117 ELAVL1 is a member of the RNA binding protein family, which binds to ARE-and U-rich element (URE)-containing sequences and stabilizes mRNAs. Long non-coding RNA is a type of RNA with a length of >200 nucleotides and non-coding protein. 122 In recent years, many reports have focused on the association of lncRNA with pyroptosis in CVDs, such as maternally expressed gene 3 (MEG3) 6 and metastasis-associated lung adenocarcinoma transcript 1 (MALAT1). 17 The KCNQ1

| Cardiac hypertrophy
The impairment of cardiac function caused by cardiac hypertrophy severely affects the quality of life of patients, but the underlying molecular mechanism remains unclear. In addition to hypertrophic cardiomyopathy, the most common cause of ventricular hypertrophy is long-term uncontrolled systolic hypertension and heart valve stenosis. 130

| ADVAN CE S IN DRUG APPLI C ATI ON TO IMPROVE C VDS BY INTERFERING PYROP TOS IS
Resveratrol (RSV) is a natural polyphenol, which protects heart tissue from damage and has anti-inflammatory, antioxidant, anti-ageing and anti-cancer properties. 85  Trimetazidine (TMZ) is an anti-ischaemic drug that significantly reduces intracellular acidosis and apoptosis, thereby protecting mitochondrial function and myocardium; TMZ is widely used to treat MI and other ischaemic heart diseases. 132 Sepsis is a life-threatening organ dysfunction syndrome caused by a host's dysfunctional response to infection and is one of the most common causes of death in hospitalized patients. 133 Cardiac dysfunction is a common complication of sepsis and an important cause of death. 134   In the cardiovascular system, the pyroptosis of VECs causes the disintegration of blood vessel walls, which consequently promotes the occurrence of As and embolism induced by lipid deposition. 143 The pyroptosis of VSMCs results in unstable atherosclerotic plaques, acute coronary syndrome and stroke; monocytes/macrophages pyroptosis aggravates the inflammatory response and promotes the development and progression of various CVDs (such as As and MI). 144 In the recent decade, research on pyroptosis and CVDs has pro- In the present review, in addition to the As, we mainly summarize the role of cardiomyocyte pyroptosis in CVDs. However, non-myocyte cell types in the myocardium, including CFs and mast cells, also play a crucial role in CVDs (eg, hypertensive heart disease and MI).
The heart is composed of 70% non-myocytes and 30% myocytes. 112 So far, few studies investigated the role of non-myocytes pyroptosis in CVDs. Thus, this research gap may be the focus of future studies. In the following years, we expect the development of new approaches on controlling the different forms of CD in clinical practice to provide new treatment strategies for patients with CVDs.

ACK N OWLED G EM ENTS
This study was supported by the Natural Science Foundation

CO N FLI C T O F I NTE R E S T
The authors declare that they have no conflict of interest.