The Role of HMGB1 in Cardiovascular Biology: Danger Signals.

Antioxidants & Redox Signaling The Role HMGB1 in Cardiovascular Biology: Danger Signals (doi: 10.1089/ars.2015.6408) This article has been peer-reviewed and accepted for publication, but has yet to undergo copyediting and proof correction. The final published version may differ from this proof.

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Forum Review Article Title: The Role HMGB1 in Cardiovascular Biology: Danger Signals Jingjing Cai#1,2,3, Juan Wen#1,3, Eileen Bauer2, Hua Zhong1,2,3, Hong Yuan1,3*, Alex F. Chen1,2* 1. The Center of Clinical Pharmacology of the Third Xiangya Hospital, Central South University, Changsha, China 2. Department of Surgery, University of Pittsburgh School of Medicine, Pittsburgh, USA 3. Department of Cardiology, the Third Xiangya Hospital, Central South University, Changsha, China # Equal contribution * Corresponding author

Running title: HMGB1 and Cardiovascular Biology Alex F. Chen Mailing address: W1114 Biomedical Science Tower, Department of Surgery, University of Pittsburgh School of Medicine, 200 Lothrop Street, Pittsburgh, PA 15213 Telephone number: 412-360-6635, 412-624-7784 Fax number: 412-383-5946 Email address: [email protected], [email protected] Hong Yuan Mailing address: 138 Tongzipo Road, the Third Xiangya Hospital, Central South University, Changsha, China, 410013 Telephone number: 86-73188618339 Fax number: 86-73188618339 Email address: [email protected] Word count: 6786(words), 40668(characters), 47398(characters and space between words) Reference numbers: 209 Number of Color illustrations: 6 Table: 1

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2 Abstract Significance: Cardiovascular disease (CVD) is the leading cause of mortality worldwide. Accumulating evidence shows that dysregulated immune response contributes to several types of cardiovascular diseases such as atherosclerosis and pulmonary hypertension (PH). Vascular intimal impairment and low-density lipoprotein oxidation trigger a complex network of innate immune responses and sterile inflammation. Recent Advances: High-mobility group box 1 (HMGB1), a nuclear DNA-binding protein, was recently discovered to function as a damage-associated molecular pattern molecule (DAMP) that initiates the innate immune responses. These findings lead to the understanding that HMGB1 plays a critical role in the inflammatory response in the pathogenesis of CVD. Critical Issues: In this review, we highlight the role of extracellular HMGB1 as a proinflammatory mediator as well as a DAMP in coronary artery disease, cerebral artery disease, peripheral artery disease and pulmonary hypertension. Future Directions: A key focus for future researches on HMGB1 location, structure, modification, and signaling will reveal HMGB1’s multiple functions and discover a targeted therapy which can eliminate HMGB1-mediated inflammation without interfering with adaptive immune responses.

Keywords HMGB1, DAMP, Inflammation, Coronary disease, Cerebral artery disease, Peripheral artery disease and Pulmonary hypertension.

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3 Introduction It becomes increasingly clear that inflammation plays an essential role in the initiation and progression of cardiovascular disease (CVD)(102,116,209). Both the innate and acquired immune systems are involved in CVD, as evidenced by the presence of macrophages, dendritic cells (DCs), and B and T lymphocytes in vascular lesions. Over the

last

decade,

a

new

class

of

pro-inflammatory

molecules

termed

damage-associated molecular pattern molecules (DAMPs) have emerged as important inflammatory mediators. DAMPs are endogenous molecules ranging from proteins to small molecules that are actively secreted or passively released by activated immune cells or damaged cells. Once released into the extracellular milieu, DAMPs promote inflammation and tissue repair. High mobility group box 1 protein (HMGB1), also known as amphoterin or HMG1, is a prototypical DAMP that has recently been implicated in the pathogenesis of CVD(169), including in atherosclerosis, acute coronary syndromes, and pulmonary hypertension. HMGB1 is a ubiquitous nuclear protein constitutively expressed in most cells. Under physiologic conditions, it acts as a structural component in the chromatin complex where it is involved in shaping nucleosomal structure and influencing multiple processes in the chromatin such as nucleosome stability and sliding, nucleosome number and genome chromatinization, nuclear catastrophe and nucleosome release, DNA binding, replication, repair and bending, and gene transcription and stability (2,161,162) (Figure 1). However, in response to cellular stress, numerous immune and non-immune cells release HMGB1 into the extracellular space where it acts as a DAMP. When

outside

the

cell,

HMGB1’s

DAMP-like

functions

range

from

pro-inflammatory effects (cytokine-like or chemotaxis) (178) to tissue regeneration (133) and even feedback suppression of cellular responses (80). This wide range of biological activities arises from different HMGB1 isoforms being able to mediate signalling through 3


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4 at least 10 different receptors. Receptors shown to interact with HMGB1 include receptor for advanced glycation end-products (RAGE), Toll-like receptors (TLRs), proteoglycans, macrophage antigen-1, syndecan-3, CD24-Siglec-10, CXCR4, integrins, and TIM-3. For some receptors, HMGB1 also interacts with coreceptors or other surface molecules (176). This review will discuss recent advances in our understanding of the DAMP-like properties of HMGB1 in the setting of vascular diseases. Recent discovery of HMGB1 as a critical mediator in vascular diseases, such as atherosclerosis (64,78,161), myocardial ischemia reperfusion injury (6,130) , heart failure (180,181), acute coronary syndrome (94,158,195), pulmonary artery disease (12,113,114), cerebral artery disease (55,97,125) and peripheral artery diseases (15,131) has stimulated tremendous interest in this field. HMGB1 can serve as an early proinflammatory mediator in the context of sterile inflammation, with release occurring as a consequence of acute cellular stressors such as hypoxia or necrosis (90). 1. Structure and Redox State of the HMGB1-Mediating Inflammatory Response HMGB1 is expressed as a single polypeptide chain of 215 amino acid residues organized into three domains that include two internal repeats of positively-charged DNA binding domains (HMG A box [9-79aa], HMG B box [95-163aa]) in the N-terminus, and a continuous stretch of negatively-charged domains in the C-terminal acidic tail (186-215aa) (4,19,171). The two HMG boxes, A and B, are similar 80 amino acid segments (29% identical, 65% similar) and form an L-shaped structure (52,141). HMGB1 is an evolutionarily highly-conserved protein in mammals and amino acid sequences of all mammalian HMGB1 proteins are virtually identical (N99%), implying similar and important biological functions in distinct organisms. The different domains of HMGB1 interact with distinct receptors and these interactions are essential for extracellular HMGB1’s biological activity. Initial studies of the structural basis for the extracellular proinflammatory cytokine activity of HMGB1 4


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5 revealed a pivotal role for residues in the B box domain (159). For example, residues 89-108 and residues 7-74 are responsible for binding to TLR4 and p53 transactivation domain, leading to inflammatory cytokine release and gene transcription, respectively (100). AA residues 150 and 183, also in the B box, are involved in the binding of HMGB1 with RAGE (10). The HMGB1 A box competes with full-length HMGB1 for binding sites, thus being able to attenuate the inflammatory cascade (119,159,199) and can function as an inhibitor of the intact HMGB1 (6). Residues 201-205 in the C-terminal acidic tail region are also responsible for the anti-inflammatory activity of HMGB1 (47). The anti-inflammatory activity of HMGB1 A box is enhanced when fused with the C-terminal acidic tail (48). The acidic amino acids within the C terminal tail may also protect the A-box and B-box during emigration from the nucleus and regulate DNA binding/bending by intramolecular interaction with the HMG boxes (17,145). (Figure 2) Recent studies have shown that the proinflammatory cytokine-stimulation response of HMGB1 is critically intertwined with a dynamic redox environment and posttranslational redox modifications. Cysteine is uniquely suited to sense a range of redox signals, as the thiol group (-SH) can be oxidized to several different reversible redox status (49). The redox status of three cysteines encoded at positions 23, 45, and 106 are critical for HMGB1’s proinflammatory activity. Specifically, all-thiol HMGB1 forms a heterocomplex with CXCL12, which signals via the CXCR4 receptor complex, causing cell migration. Studies have shown that during inflammation, reactive oxygen species (ROS) can result in the formation of disulfide bonds between C23 and C45 (20,148). The disulfide HMGB1 exerts cytokine’s activity, triggering inflammation through TLR4/MD2/CD14 receptor complex in macrophages and other cells (87,197). Continuous and high ROS levels eventually induce the terminal oxidation of HMGB1, which abrogates its cytokine-like activity (80) (Figure 3). Disulfide -HMGB1 does not compete with all-thiol HMGB1 for cell migration and all-thiol HMGB1 does not compete

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6 with disulfide -HMGB1 in cytokine stimulation. Thus, thiol modification can serve as a molecular switch for HMGB1 to function as a chemoattractant or as a proinflammatory cytokine through interaction with different receptors, CXCR4 (153) or TLR4 (198), respectively (Figure 3). C106 in Box B is required for HMGB1 nuclear location because C106 mutation, but not C23 and C45 mutations, promotes HMGB1 translocation from the nucleus to the cytosol. The native state of HMGB1 is rapidly lost via oxidation of sulfhydryl groups during storage (92). Compared with oxidized HMGB1, reduced HMGB1 exhibits a stronger affinity for distorted DNA structures (137). ROS is a major signal that decreases nuclear HMGB1 DNA binding activation, which in turn promotes cytoplasmic translocation and release. 2. HMGB1 Release As both a nuclear factor and a secreted protein, HMGB1 resides in the nucleus of almost all cells. At baseline, it can be rapidly mobilized to different compartments within the cell or be released into the extracellular space. Translocation of HMGB1 from the nucleus to the cytosol, mitochondria, or lysosomes following various stressors regulates cellular processes such as autophagy and apoptosis (14,21,43,109,166-168,190) (Figure 1). HMGB1, released into the extracellular space, results in a similarly large number of interactions and functions. HMGB1 can be actively secreted from multiple cell types, including macrophages, monocytes, hepatocytes (174,175), natural killer (NK) cells, DCs, endothelial cells, and platelets (53). HMGB1 can be released into the extracellular space by an active process or passively released from cells that are undergoing necrosis, apoptosis, pyroptosis, and NETosis. (5,175). In the extracellular milieu, HMGB1 interacts with other soluble molecules, cellular receptors, and other surface molecules, endowing it with the chemokine- or cytokine-like activity. In this capacity, HMGB1 has been shown to influence cell migration, proliferation, and differentiation; tissue regeneration, angiogenesis, bacterial killing, and cell senescence (79) (Figure 1). 6


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7 2.1 Active Release of HMGB1 Active release of HMGB1 occurs when monocytes, macrophages (17), NK cells (155), DCs (16), pituicytes (183), endothelial cells (38,173), platelets (70,145) and other immunological cells are exposed to microbe-associated molecular patterns (MAMPs), pathogen-associated molecular patterns (PAMPs), DAMPs, or inflammatory cytokines such as IL-1 and TNF (Figure 4) . Active secretion of HMGB1 requires the translocation of HMGB1 from the nucleus to the cytosol and the prevention of newly-synthesized HMGB1 from being shuttled from the cytoplasm into the nucleus prior to its release from the cell. HMGB1 can not translocate directly from the endoplasmic reticulum and Golgi apparatus to the cell membrane after synthesis due to the lack of a leader peptide, which is required for transport through the classical secretory pathway. Post-translational modifications such as phosphorylation, methylation, and acetylation are involved in the nucleo-cytoplasmic shuttling of HMGB1 (17,69,203). Acetylation of the lysine residues and phosphorylation of both nuclear localization signals for controlled nuclear transport regions of HMGB1 was found to prevent HMGB1 from interacting with the nuclear-importer protein complex and re-entering the nucleus. Cytoplasmic HMGB1 is taken up by secretory lysosomes and then actively secreted by activated cells during immune responses (17,35,109). 2.2 Passive Release of HMGB1 2.2.1 Necrosis Biologically active HMGB1 can be passively released by necrotic and damaged cells following disruption of nuclear and cell membranes (28,151). As a chromatin protein, HMGB1 binds tightly to nucleosomes reconstituted from purified DNA and histones under physiological conditions. However, HMGB1 is bound loosely to the chromatin of 7


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8 interphase cells, mechanically damaged cells, or under necrosis. This allows HMGB1 to diffuse into the extracellular space and stimulate production of TNF and other cytokines (151,174) (Figure 3). Previous studies have demonstrated that post-translational modification of HMGB1 by methylation at Lys42 and the acidic tail modified by poly (ADP)-ribosylation is required for relocation of HMGB1 from the nucleus to the cytoplasm during necrosis (30,69). The extracellular environment during necrosis may impact the biochemical and oxidative properties of released HMGB1. HMGB1 is reduced inside the cell nucleus. However, it can be oxidized extracellularly to disulfide–HMGB1 form by the oxidative milieu following inflammation and the immune response (178). 2.2.2 Apoptosis Early studies indicated that apoptotic cells failed to release HMGB1 even after the secondary necrosis, as apoptotic cells fail to initiate an inflammatory response in macrophages [19]. However, recent studies reveal that cells undergoing apoptosis release large amounts of HMGB1. However, HMGB1 released from apoptotic cells has less immunological activity compared with HMGB1 released passively during necrosis (80). The disparity between the modes of cell death underlies the important differences in the ability of necrotic cells and apoptotic cells to activate an inflammatory response. Studies have shown that the ROS generated by mitochondria in apoptotic cells oxidize the cysteine at position 106 within Box B and suppress the proinflammatory activities of HMGB1 (80). 2.2.3 Pyroptosis Pyroptosis is a form of programmed cell death associated with antimicrobial responses, which requires the function of the enzyme caspase-1. In this process, HMGB1 is actively released from immune cells through protein kinase R (PKR), RAGE, and dynamin-dependent signalling (109,190). Initial studies showed that HMGB1 released by pyroptosis contains active (disulfide-bonded) and terminally oxidized forms, whereas 8


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9 HMGB1 released during necrosis contains both disulfide- and all-thiol forms (109). Recent work from Nystrom et al., however, showed that macrophages undergoing unprimed NLRC4 inflammasome pyroptosis release HMGB1 in the all-thiol redox form, as this process is independent of mitochondrial involvement and does not produce inflammatory cytokines, nitric oxide, or ROS. In contrast, NLRC4-induced cells primed with cell surface TLR ligands (but not endosomal TLRs) can release both all-thiol HMGB1 and disulfide HMGB1. Of note, HMGB1 showed acetylation with or without LPS priming (129). These results suggest that TLR or inflammasome activation determines the different isoform of HMGB1 released from the cell in immune responses. 2.2.4 NETosis The formation of extracellular traps (ETs) by neutrophils and mast cells is an important mechanism in the innate immune response. These structures consist of a chromatin-DNA backbone with attached antimicrobial peptides and enzymes that trap and kill microbes. After stimulation of neutrophils and mast cells with phorbol esters, chemoattractant peptides, or chemokines, the generation of ROS by NAPDH (nicotinamide adenine dinucleotide phosphate [reduced form]) oxidase initiates a signaling cascade that leads to the disintegration of the nuclear and cellular membranes and the formation of ETs. This form of cell death is termed "ETosis” (189). Neutrophils can display another, more dramatic response to stimuli such as bacteria, LPS, and cytokines, undergoing of a process termed NETosis. This process culminates in cell death, releasing structures called neutrophil extracellular traps (NETs). NETosis is another form of regulated cell death that occurs primarily with neutrophils (18). HMGB1 is a component of NETs, which also showed great ability to produce NET formation in vitro and in vivo (81,122). The presence of HMGB1 in NETs underlies antibacterial action which providing both a physical trapping mechanism and localized killing. The process of NETosis, with or without cell death, can be a source of extracellular HMGB1 9


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10 release into the tissue and serve as a biomarker. NET formation occurs via both NADPH oxidase-dependent and –independent mechanisms. In the context of NADPH oxidase activation, the highly oxidizing environment may lead to HMGB1 inactivation; however, the biochemistry of HMGB1 released during NETosis has not yet been analyzed in detail and its activity remains unknown (18). 3. Vascular disease and HMGB1 3. 1 Coronary Artery Disease and HMGB1 In light of the cytokine activities of HMGB1 in the proinflammatory response discussed above, accumulating evidence suggests that HMGB1 evoked by injury plays a pathogenic role in all stages of coronary artery disease (CAD), from initiation to plaque rupture and associated thrombotic complications. We reviewed both experimental and clinical studies of the role of HMGB1 in the pathogenesis and progression in CAD. Here, we illustrate this effect from subclinical CAD to acute coronary syndrome. 3.1.1 HMGB1 in Atherosclerosis Atherosclerosis can result in CVD, which is still the primary cause of morbidity and mortality in the world. Rather than just a process of lipoprotein accumulation, chronic inflammation evoked by injury is now believed to be the major pathogenic stimulus of atherogenesis (135). Inflammation plays a pivotal role in all stages of atherosclerosis, from initiation through progression and, ultimately, associated thrombotic complications (117). Atherosclerosis begins with endothelial dysfunction as a result of endothelial injury, and is followed by macrophage infiltration and fatty streak accumulation. Endothelial injury leads to compensatory responses that alter hemostatic properties of the endothelium and results in increased endothelial permeability and adhesiveness. This allows for the deposition of lipids into the intima, and induces the attachment and accumulation of monocytes, macrophages, and platelets on the vessel wall (143). 10


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11 Immune cells in turn produce and release proinflammatory cytokines and chemokines. The formation of advanced atherosclerotic lesions requires the mobilization of smooth muscles cells and platelet aggregation, leading to thrombosis and the clinical manifestations of atherosclerotic disease (103). As an important cytokine in the innate immunity, HMGB1 is shown to act as a core mediator in the pathogenesis of atherosclerosis. In response to primary endothelial cell injury, HMGB1 release initiates a pronounced inflammatory response driven by endothelial cells as well as infiltrated innate immune cells. HMGB1 seems to join in the formation of atherosclerotic lesion, as increased expression of HMGB1 is observed in different cell types, smooth muscle cells, endothelial cells, foam cells, macrophages, and activated platelets (76,138,145) in atherosclerotic lesions derived from both human autopsy specimens and experimental tissue in animals (68,76,78,138,145). Its function in plaque formation in the initial stages may be mediated by stimulating macrophage migration and modulating expression of proinflammatory mediators in endothelial cells such proinflammatory cytokines (TNF-α), chemokines (IL-8, MCP-1), adhesion molecules (ICAM-1 and VCAM-1), and macrophage inflammatory proteins (MIP-1α and MIP-1β) (9,68,78). Intriguingly, neutralizing HMGB1 treatment only had small effects on the expression of proinflammatory cytokines, which is not consistent with its effect on atherosclerotic plaque, suggesting that immune cells but not cytokines are the direct target of HMGB1 (78). Experimental studies show that HMGB1 stimulates chemotaxis in rat smooth muscle cells (28) and promotes cell proliferation, cell migration, and release of HMGB1 as well as C-reactive protein in smooth muscle cells (68,138). In addition, HMGB1 released by activated platelets may contribute to the adherence of platelets to vascular endothelial cells, an essential step in the formation of thrombus (145). HMGB1 also drives the process of plaque remodeling. Kanellakis et al. reported that monoclonal anti-HMGB1

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12 neutralizing antibodies attenuated atherosclerosis by 55% in apolipoprotein E-deficient mice. Furthermore, a decrease in macrophage, DC, and CD4+ T-cell accumulation in atherosclerotic lesions was observed when treated with anti-HMGB1 neutralizing antibodies (78). A recent clinical study also suggests that HMGB1 was higher in patients with non-calcified and remodeled plaques (8), which may cause chronic sub-clinical embolization of atherothrombotic debris, resulting in myocardial micro-necrosis and further release of HMGB1 by “stressed” cardiomyocytes. In addition to its priming role in the pathogenesis of atherosclerosis, clinical studies show that serum HMGB1 levels correlate with the severity of coronary artery stenosis. In one clinical trial, 104 patients were recruited and allocated into three groups according to symptoms, including stable angina pectoris (SAP), unstable angina pectoris (USAP), and control (without angina pectoris symptom). Results showed that serum HMGB1 level was markedly increased with severe coronary artery stenosis in patients with SAP and USAP, especially in SAP patients (64). 3.1.2 HMGB1 in Acute Coronary Syndrome 3.1.2.1 Experimental Studies To date, studies addressing whether HMGB1 exerts salutary or detrimental effects after myocardial infarction (MI) have generated conflicting results. In vitro, anoxia-reoxygenation challenge induced an increased myocardial expression of cytoplasmic HMGB1 in isolated cardiomyocytes. In mouse and rat models, increased heart and/or circulating HMGB1 levels and inflammatory responses have been demonstrated after experimental MI and ischemia reperfusion (I/R) injury (6,89,130). Some recent pre-clinical studies demonstrated a beneficial effect in ischemic heart disease, which not only acts as a cytokine in the process of myocardial I/R, but is also released as a “damage signal” in myocardial regeneration, promoting proliferation of resident cardiac c-kit+ stem cells and their differentiation into myocytes (104). Hu et al. 12


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13 showed that HMGB1 preconditioning significantly reduces the infarct size induced by I/R and attenuates proinflammatory response (65). Abarbanell et al. induced global myocardial I/R in rats with or without local injection of HMGB1 into the infarcted area at the start of reperfusion; a lower dose of HMGB1 manifested cardioprotective function in association with suppressed myocardial inflammation. Moreover, the administration of a HMGB1-neutralizing antibody before reperfusion resulted in an increased infarcted area and increased myocardial apoptosis (130,194). Another parallel experiment in mice with MI demonstrated the benefit of HMGB1 secreted from the infarcted, necrotic myocardium. Transgenic mice with cardiac-specific overexpression of HMGB1 were also shown to exhibit enhanced angiogenesis, improved cardiac function, and improved survival compared to wild-type mice after myocardial infarction (89). In contrast, some studies show a deleterious effect of HMGB1 in myocardial I/R injury. Andrassy et al. (6) reported that administration of HMGB1 in mice exposed to myocardial I/R injury resulted in increased inflammation and enhanced myocardial injury, which was attenuated by a functional HMGB1 antagonist. In particular, Tzeng et al. (177) demonstrated negative inotropic effects of HMGB1, which may contribute to excessive and/or sustained inflammation and/or profound myocardial depression and myocardial collapse. It is worth noting that total blockade of HMGB1 results in impaired healing (130). The mechanisms by which HMGB1 might be involved in the negative effects on myocardial function have been partially explained, with NF-κB, ERK, and PKCε suggested as potential cellular pathways. Along these lines, a recent study suggested that the heterogeneity in the reported effects of HMGB1 might be due to different mechanisms during conditions characterized either by permanent ischemia or I/R (45) and the dose of HMGB1 used in experiments.

3.1.2.2 Clinical studies

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14 Accumulating clinical data validate that HMGB1 acts as biomarker and therapeutic target in defined acute coronary syndrome (ACS). Serum HMGB1 levels are significantly increased in patients with acute MI. Elevated levels of serum HMGB1 were reported for the first time in a prospective observational study evaluating nine ACS patients, and was independent of creatine phosphokinase or troponin levels (46) (104). Kohno et al. (94) investigated the potential role of HMGB1 as a predictor of cardiovascular death in 35 patients presenting in the hospital with ST elevated MI (STEMI), compared to a control group of patients with chronic SAP. Serum HMGB1 levels were significantly increased in patients with MI, as detected at six, 12, 18, 24, 72 hours, and seven days after admission, and peaked at 12 hours after MI. A higher peak serum HMGB1 level was associated with pump failure, cardiac rupture, and in-hospital cardiac death. Another larger study investigated the potential role of HMGB1 in STEMI patients with occluded left anterior descending coronary artery successfully treated with primary percutaneous coronary intervention (PCI). The researchers recruited a highly homogeneous group of 141 patients with a 10 month follow up and healthy subjects serving as the control group. During the PCI procedure, plasma HMGB1 levels were higher in patients with MI (although not age-matched) compared to the levels in patients who died and who survived (4.8 g/mL vs. 2.9 g/mL). In a multivariate Cox regression analysis, plasma levels of HMGB1 were strongly associated with cardiovascular death after adjustment for age, sex, CK-MB, and TnI (hazard ratio: 1.75, 95% confidence interval: 1.1–2.8, P = 0.022) (157). HMGB1 functions as a risk marker and has also recently been reported in patients with non ST-elevation MI (NSTEMI) and UAP. A total of 258 patients were included in a parallel clinical trial and patients hospitalized for UAP or NSTEMI within 24 hours of the onset of chest symptoms were included. Within a median follow-up time period of 49 months, 38 patients (15%) suffered cardiovascular death. In a stepwise Cox regression

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15 analysis including 19 well-known clinical and biochemical predictors of ACS, HMGB1 [relative risk (RR) 3.24 per 10-fold increment; P = 0.0003], as well as cardiac troponin I, Killip class > 1, and age, but not hsCRP, were independently associated with cardiovascular mortality. The group concluded that a cut-off serum HMGB1 value of ≥2.4 ng/mL in patients with UA/NSTEMI serves as a high-risk predictor for cardiovascular death (54). Additional studies further support the notion of using HMGB1 levels as risk factor to predict infarct transmurality and functional recovery in patients after MI for NSTEMI and STEMI. Andrassy et al. (7) applied cardiac magnetic resonance imaging to estimate residual ventricular function at two to four days and six months after MI. An inverse correlation was observed between HMGB1 levels during MI and the residual ejection fraction both in STEMI and NSTEMI, respectively (r2=−0.40 and r2=−0.25; P

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