Acute liver failure (ALF) is marked by extensive inflammation and immune dysregulation, which are closely associated with neutrophil infiltration and NETosis. However, the specific mechanisms that drive NETosis in ALF remain poorly understood.

We employed flow cytometry, western blot, qRT-PCR, and cf-DNA assay to investigate the link between NETosis and ALF. The role of HDAC6-mediated deacetylation of histidine triad nucleotide-binding protein 2 (Hint2) was assessed, along with the effects of lentiviral vector-based overexpression and knockdown of Hint2 on mitochondrial function and NETosis. Additionally, CO-IP, IF, protein docking analysis, mCa2+ uptake assay, and mtROS measurement were used to explore the interaction between Hint2 and mitochondrial calcium uniporter complex (MCUc). Finally, experimental neutrophil depletion in mice was conducted to confirm the protective effect of NETosis inhibition in ALF.

Our study demonstrated that Hint2 undergoes HDAC6-mediated deacetylation, disrupting mitochondrial dynamics and triggering NETosis during ALF. Furthermore, MICU1 bridges Hint2 and NETosis by regulating mCa2+ homeostasis and mtROS production. Activation of Hint2, either through the HDAC6 inhibitor ACY1215 or via overexpression, increased the level of MICU1 to suppress the opening of the MCUc and the associated mtROS release, thereby inhibiting NETosis. Conversely, Hint2 knockdown induced NETosis by surging mCa2+ overload and mtROS production, while the MCUc inhibitor RU265 mitigates NETosis by blocking mCa2+ influx.

Our findings recognized the HDAC6/Hint2/MICU1 axis as a novel pathway in neutrophils, the inhibition of which intercepts mCa2+ overload and mtROS accumulation, thereby reducing NETosis and facilitating liver recovery during ALF.

The global rise in diabetes prevalence has significantly contributed to the increasing burden of atherosclerotic cardiovascular disease (ASCVD), a leading cause of morbidity and mortality in this population. Diabetes accelerates atherosclerosis through mechanisms such as hyperglycemia, oxidative stress, chronic inflammation, and epigenetic dysregulation, leading to unstable plaques and an elevated risk of cardiovascular events. Despite advancements in controlling traditional risk factors like dyslipidemia and hypertension, a considerable residual cardiovascular risk persists, highlighting the need for innovative therapeutic approaches. Emerging treatments, including sodium-glucose cotransporter 2 (SGLT2) inhibitors, glucagon-like peptide-1 (GLP-1) receptor agonists, epigenetic modulators, and RNA-based therapies, are showing promise in addressing the unique challenges of diabetes-associated ASCVD. Precision medicine strategies, such as nanoparticle-based drug delivery and cell-specific therapies, offer further potential for mitigating cardiovascular complications. Advances in multiomics and systems biology continue to deepen our understanding of the molecular mechanisms driving diabetes-associated atherosclerosis. This review synthesizes recent advances in understanding the pathophysiology and treatment of diabetes-related atherosclerosis, offering a roadmap for future research and precision medicine approaches to mitigate cardiovascular risk in this growing population.

Atherosclerosis, the primary cause of coronary artery disease (CAD), remains a leading global cause of mortality. It is characterized by the accumulation of cholesterol-rich plaques and inflammation, which narrow the coronary arteries and increase the risk of rupture. To elucidate this complex biological process and improve therapeutic strategies, CAD has been extensively explored from an epigenetic perspective over the past two decades. Epigenetics is a field investigating heritable alterations in gene expression without DNA sequence changes, such as DNA methylation, histone modifications, and non-coding RNAs. Increasing evidence has indicated that the development of CAD is significantly influenced by epigenetic changes. Meanwhile, the impact of epigenetics in CAD is now transitioning from pathophysiology to therapeutics. Focusing on the key epigenetic enzymes and their target genes will help to facilitate the diagnosis and treatment of CAD. This review synthesizes novel epigenetic insights into CAD, addressing the pathological processes, key molecular mechanisms, and potential biomarkers. Furthermore, we discuss emerging therapeutic strategies targeting epigenetic pathways. By focusing on pivotal enzymes and their associated genes, this work aims to advance CAD diagnostics and interventions.

Atherosclerosis is a disease of large and medium arteries that can lead to life-threatening cardiovascular and cerebrovascular consequences, such as myocardial infarction and stroke. Moreover, atherosclerosis is a major contributor to cardiovascular-related mortality in individuals with diabetes mellitus. Diabetes aggravates the pathobiological mechanisms that underlie the development of atherosclerosis. Currently available anti-atherosclerotic drugs or strategies solely focus on optimal control of systemic risk factors, including hyperglycaemia and dyslipidaemia, but do not adequately target the diabetes-exacerbated mechanisms of atherosclerotic cardiovascular disease, highlighting the need for targeted, mechanism-based therapies. This Review focuses on emerging pathological mechanisms and related novel therapeutic targets in atherosclerotic cardiovascular disease in patients with diabetes.

Atherosclerosis is a complex and multigenic pathology associated with significant epigenetic reprogramming. Traditional factors (age, sex, obesity, hyperglycaemia, dyslipidaemia, hypertension) and non-traditional factors (foetal indices, microbiome alteration, clonal hematopoiesis, air pollution, sleep disorders) induce endothelial dysfunction, resulting in reduced vascular tone and increased vascular permeability, inflammation and shear stress. These factors induce paracrine and autocrine interactions between several cell types, including vascular smooth muscle cells, endothelial cells, monocytes/macrophages, dendritic cells and T cells. Such cellular interactions lead to tissue-specific epigenetic reprogramming regulated by DNA methylation, histone modifications and microRNAs, which manifests in atherosclerosis. Our review outlines epigenetic signatures during atherosclerosis, which are viewed as potential clinical biomarkers that may be adopted as new therapeutic targets. Additionally, we emphasize epigenetic modifiers referred to as 'epidrugs' as potential therapeutic molecules to correct gene expression patterns and restore vascular homeostasis during atherosclerosis. Further, we suggest nanomedicine-based strategies involving the use of epidrugs, which may selectively target cells in the atherosclerotic microenvironment and reduce off-target effects.

The NOD-like receptor pyrin domain-containing protein 3 (NLRP3) inflammasome is a central regulator of innate immunity, essential for processing and release of interleukin-1β and pyroptotic cell death. As endogenous NLRP3 activating triggers are hallmarks of many human chronic inflammatory diseases, inhibition of NLRP3 has emerged as a therapeutic target. Here we identify NDT-19795 as a novel carboxylic acid-containing NLRP3 activation inhibitor in both human and mouse monocytes and macrophages. Remarkably, conversion of the carboxylate to an isopropyl-ester (NT-0796) greatly enhances NLRP3 inhibitory potency in human monocytes. This increase is attributed to the ester-containing pharmacophore being more cell-penetrant than the acid species and, once internalized, the ester being metabolized to NDT-19795 by carboxylesterase-1 (CES-1). Mouse macrophages do not express CES-1, and NT-0796 is ineffective in these cells. Mice also contain plasma esterase (Ces1c) activity which is absent in humans. To create a more human-like model, we generated a mouse line in which the genome was modified, removing Ces1c and replacing this segment of DNA with the human CES-1 gene driven by a mononuclear phagocyte-specific promoter. We show human CES-1 presence in monocytes/macrophages increases the ability of NT-0796 to inhibit NLRP3 activation both in vitro and in vivo. As NLRP3 is widely expressed by monocytes/macrophages, the co-existence of CES-1 in these same cells affords a unique opportunity to direct ester-containing NLRP3 inhibitors precisely to target cells of interest. Profiling NT-0796 in mice humanized with respect to CES-1 biology enables critical modeling of the pharmacokinetics and pharmacodynamics of this novel therapeutic candidate. SIGNIFICANCE STATEMENT: Inhibition of NLRP3 represents a desirable therapeutic strategy for the treatment of multiple human disorders. In this study pharmacological properties of a structurally-novel, ester-containing NLRP3 inhibitor NT-0796 are characterized. To study pharmacodynamics of NT-0796 in vivo, a mouse line was engineered possessing more human-like traits with respect to carboxylesterase biology. In the context of these hCES-1 mice, NT-0796 serves as a more effective inhibitor of NLRP3 activation than the corresponding acid, highlighting the full translational potential of the ester strategy.

To improve the tolerability and therapeutic application of histone deacetylase inhibitors (HDACi), by application of an esterase-sensitive motif (ESM), to target pharmacological activity directly to mononuclear myeloid cells expressing the processing enzyme carboxylesterase-1 (CES1).

This first-in-human study comprised single and multiple ascending dose cohorts to determine safety and tolerability. Pharmacodynamic parameters included acetylation, cytokine inhibition and intracellular concentrations of processed acid metabolite in isolated monocytes. Mechanistic work was conducted in vitro and in a CES1/Es1e^lo mouse strain.

ESM-HDAC391 showed transient systemic exposure (plasma half-life of 21-30 min) but selective retention of processed acid for at least 12 hours, resulting in robust targeted mechanistic engagement (increased acetylation in monocytes plus inhibition of ex vivo stimulated cytokine production). ESM-HDAC391 was well tolerated and clinical toxicities common to non-targeted HDACi were not observed. ESM-HDAC391 treatment was accompanied by the novel finding of a dose-dependent monocyte depletion that was transient and reversible and which plateaued at 0.06 × 10⁹ monocytes/L after repeat dosing with 20 or 40 mg. Characterisation of monocyte depletion in transgenic mice (CES1/Es1e^lo ) suggested that colony stimulating factor 1 receptor loss on circulating cells contributed to ESM-HDAC-mediated depletion. Further mechanistic investigations using human monocytes in vitro demonstrated HDACi-mediated change in myeloid fate through modulation of colony stimulating factor 1 receptor and downstream effects on cell differentiation.

These findings demonstrate selective targeting of monocytes in humans using the ESM approach and identify monocytopaenia as a novel outcome of ESM-HDACi treatment, with implications for potential benefit of these molecules in myeloid-driven diseases.

Atherosclerosis is a chronic inflammatory response mediated by various factors, where epigenetic regulation involving histone deacetylation is envisaged to modulate the expression of related proteins by regulating the binding of transcription factors to DNA, thereby influencing the development of atherosclerosis. The mechanism of atherosclerosis by histone deacetylation is partly known; hence, this project aimed at investigating the role of histone deacetylase 9 (HDAC9) in atherosclerosis. For this purpose, serum was separated from blood samples following clotting and centrifugation from atherosclerotic and healthy patients (n = 40 each), and then, various tests were performed. The results indicated that toll-like receptor 4 (TLR4) was not only positively correlated to the HDAC9 gene, but was also upregulated in atherosclerosis, where it was also significantly upregulated in the atherosclerosis cell model of oxidized low-density lipoprotein-induced macrophages. Conversely, the TLR4 was significantly downregulated in instances of loss of HDAC9 function, cementing the bridging relationship between HDAC9 and macrophage polarization, where the HDAC9 was found to upregulate M1 macrophage polarization which translated into the release of higher content of proinflammatory cytokines such as interleukin-1beta (IL-1β) and tumor necrosis factor-alpha (TNF-α), which tend to significantly decrease following the deletion of TLR4. Hence, this study reports novel relation between epigenetic control and atherosclerosis, which could partly be explained by histone deacetylation.

Histone deacetylase inhibitors [HDACi] exert potent anti-inflammatory effects. Because of the ubiquitous expression of HDACs, clinical utility of HDACi is limited by off-target effects. Esterase-sensitive motif [ESM] technology aims to deliver ESM-conjugated compounds to human mononuclear myeloid cells, based on their expression of carboxylesterase 1 [CES1]. This study aims to investigate utility of an ESM-tagged HDACi in inflammatory bowel disease [IBD].

CES1 expression was assessed in human blood, in vitro differentiated macrophage and dendritic cells, and Crohn's disease [CD] colon mucosa, by mass cytometry, quantitative polymerase chain reaction [PCR], and immunofluorescence staining, respectively. ESM-HDAC528 intracellular retention was evaluated by mass spectrometry. Clinical efficacy of ESM-HDAC528 was tested in dextran sulphate sodium [DSS]-induced colitis and T cell transfer colitis models using transgenic mice expressing human CES1 under the CD68 promoter.

CES1 mRNA was highly expressed in human blood CD14+ monocytes, in vitro differentiated and lipopolysaccharide [LPS]-stimulated macrophages, and dendritic cells. Specific hydrolysis and intracellular retention of ESM-HDAC528 in CES1+ cells was demonstrated. ESM-HDAC528 inhibited LPS-stimulated IL-6 and TNF-α production 1000 times more potently than its control, HDAC800, in CES1high monocytes. In healthy donor peripheral blood, CES1 expression was significantly higher in CD14++CD16- monocytes compared with CD14+CD16++ monocytes. In CD-inflamed colon, a higher number of mucosal CD68+ macrophages expressed CES1 compared with non-inflamed mucosa. In vivo, ESM-HDAC528 reduced monocyte differentiation in the colon and significantly improved colitis in a T cell transfer model, while having limited potential in ameliorating DSS-induced colitis.

We demonstrate that monocytes and inflammatory macrophages specifically express CES1, and can be preferentially targeted by ESM-HDAC528 to achieve therapeutic benefit in IBD.

Bladder cancer (BC) is the second most frequent cancer of the genitourinary system. The most successful therapy since the 1970s has consisted of intravesical instillations of Bacillus Calmette-Guérin (BCG) in which the tumor microenvironment (TME), including macrophages, plays an important role. However, some patients cannot be treated with this therapy due to comorbidities and severe inflammatory side effects. The overexpression of histone deacetylases (HDACs) in BC has been correlated with macrophage polarization together with higher tumor grades and poor prognosis. Herein we demonstrated that phenylbutyrate acid (PBA), a HDAC inhibitor, acts as an antitumoral compound and immunomodulator. In BC cell lines, PBA induced significant cell cycle arrest in G1, reduced stemness markers and increased PD-L1 expression with a corresponding reduction in histone 3 and 4 acetylation patterns. Concerning its role as an immunomodulator, we found that PBA reduced macrophage IL-6 and IL-10 production as well as CD14 downregulation and the upregulation of both PD-L1 and IL-1β. Along this line, PBA showed a reduction in IL-4-induced M2 polarization in human macrophages. In co-cultures of BC cell lines with human macrophages, a double-positive myeloid-tumoral hybrid population (CD11b+EPCAM+) was detected after 48 h, which indicates BC cell-macrophage fusions known as tumor hybrid cells (THC). These THC were characterized by high PD-L1 and stemness markers (SOX2, NANOG, miR-302) as compared with non-fused (CD11b-EPCAM+) cancer cells. Eventually, PBA reduced stemness markers along with BMP4 and IL-10. Our data indicate that PBA could have beneficial properties for BC management, affecting not only tumor cells but also the TME.

ATP-citrate lyase (Acly) is the target of the new class low-density lipoprotein-cholesterol (LDL-C)-lowering drug bempedoic acid (BA). Acly is a key metabolic enzyme synthesizing acetyl-CoA as the building block of cholesterol and fatty acids. Treatment with BA lowers circulating lipid levels and reduces systemic inflammation, suggesting a dual benefit of this drug for atherosclerosis therapy. Recent studies have shown that targeting Acly in macrophages can attenuate inflammatory responses and decrease atherosclerotic plaque vulnerability. Therefore, it could be beneficial to extend the application of Acly inhibition from solely lipid-lowering by liver-specific inhibition to also targeting macrophages in atherosclerosis. Here, we outline the possibilities of targeting Acly and describe the future needs to translate these findings to the clinic.

Epigenomic and epigenetic research has been providing several new insights into a variety of diseases caused by non-resolving inflammation, including cardiovascular diseases. Atherosclerosis (AS) has long been recognized as a chronic inflammatory disease of the arterial walls, characterized by local persistent and stepwise accelerating inflammation without resolution, also known as uncontrolled inflammation. The pathogenesis of AS is driven primarily by highly plastic macrophages via their polarization to pro- or anti-inflammatory phenotypes as well as other novel subtypes recently identified by single-cell sequencing. Although emerging evidence has indicated the key role of the epigenetic machinery in the regulation of macrophage plasticity, the investigation of epigenetic alterations and modifiers in AS and related inflammation is still in its infancy. An increasing number of the epigenetic modifiers (e.g. TET2, DNMT3A, HDAC3, HDAC9, JMJD3, KDM4A) have been identified in epigenetic remodelling of macrophages through DNA methylation or histone modifications (e.g. methylation, acetylation, and recently lactylation) in inflammation. These or many unexplored modifiers function to determine or switch the direction of macrophage polarization via transcriptional reprogramming of gene expression and intracellular metabolic rewiring upon microenvironmental cues, thereby representing a promising target for anti-inflammatory therapy in AS. Here, we review up-to-date findings involving the epigenetic regulation of macrophages to shed light on the mechanism of uncontrolled inflammation during AS onset and progression. We also discuss current challenges for developing an effective and safe anti-AS therapy that targets the epigenetic modifiers and propose a potential anti-inflammatory strategy that repolarizes macrophages from pro- to anti-inflammatory phenotypes.

Atherosclerosis is a disease of large and medium arteries that can lead to life-threatening cerebrovascular and cardiovascular consequences such as heart failure and stroke and is a major contributor to cardiovascular-related mortality worldwide. Atherosclerosis development is a complex process that involves specific structural, functional and transcriptional changes in different vascular cell populations at different stages of the disease. The application of single-cell RNA sequencing (scRNA-seq) analysis has discovered not only disease-related cell-specific transcriptomic profiles but also novel subpopulations of cells once thought as homogenous cell populations. Vascular cells undergo specific transcriptional changes during the entire course of the disease. Epigenetics is the instruction-set-architecture in living cells that defines and maintains the cellular identity by regulating the cellular transcriptome. Although different cells contain the same genetic material, they have different epigenomic signatures. The epigenome is plastic, dynamic and highly responsive to environmental stimuli. Modifications to the epigenome are driven by an array of epigenetic enzymes generally referred to as writers, erasers and readers that define cellular fate and destiny. The reversibility of these modifications raises hope for finding novel therapeutic targets for modifiable pathological conditions including atherosclerosis where the involvement of epigenetics is increasingly appreciated. This article provides a critical review of the up-to-date research in the field of epigenetics mainly focusing on in vivo settings in the context of the cellular role of individual vascular cell types in the development of atherosclerosis.

Histone deacetylases (HDACs) and bromodomain-containing proteins (BCPs) play a key role in chromatin remodeling. Based on their ability to regulate inducible gene expression in the context of inflammation and cancer, HDACs and BCPs have been the focus of drug discovery efforts, and numerous small-molecule inhibitors have been developed. However, dose-limiting toxicities of the first generation of inhibitors, which typically target multiple HDACs or BCPs, have limited translation to the clinic. Over the last decade, an increasing effort has been dedicated to designing class-, isoform-, or domain-specific HDAC or BCP inhibitors, as well as developing strategies for cell-specific targeted drug delivery. Selective inhibition of the epigenetic modulators is helping to elucidate the functions of individual epigenetic proteins and has the potential to yield better and safer therapeutic strategies. In accordance with this idea, several in vitro and in vivo studies have reported the ability of more selective HDAC/BCP inhibitors to recapitulate the beneficial effects of pan-inhibitors with less unwanted adverse events. In this review, we summarize the most recent advances with these strategies, discussing advantages and limitations of these approaches as well as some therapeutic perspectives, focusing on autoimmune and inflammatory diseases.

Urotensin II (UII), a vital vasoconstrictor peptide, causes an inflammatory response in the pathogenesis of atherosclerosis. Previous studies have reported that the Ras homolog gene family, member A (RhoA)/Rho kinases (ROCK) pathway modulates the inflammatory response of the atherosclerotic process. However, to the best of our knowledge, whether the RhoA/ROCK pathway mediates the inflammatory effect of UII has not been previously elucidated. Salidroside and isorhamnetin are two early developed antioxidant Tibetan drugs, both displaying cardioprotective effects against atherosclerosis. Therefore, the aim of the present study was to investigate the protective effects of salidroside, isorhamnetin or combination of these two drugs on the UII-induced inflammatory response in vivo (rats) or in vitro [primary vascular smooth muscle cells (VSMCs)], as well as to examine the role of the RhoA/ROCK pathway in these processes. The levels of inflammatory markers were measured via ELISA. The mRNA and protein expression levels of RhoA and ROCK II were detected using reverse transcription-quantitative PCR assay and western blot analysis. It was demonstrated that salidroside, isorhamnetin and both in combination decreased the levels of the serum pro-inflammatory cytokines TNF-α and IL-1β, as well as increased the levels of the anti-inflammatory cytokine IL-10 and macrophage migration inhibitory factor in rats with subacute infusion of UII and in the culture supernatant from primary VSMCs-exposed to UII. Moreover, salidroside, isorhamnetin and both in combination attenuated the mRNA and protein expression levels of RhoA and ROCK II in vivo and in vitro, at concentrations corresponding to human therapeutic blood plasma concentrations. Thus, these drugs could inhibit the RhoA/ROCK II pathway under UII conditions. The combination of salidroside and isorhamnetin did not display a stronger inhibitory effect on the inflammatory response and the RhoA/ROCK II pathway compared with salidroside and isorhamnetin in isolation. Collectively, the results indicated that salidroside, isorhamnetin and both in combination inhibited the RhoA/ROCK II pathway, which then attenuated the inflammatory response under UII-induced conditions, resulting in cardioprotection in atherosclerosis.

This review discusses the current developments on epigenetic inhibition as treatment for atherosclerosis.

The first phase III clinical trial targeting epigenetics in cardiovascular disease (CVD), BETonMACE, using the bromodomain inhibitor apabetalone (RVX-208) showed no significant effect on major adverse cardiovascular events (MACE) in patients with type II diabetes, low HDL-c and a recent acute coronary artery event compared with its placebo arm.

Preclinical and clinical studies suggest that targeting epigenetics in atherosclerosis is a promising novel therapeutic strategy against CVD. Interfering with histone acetylation by targeting histone deacetylates (HDACs) and bromodomain and extraterminal domain (BET) proteins demonstrated encouraging results in modulating disease progression in model systems. Although the first phase III clinical trial targeting BET in CVD showed no effect on MACE, we suggest that there is sufficient potential for future clinical usage based on the outcomes in specific subgroups and the fact that the study was slightly underpowered. Lastly, we propose that there is future window for targeting repressive histone modifications in atherosclerosis.

Metabolic associated fatty liver disease (MAFLD), formerly named non-alcoholic fatty liver disease is the most common liver disorder in many countries. The inflammatory subtype termed steatohepatitis is a driver of disease progression to cirrhosis, hepatocellular carcinoma, liver transplantation, and death, but also to extrahepatic complications including cardiovascular disease, diabetes and chronic kidney disease. The plasticity of macrophages in response to various environmental cues and the fact that they can orchestrate cross talk between different cellular players during disease development and progression render them an ideal target for drug development. This report reviews recent advances in our understanding of macrophage biology during the entire spectrum of MAFLD including steatosis, inflammation, fibrosis, and hepatocellular carcinoma, as well as for the extra-hepatic manifestations of MAFLD. We discuss the underlying molecular mechanisms of macrophage activation and polarization as well as cross talk with other cell types such as hepatocytes, hepatic stellate cells, and adipose tissue. We conclude with a discussion on the potential translational implications and challenges for macrophage based therapeutics for MAFLD.