Resident and recruited macrophages differentially contribute to cardiac healing after myocardial ischemia.

Cardiac macrophages are heterogenous in phenotype and functions, which has been associated with differences in their ontogeny. Despite extensive research, our understanding of the precise role of different subsets of macrophages in ischemia/reperfusion (I/R) injury remains incomplete. We here investigated macrophage lineages and ablated tissue macrophages in homeostasis and after I/R injury in a CSF1R-dependent manner. Genomic deletion of a fms-intronic regulatory element (FIRE) in the Csf1r locus resulted in specific absence of resident homeostatic and antigen-presenting macrophages, without affecting the recruitment of monocyte-derived macrophages to the infarcted heart. Specific absence of homeostatic, monocyte-independent macrophages altered the immune cell crosstalk in response to injury and induced proinflammatory neutrophil polarization, resulting in impaired cardiac remodeling without influencing infarct size. In contrast, continuous CSF1R inhibition led to depletion of both resident and recruited macrophage populations. This augmented adverse remodeling after I/R and led to an increased infarct size and deterioration of cardiac function. In summary, resident macrophages orchestrate inflammatory responses improving cardiac remodeling, while recruited macrophages determine infarct size after I/R injury. These findings attribute distinct beneficial effects to different macrophage populations in the context of myocardial infarction.

CD47 blockade reduces ischemia/reperfusion injury in murine heart transplantation and improves donor heart preservation.

BACKGROUND: Myocardial ischemia-reperfusion injury (MIRI) is an important cause of early dysfunction and exacerbation of immune rejection in transplanted hearts. The integrin-related protein CD47 exacerbates myocardial ischemia-reperfusion injury by inhibiting the nitric oxide signaling pathway through interaction with thrombospondin-1 (TSP-1). In addition, the preservation quality of the donor hearts is a key determinant of transplant success. Preservation duration beyond four hours is associated with primary graft dysfunction. We hypothesized that blocking the CD47-TSP-1 system would attenuate ischemia-reperfusion injury in the transplanted heart and, thus, improve the preservation of donor hearts. METHODS: We utilized a syngeneic mouse heart transplant model to assess the effect of CD47 monoclonal antibody (CD47mAb) to treat MIRI. Donor hearts were perfused with CD47mAb or an isotype-matched control immunoglobulin (IgG2a) and were implanted into the abdominal cavity of the recipients after being stored in histidine-tryptophan-ketoglutarate (HTK) solution at 4 °C for 4 h or 8 h. RESULTS: At both the 4-h and 8-h preservation time points, mice in the experimental group perfused with CD47mAb exhibited prolonged survival in the transplanted heart, reduced inflammatory response and oxidative stress, significantly decreased inflammatory cell infiltration, and fewer apoptosis-related biomarkers. CONCLUSION: The application of CD47mAb for the blocking of CD47 attenuates MIRI as well as improves the preservation and prognosis of the transplanted heart in a murine heart transplant model.

Selectin-targeting glycosaminoglycan-peptide conjugate limits neutrophil-mediated cardiac reperfusion injury.

AIMS: One of the hallmarks of myocardial infarction (MI) is excessive inflammation. During an inflammatory insult, damaged endothelial cells shed their glycocalyx, a carbohydrate-rich layer on the cell surface which provides a regulatory interface to immune cell adhesion. Selectin-mediated neutrophilia occurs as a result of endothelial injury and inflammation. We recently designed a novel selectin-targeting glycocalyx mimetic (termed DS-IkL) capable of binding inflamed endothelial cells. This study examines the capacity of DS-IkL to limit neutrophil binding and platelet activation on inflamed endothelial cells, as well as the cardioprotective effects of DS-IkL after acute myocardial infarction. METHODS AND RESULTS: In vitro, DS-IkL diminished neutrophil interactions with both recombinant selectin and inflamed endothelial cells, and limited platelet activation on inflamed endothelial cells. Our data demonstrated that DS-IkL localized to regions of vascular inflammation in vivo after 45 min of left anterior descending coronary artery ligation-induced MI. Further, findings from this study show DS-IkL treatment had short- and long-term cardioprotective effects after ischaemia/reperfusion of the left anterior descending coronary artery. Mice treated with DS-IkL immediately after ischaemia/reperfusion and 24 h later exhibited reduced neutrophil extravasation, macrophage accumulation, fibroblast and endothelial cell proliferation, and fibrosis compared to saline controls. CONCLUSIONS: Our findings suggest that DS-IkL has great therapeutic potential after MI by limiting reperfusion injury induced by the immune response.

Innate Lymphoid Cells and Myocardial Infarction.

Myocardial infarction results from obstruction of a coronary artery that causes insufficient blood supply to the myocardium and leads to ischemic necrosis. It is one of the most common diseases threatening human health and is characterized by high morbidity and mortality. Atherosclerosis is the pathological basis of myocardial infarction, and its pathogenesis has not been fully elucidated. Innate lymphoid cells (ILCs) are an important part of the human immune system and participate in many processes, including inflammation, metabolism and tissue remodeling, and play an important role in atherosclerosis. However, their specific roles in myocardial infarction are unclear. This review describes the current understanding of the relationship between innate lymphoid cells and myocardial infarction during the acute phase of myocardial infarction, myocardial ischemia-reperfusion injury, and heart repair and regeneration following myocardial infarction. We suggest that this review may provide new potential intervention targets and ideas for treatment and prevention of myocardial infarction.

Pharmacokinetic study of the novel phosphocholine derivative 3-dibutylaminopropylphosphonic acid by LC-MS coupling.

CRP is an important mediator of the inflammatory response. Pro-inflammatory CRP effects are mediated by pCRP* and mCRP, dissociation products of the native pCRP. The concentration of pCRP during inflammation may rise up to concentrations 1000-fold from baseline. By prevention of the conformational change from pCRP to pCRP*, pro-inflammatory immune responses can be inhibited and local tissue damage reduced. 3-(Dibutylamino)propylphosphonic acid (C10m) is a new substance that can suppress ischemic-reperfusion injury by targeting CRP in the complement cascade. It hampers dissociation of pCRP into its monomers, thus preventing exacerbation of tissue inflammation subsequent to reperfusion injury. In this study, the pharmacokinetics and metabolism of the new drug candidate C10m was investigated. A sensitive and selective method for detection of C10m and its metabolites from plasma and urine was developed with LC-MS and LC-MS/MS coupling. The LLOQ is at 0.1 µg mL-1 and recovery at 87.4% ± 2.8%. Accuracy and precision were within 15% coefficient of variation and nominal concentrations, respectively. Concentration time profile after i.v. bolus injection of C10m was analyzed by LC-MS/MS. Bioavailability has shown to be below 30%. Most likely due to the compounds' very polar chemical properties, no phase-I or phase-II metabolism could be observed. Absence of phase-I metabolism was cross-checked by performing microsomal incubations. Our study revealed that C10m is rapidly eliminated via urine excretion and that half-times appear to be increased with coadministration of the target pCRP.

Hydrogen Attenuates Myocardial Injury in Rats by Regulating Oxidative Stress and NLRP3 Inflammasome Mediated Pyroptosis.

Purpose: Hydrogen (H2) is an antioxidant with anti-inflammatory and apoptosis functions.This study aimed to estimate the effects of H2 on acute myocardial infarction (AMI) in rats and its association with the inhibition of oxidative stress and cardiomyocyte pyroptosis. Methods: Sixty-four rats were randomly divided into three groups (Sham, AMI, and H2). The left anterior descending coronary artery (LAD) of rats in the AMI and H2 groups was ligated, while rats in the Sham group were threaded without ligation. In addition, 2% H2 was administered by inhalation for 24 h after ligation in the H2 group. Transthoracic echocardiography was performed after H2 inhalation, followed by collection of the serum and cardiac tissue of all rats. Results: H2 inhalation ameliorated the cardiac dysfunction, infarct size and inflammatory cell infiltration caused by AMI. Meanwhile, H2 inhalation reduced the concentration of serum Troponin I (TnI), brain natriuretic peptide (BNP), reactive oxygen species (ROS), cardiac malondialdehyde (MDA), and 8-OHdG. In addition, H2 inhalation inhibited cardiac inflammation and pyroptosis relative proteins expression. Conclusion: H2 effectively promoted heart functions in AMI rats by regulating oxidative stress and pyroptosis.

Polypeptide Globular Adiponectin Ameliorates Hypoxia/Reoxygenation-Induced Cardiomyocyte Injury by Inhibiting Both Apoptosis and Necroptosis.

Adiponectin is a small peptide secreted and a key component of the endocrine system and immune system. Although globular adiponectin protects myocardial ischemia/reperfusion-induced cardiomyocyte injury, the protective mechanisms remain largely unresolved. Using a neonatal rat ventricular myocyte hypoxia/reoxygenation model, we investigated the role of its potential mechanisms of necroptosis in globular adiponectin-mediated protection in hypoxia/reoxygenation-induced cardiomyocyte injury as compared to apoptosis. We found that globular adiponectin treatment attenuated cardiomyocyte injury as indicated by increased cell viability and reduced lactate dehydrogenase release following hypoxia/reoxygenation. Immunofluorescence staining and Western blotting demonstrated that both necroptosis and apoptosis were triggered by hypoxia/reoxygenation and diminished by globular adiponectin. Necrostatin-1 (RIP1-specific inhibitor) and Z-VAD-FMK (pan-caspase inhibitor) only mimicked the inhibition of necroptosis and apoptosis, respectively, by globular adiponectin in hypoxia/reoxygenation-treated cardiomyocytes. Globular adiponectin attenuated reactive oxygen species production, oxidative damage, and p38MAPK and NF-κB signaling, all important for necroptosis and apoptosis. Collectively, our study suggests that globular adiponectin inhibits hypoxia/reoxygenation-induced necroptosis and apoptosis in cardiomyocytes probably by reducing oxidative stress and interrupting p38MAPK signaling.

Acacetin Protects Myocardial Cells against Hypoxia-Reoxygenation Injury through Activation of Autophagy.

Ischemic heart disease is a leading cause of mortality and morbidity worldwide. We previously demonstrated that acacetin protects against myocardial ischemia reperfusion injury in rats, although the underlying mechanism remains to be elucidated. In the present study, we investigated the effects of acacetin on autophagy during hypoxia/reoxygenation (H/R) injury by exposing H9c2 myocardial cells to H/R with or without acacetin pretreatment during hypoxia. Our results show that acacetin significantly increased cell viability in a dose-dependent manner, enhanced antioxidant capacity, and suppressed protein apoptosis of rat cardiomyocytes H9c2 cells following H/R injury. In addition, lentiviral infection of H9c2 cardiomyocytes revealed that acacetin pretreatment significantly enhanced the fluorescence intensity of autophagy proteins Beclin 1, LC3-II, and p62. These results indicate that acacetin protected H9c2 cardiomyocytes from H/R damage by enhancing autophagy. Moreover, we found that application of acacetin increased activation of the PI3K/Akt signaling pathway, whereas cotreatment with the PI3K inhibitor LY294002 reversed the inhibition of apoptosis and autophagy induced by acacetin. In conclusion, acacetin mitigated H/R injury by promoting autophagy through activating the PI3K/Akt/mTOR signaling pathway.

Bone marrow-derived mesenchymal stem cells attenuate myocardial ischemia-reperfusion injury via upregulation of splenic regulatory T cells.

BACKGROUND: Myocardial ischemia-reperfusion injury (MIRI) is the main pathological manifestation of cardiovascular diseases such as myocardial infarction. The potential therapeutic effects of bone marrow-derived mesenchymal stem cells (BM-MSCs) and the participation of regulatory T cells (Tregs) in MIRI remains to be defined. METHODS: We used the experimental acute MIRI that was induced in mice by left ascending coronary ischemia, which were subsequently randomized to receive immunoglobulin G (IgG) or anti-CD25 antibody PC61 with or without intravenously injected BM-MSCs. The splenectomized mice underwent prior to experimental MIRI followed by intravenous administration of BM-MSCs. At 72 h post-MIRI, the hearts and spleens were harvested and subjected to cytometric and histologic analyses. RESULTS: CD25+Foxp3+ regulatory T cells were significantly elevated after MIRI in the hearts and spleens of mice receiving IgG + BM-MSCs and PC61 + BM-MSCs compared to the respective control mice (all p < 0.01). This was accompanied by upregulation of interleukin 10 and transforming growth factor ß1 and downregulation of creatinine kinase and lactate dehydrogenase in the serum. The post-MIRI mice receiving BM-MSCs showed attenuated inflammation and cellular apoptosis in the heart. Meanwhile, splenectomy compromised all therapeutic effects of BM-MSCs. CONCLUSION: Administration of BM-MSCs effectively alleviates MIRI in mice through inducing Treg activation, particularly in the spleen.

Does remote ischaemic conditioning reduce inflammation? A focus on innate immunity and cytokine response.

The benefits of remote ischaemic conditioning (RIC) have been difficult to translate to humans, when considering traditional outcome measures, such as mortality and heart failure. This paper reviews the recent literature of the anti-inflammatory effects of RIC, with a particular focus on the innate immune response and cytokine inhibition. Given the current COVID-19 pandemic, the inflammatory hypothesis of cardiac protection is an attractive target on which to re-purpose such novel therapies. A PubMed/MEDLINE™ search was performed on July 13th 2020, for the key terms RIC, cytokines, the innate immune system and inflammation. Data suggest that RIC attenuates inflammation in animals by immune conditioning, cytokine inhibition, cell survival and the release of anti-inflammatory exosomes. It is proposed that RIC inhibits cytokine release via a reduction in nuclear factor kappa beta (NF-κB)-mediated NLRP3 inflammasome production. In vivo, RIC attenuates pro-inflammatory cytokine release in myocardial/cerebral infarction and LPS models of endotoxaemia. In the latter group, cytokine inhibition is associated with a profound survival benefit. Further clinical trials should establish whether the benefits of RIC in inflammation can be observed in humans. Moreover, we must consider whether uncomplicated MI and elective surgery are the most suitable clinical conditions in which to test this hypothesis.

Neohesperidin alleviated pathological damage and immunological imbalance in rat myocardial ischemia-reperfusion injury via inactivation of JNK and NF-κB p65.

Neohesperidin (NEO) exerts antiviral, antioxidant, anti-inflammation, and antitumor effects in some diseases. The purpose of this study was to investigate the effect and mechanism of NEO on myocardial ischemia-reperfusion (I/R) injury. Results indicated that NEO suppressed the levels of serum inflammatory cytokines, myocardial damage markers, and oxidative stress markers, and increased the levels of antioxidant in myocardial I/R rats. NEO also inhibited cell apoptosis. Besides, NEO also inhibited the phosphorylation of c-Jun N-terminal kinases (JNK) and nuclear factor kappa B (NF-κB) p65. Furthermore, the protective effects of NEO on myocardial tissue damage, inflammatory cytokines, myocardial injury markers, oxidative stress markers, cell apoptosis, spleen, thymus and liver indices, and phagocytic indices were reversed by JNK activator and NF-κB activator, respectively. In conclusion, NEO alleviates myocardial damage, oxidative stress, cell apoptosis, and immunological imbalance in I/R injury via the inactivation of JNK and NF-κB, making NEO a potential agent for myocardial I/R therapy.

A double-edged sword of immuno-microenvironment in cardiac homeostasis and injury repair.

The response of immune cells in cardiac injury is divided into three continuous phases: inflammation, proliferation and maturation. The kinetics of the inflammatory and proliferation phases directly influence the tissue repair. In cardiac homeostasis, cardiac tissue resident macrophages (cTMs) phagocytose bacteria and apoptotic cells. Meanwhile, NK cells prevent the maturation and transport of inflammatory cells. After cardiac injury, cTMs phagocytose the dead cardiomyocytes (CMs), regulate the proliferation and angiogenesis of cardiac progenitor cells. NK cells prevent the cardiac fibrosis, and promote vascularization and angiogenesis. Type 1 macrophages trigger the cardioprotective responses and promote tissue fibrosis in the early stage. Reversely, type 2 macrophages promote cardiac remodeling and angiogenesis in the late stage. Circulating macrophages and neutrophils firstly lead to chronic inflammation by secreting proinflammatory cytokines, and then release anti-inflammatory cytokines and growth factors, which regulate cardiac remodeling. In this process, dendritic cells (DCs) mediate the regulation of monocyte and macrophage recruitment. Recruited eosinophils and Mast cells (MCs) release some mediators which contribute to coronary vasoconstriction, leukocyte recruitment, formation of new blood vessels, scar formation. In adaptive immunity, effector T cells, especially Th17 cells, lead to the pathogenesis of cardiac fibrosis, including the distal fibrosis and scar formation. CMs protectors, Treg cells, inhibit reduce the inflammatory response, then directly trigger the regeneration of local progenitor cell via IL-10. B cells reduce myocardial injury by preserving cardiac function during the resolution of inflammation.

Covalent modification of Keap1 at Cys77 and Cys434 by pubescenoside a suppresses oxidative stress-induced NLRP3 inflammasome activation in myocardial ischemia-reperfusion injury.

Background and Purpose: Kelch ECH-associating protein 1 (Keap1) is a crucial chaperonin for E3 ubiquitin ligases. Modification of the key reactive cysteine residues in Keap1 affects the interaction between Keap1 and its substrate nuclear factor erythroid 2-related factor 2 (Nrf2), subsequently regulating oxidative stress and NLPR3 inflammasome activation, which are important factors for myocardial ischemia-reperfusion injury (MI/RI). Pubescenoside A (PBA), an active compound from Ilex pubescens, has antithrombotic and anti-inflammatory effects. However, the effect of PBA on MI/RI is still unknown. In the present study, we aimed to determine whether PBA can protect the heart against MI/RI and clarify the direct target and the underlying mechanism of PBA. Methods: The left anterior descending artery (LAD) ligation-induced MI/RI mice model or oxygen and glucose deprivation/reperfusion (OGD/R) were used to evaluate the cardioprotective effect of PBA. Pull-down assays, co-immunoprecipitation (Co-IP) assays, LC/MS/MS, isothermal calorimetry (ITC) experiments and covalent docking were used to identify the target of PBA. Results: PBA protected cardiomyocytes against OGD/R in vitro and LAD-induced MI/RI in vivo. PBA suppressed NLRP3 inflammation activation and induced the Nrf2 signaling pathway. Interestingly, PBA targeted Keap1 by selectively covalently binding to conserved cysteine residues, cysteine 77 (Cys77) in the BTB domain and cysteine 434 (Cys434) in the Kelch domain of Keap1, subsequently inhibiting ubiquitination of Nrf2 and activating antioxidant enzymes. Additionally, the cysteines of Keap1 has different degree of activation by PBA as follows: Cys77 > Cys434 > Cys23 > Cys38 > Cys226 > Cys273, which further elucidates the cysteine sensitivity of Keap1. Conclusions: Our results indicated that PBA might be a new Nrf2 activator that covalently binds to two critical domains of Keap1, and shows cardioprotective activities against ischemia-reperfusion injury.

Betulin Alleviates Myocardial Ischemia-Reperfusion Injury in Rats via Regulating the Siti1/NLRP3/NF-κB Signaling Pathway.

To study the effects of betulin (BE) on myocardial ischemia-reperfusion (I/R) injury in rats, electrocardiogram (ECG) was detected by an electrocardiograph; myocardial infarction was evaluated by triphenyltetrazolium (TTC) staining, serum biochemical indicators myocardial enzymes creatine kinase (CK), lactate dehydrogenase (LDH), aspartate aminotransferase (AST), serum superoxide dismutase (SOD), glutathione (GSH), nitric oxide (NO), and malondialdehyde (MDA); and inflammatory cytokines were tested by using commercial kits. The expression of the Siti1/NLRP3/NF-κB signaling pathway was detected by western blotting and immunohistochemistry experiments. BE improved ECG; reduced myocardial infarction area; decreased CK, LDH, AST, MDA, NO, and inflammatory cytokines; and increased SOD and GSH in I/R rats. In addition, BE also increased Siti1 and decreased the NLRP3/NF-κB signaling pathway in I/R rats. This study shows that the protection of BE is associated with changes in the Siti1/NLRP3/NF-κB pathway.

miR-330-5p inhibits NLRP3 inflammasome-mediated myocardial ischaemia-reperfusion injury by targeting TIM3.

BACKGROUND/AIMS: The Nod-like receptor protein-3 (NLRP3) inflammasome signalling pathway is involved in the inflammatory reaction of myocardial ischaemia-reperfusion (I/R) injury. Our previous study showed that miR-330-5p was differentially expressed in both cerebral and myocardial I/R injury, and thus might be a biomarker for I/R injury-related diseases. Another study also indicated that miR-330-5p could promote NLRP3 inflammasome activation in renal IRI. However, the role of miR-330-5p in myocardial I/R injury-induced inflammatory responses is unknown. This study aimed to investigate the role of miR-330-5p in NLRP3 inflammasome-mediated myocardial I/R injury. METHODS: Myocardial I/R injury was induced in mice by occlusion of the left anterior descending coronary artery for 45 min followed by reperfusion. For NLRP3 inflammasome stimulation in vitro, cardiomyocytes were treated with 2 h of oxygen and glucose deprivation (OGD) or LPS (100 ng/ml). Myocardial miR-330-5p expression was examined by PCR at different treatment times. A miR-330-5p antagomir and an agomir were used to regulate miR-330-5p expression. To evaluate the role of miR-330-5p in myocardial I/R injury, 2,3,5-triphenyltetrazolium chloride (TTC) staining, echocardiography, and immunoblotting were used to assess infarct volume, cardiac function, and NLRP3 inflammasome activation respectively. A luciferase binding assay was used to examine whether miR-330-5p could directly bind to the T cell immunoglobulin domain and mucin domain-containing molecule-3 (TIM3). Finally, the role of the miR-330-5p/TIM3 axis in regulating apoptosis and NLRP3 inflammasome formation was evaluated with flow cytometry assays and immunofluorescence staining. RESULTS: Compared to that in the model group, the inhibition of miR-330-5p significantly aggravated myocardial I/R injury, resulting in increased infarct volume and more severe cardiac dysfunction. Moreover, inhibition of miR-330-5p significantly increased the levels of NLRP3 inflammasome-related proteins, including caspase-1, IL-1ß, IL-18 and TNF-α, in both in-vivo and in-vitro models. Furthermore, TIM3 was confirmed as a potential target of miR-330-5p. As predicted, suppression of TIM3 by siRNA ameliorated the anti-miR-330-5p-mediated activation of the NLRP3 inflammasome induced by OGD and LPS, thus decreasing cardiomyocyte apoptosis. CONCLUSIONS: Our study indicated that the miR-330-5p/TIM3 axis was involved in the regulatory mechanism of NLRP3 inflammasome-mediated myocardial inflammation.

Damage-Associated Molecular Patterns in Myocardial Infarction and Heart Transplantation: The Road to Translational Success.

In the setting of myocardial infarction (MI), ischemia reperfusion injury (IRI) occurs due to occlusion (ischemia) and subsequent re-establishment of blood flow (reperfusion) of a coronary artery. A similar phenomenon is observed in heart transplantation (HTx) when, after cold storage, the donor heart is connected to the recipient's circulation. Although reperfusion is essential for the survival of cardiomyocytes, it paradoxically leads to additional myocardial damage in experimental MI and HTx models. Damage (or danger)-associated molecular patterns (DAMPs) are endogenous molecules released after cellular damage or stress such as myocardial IRI. DAMPs activate pattern recognition receptors (PRRs), and set in motion a complex signaling cascade resulting in the release of cytokines and a profound inflammatory reaction. This inflammatory response is thought to function as a double-edged sword. Although it enables removal of cell debris and promotes wound healing, DAMP mediated signalling can also exacerbate the inflammatory state in a disproportional matter, thereby leading to additional tissue damage. Upon MI, this leads to expansion of the infarcted area and deterioration of cardiac function in preclinical models. Eventually this culminates in adverse myocardial remodeling; a process that leads to increased myocardial fibrosis, gradual further loss of cardiomyocytes, left ventricular dilation and heart failure. Upon HTx, DAMPs aggravate ischemic damage, which results in more pronounced reperfusion injury that impacts cardiac function and increases the occurrence of primary graft dysfunction and graft rejection via cytokine release, cardiac edema, enhanced myocardial/endothelial damage and allograft fibrosis. Therapies targeting DAMPs or PRRs have predominantly been investigated in experimental models and are potentially cardioprotective. To date, however, none of these interventions have reached the clinical arena. In this review we summarize the current evidence of involvement of DAMPs and PRRs in the inflammatory response after MI and HTx. Furthermore, we will discuss various current therapeutic approaches targeting this complex interplay and provide possible reasons why clinical translation still fails.

Innate Immunity Effector Cells as Inflammatory Drivers of Cardiac Fibrosis.

Despite relevant advances made in therapies for cardiovascular diseases (CVDs), they still represent the first cause of death worldwide. Cardiac fibrosis and excessive extracellular matrix (ECM) remodeling are common end-organ features in diseased hearts, leading to tissue stiffness, impaired myocardial functional, and progression to heart failure. Although fibrosis has been largely recognized to accompany and complicate various CVDs, events and mechanisms driving and governing fibrosis are still not entirely elucidated, and clinical interventions targeting cardiac fibrosis are not yet available. Immune cell types, both from innate and adaptive immunity, are involved not just in the classical response to pathogens, but they take an active part in "sterile" inflammation, in response to ischemia and other forms of injury. In this context, different cell types infiltrate the injured heart and release distinct pro-inflammatory cytokines that initiate the fibrotic response by triggering myofibroblast activation. The complex interplay between immune cells, fibroblasts, and other non-immune/host-derived cells is now considered as the major driving force of cardiac fibrosis. Here, we review and discuss the contribution of inflammatory cells of innate immunity, including neutrophils, macrophages, natural killer cells, eosinophils and mast cells, in modulating the myocardial microenvironment, by orchestrating the fibrogenic process in response to tissue injury. A better understanding of the time frame, sequences of events during immune cells infiltration, and their action in the injured inflammatory heart environment, may provide a rationale to design new and more efficacious therapeutic interventions to reduce cardiac fibrosis.

Extracellular vesicles derived from Krüppel-Like Factor 2-overexpressing endothelial cells attenuate myocardial ischemia-reperfusion injury by preventing Ly6Chigh monocyte recruitment.

Background: The ischemia/reperfusion (I/R) process in patients with ST-segment elevation myocardial infarction (STEMI) triggers an immune response, resulting in myocyte death. Krüppel-Like Factor 2 (KLF2), which is highly expressed in endothelial cells (ECs) under laminar flow, exerts anti-inflammatory effects. In this study, we explored the role of small extracellular vesicles (EVs) from KLF2-overexpressing ECs (KLF2-EVs) in the immunomodulation and its implications in myocardial I/R injury. Methods and Results: The small EVs were isolated from KLF2-overexpressing ECs' supernatant using gradient centrifugation. Mice were subjected to 45 min of ischemia followed by reperfusion, and KLF2-EVs were administrated through intravenous injection. KLF2-EVs ameliorated I/R injury and alleviated inflammation level in the serum and heart. We employed the macrophage depletion model and splenectomy and showed that Ly6Chigh monocyte recruitment from bone marrow was the main target of KLF2-EVs. miRNA-sequencing of KLF2-EVs and bioinformatics analysis implicated miRNA-24-3p (miR-24-3p) as a potent candidate mediator of monocyte recruitment and CCR2 as a downstream target. miR-24-3p mimic inhibited the migration of Ly6Chigh monocytes, and miR-24-3p antagomir reversed the effect of KLF2-EVs in myocardial I/R. Conclusion: Our data demonstrated that KLF2-EVs attenuated myocardial I/R injury in mice via shuttling miR-24-3p that restrained the Ly6Chigh monocyte recruitment. Thus, KLF2-EVs could be a potential therapeutic agent for myocardial I/R injury.

Neuraminidase-1 promotes heart failure after ischemia/reperfusion injury by affecting cardiomyocytes and invading monocytes/macrophages.

Neuraminidase (NEU)1 forms a multienzyme complex with beta-galactosidase (ß-GAL) and protective-protein/cathepsin (PPC) A, which cleaves sialic-acids from cell surface glycoconjugates. We investigated the role of NEU1 in the myocardium after ischemia/reperfusion (I/R). Three days after inducing I/R, left ventricles (LV) of male mice (3 months-old) displayed upregulated neuraminidase activity and increased NEU1, ß-GAL and PPCA expression. Mice hypomorphic for neu1 (hNEU1) had less neuraminidase activity, fewer pro-inflammatory (Lin-CD11b+F4/80+Ly-6Chigh), and more anti-inflammatory macrophages (Lin-CD11b+F4/80+Ly-6Clow) 3 days after I/R, and less LV dysfunction 14 days after I/R. WT mice transplanted with hNEU1-bone marrow (BM) and hNEU1 mice with WT-BM showed significantly better LV function 14 days after I/R compared with WT mice with WT-BM. Mice with a cardiomyocyte-specific NEU1 overexpression displayed no difference in inflammation 3 days after I/R, but showed increased cardiomyocyte hypertrophy, reduced expression and mislocalization of Connexin-43 in gap junctions, and LV dysfunction despite a similar infarct scar size to WT mice 14 days after I/R. The upregulation of NEU1 after I/R contributes to heart failure by promoting inflammation in invading monocytes/macrophages, enhancing cardiomyocyte hypertrophy, and impairing gap junction function, suggesting that systemic NEU1 inhibition may reduce heart failure after I/R.

Monocyte recruitment and fate specification after myocardial infarction.

Monocytes are critical mediators of the inflammatory response following myocardial infarction (MI) and ischemia-reperfusion injury. They are involved in both initiation and resolution of inflammation and play an integral role in cardiac repair. The antagonistic nature of their function is dependent on their subset heterogeneity and biphasic response following injury. New advancements in single-cell transcriptomics and mass cytometry have allowed us to identify smaller, transcriptionally distinct clusters that may have functional relevance in disease and homeostasis. Additionally, recent insights into the spatiotemporal dynamics of monocytes following ischemic injury and their subsequent interactions with the endothelium and other immune cells reveal a complex interplay between monocytes and the cardiac milieu. In this review, we highlight recent findings on monocyte functional heterogeneity, present new mechanistic insight into monocyte recruitment and fate specification following MI, and discuss promising therapeutic avenues targeting monocytes for the treatment of ischemic heart disease.