Mitochondrial Permeability Uncouples Elevated Autophagy and Lifespan Extension.

Autophagy is required in diverse paradigms of lifespan extension, leading to the prevailing notion that autophagy is beneficial for longevity. However, why autophagy is harmful in certain contexts remains unexplained. Here, we show that mitochondrial permeability defines the impact of autophagy on aging. Elevated autophagy unexpectedly shortens lifespan in C. elegans lacking serum/glucocorticoid regulated kinase-1 (sgk-1) because of increased mitochondrial permeability. In sgk-1 mutants, reducing levels of autophagy or mitochondrial permeability transition pore (mPTP) opening restores normal lifespan. Remarkably, low mitochondrial permeability is required across all paradigms examined of autophagy-dependent lifespan extension. Genetically induced mPTP opening blocks autophagy-dependent lifespan extension resulting from caloric restriction or loss of germline stem cells. Mitochondrial permeability similarly transforms autophagy into a destructive force in mammals, as liver-specific Sgk knockout mice demonstrate marked enhancement of hepatocyte autophagy, mPTP opening, and death with ischemia/reperfusion injury. Targeting mitochondrial permeability may maximize benefits of autophagy in aging.

Nitric Oxide Signaling and Regulation in the Cardiovascular System: Recent Advances.

Nitric oxide (NO) from endothelial NO synthase (eNOS) importantly contributes to vascular homeostasis. Reduced NO production or increased scavenging during disease conditions with oxidative stress contribute to endothelial dysfunction and NO deficiency. In addition to the classical enzymatic NOS system, NO can also be generated via the nitrate-nitrite-NO pathway. Dietary and pharmacological approaches aimed at increasing NO bioactivity, especially in the cardiovascular system, have been the focus of much research since the discovery of this small gaseous signaling molecule. Despite wide appreciation of the biological role of NOS/NO signaling, questions still remain about the chemical nature of NOS-derived bioactivity. Recent studies show that NO-like bioactivity can be efficiently transduced by mobile NO-ferroheme species which can transfer between proteins, partition into a hydrophobic phase, and directly activate the sGC-cGMP-PKG pathway without intermediacy of free NO. Moreover, interaction between red blood cells and the endothelium in the regulation of vascular NO homeostasis have gained much attention, especially in conditions with cardiometabolic disease. In this review we discuss both classical and non-classical pathways for NO generation in the cardiovascular system, and how these can be modulated for therapeutic purposes. Significance Statement After four decades of intensive research, questions persist about the transduction and control of NO synthase bioactivity. Here we discuss NO signaling in cardiovascular health and disease, highlighting new findings, such as the important role of red blood cells in cardiovascular NO homeostasis. Non-classical signaling modes, like the nitrate-nitrite-NO pathway, and therapeutic opportunities related to the NO system are discussed. Existing and potential pharmacological treatments/strategies, as well as dietary components influencing NO generation and signaling are covered.

Transient Inhibition of Translation Improves Cardiac Function After Ischemia/Reperfusion by Attenuating the Inflammatory Response.

BACKGROUND: The myocardium adapts to ischemia/reperfusion (I/R) by changes in gene expression, determining the cardiac response to reperfusion. mRNA translation is a key component of gene expression. It is largely unknown how regulation of mRNA translation contributes to cardiac gene expression and inflammation in response to reperfusion and whether it can be targeted to mitigate I/R injury. METHODS: To examine translation and its impact on gene expression in response to I/R, we measured protein synthesis after reperfusion in vitro and in vivo. Underlying mechanisms of translational control were examined by pharmacological and genetic targeting of translation initiation in mice. Cell type-specific ribosome profiling was performed in mice that had been subjected to I/R to determine the impact of mRNA translation on the regulation of gene expression in cardiomyocytes. Translational regulation of inflammation was studied by quantification of immune cell infiltration, inflammatory gene expression, and cardiac function after short-term inhibition of translation initiation. RESULTS: Reperfusion induced a rapid recovery of translational activity that exceeds baseline levels in the infarct and border zone and is mediated by translation initiation through the mTORC1 (mechanistic target of rapamycin complex 1)-4EBP1 (eIF4E-binding protein 1)-eIF (eukaryotic initiation factor) 4F axis. Cardiomyocyte-specific ribosome profiling identified that I/R increased translation of mRNA networks associated with cardiac inflammation and cell infiltration. Short-term inhibition of the mTORC1-4EBP1-eIF4F axis decreased the expression of proinflammatory cytokines such as Ccl2 (C-C motif chemokine ligand 2) of border zone cardiomyocytes, thereby attenuating Ly6Chi monocyte infiltration and myocardial inflammation. In addition, we identified a systemic immunosuppressive effect of eIF4F translation inhibitors on circulating monocytes, directly inhibiting monocyte infiltration. Short-term pharmacological inhibition of eIF4F complex formation by 4EGI-1 or rapamycin attenuated translation, reduced infarct size, and improved cardiac function after myocardial infarction. CONCLUSIONS: Global protein synthesis is inhibited during ischemia and shortly after reperfusion, followed by a recovery of protein synthesis that exceeds baseline levels in the border and infarct zones. Activation of mRNA translation after reperfusion is driven by mTORC1/eIF4F-mediated regulation of initiation and mediates an mRNA network that controls inflammation and monocyte infiltration to the myocardium. Transient inhibition of the mTORC1-/eIF4F axis inhibits translation and attenuates Ly6Chi monocyte infiltration by inhibiting a proinflammatory response at the site of injury and of circulating monocytes.

Inhibition of Hmbox1 Promotes Cardiomyocyte Survival and Glucose Metabolism Through Gck Activation in Ischemia/Reperfusion Injury.

BACKGROUND: Exercise-induced physiological cardiac growth regulators may protect the heart from ischemia/reperfusion (I/R) injury. Homeobox-containing 1 (Hmbox1), a homeobox family member, has been identified as a putative transcriptional repressor and is downregulated in the exercised heart. However, its roles in exercise-induced physiological cardiac growth and its potential protective effects against cardiac I/R injury remain largely unexplored. METHODS: We studied the function of Hmbox1 in exercise-induced physiological cardiac growth in mice after 4 weeks of swimming exercise. Hmbox1 expression was then evaluated in human heart samples from deceased patients with myocardial infarction and in the animal cardiac I/R injury model. Its role in cardiac I/R injury was examined in mice with adeno-associated virus 9 (AAV9) vector-mediated Hmbox1 knockdown and in those with cardiac myocyte-specific Hmbox1 ablation. We performed RNA sequencing, promoter prediction, and binding assays and identified glucokinase (Gck) as a downstream effector of Hmbox1. The effects of Hmbox1 together with Gck were examined in cardiomyocytes to evaluate their cell size, proliferation, apoptosis, mitochondrial respiration, and glycolysis. The function of upstream regulator of Hmbox1, ETS1, was investigated through ETS1 overexpression in cardiac I/R mice in vivo. RESULTS: We demonstrated that Hmbox1 downregulation was required for exercise-induced physiological cardiac growth. Inhibition of Hmbox1 increased cardiomyocyte size in isolated neonatal rat cardiomyocytes and human embryonic stem cell-derived cardiomyocytes but did not affect cardiomyocyte proliferation. Under pathological conditions, Hmbox1 was upregulated in both human and animal postinfarct cardiac tissues. Furthermore, both cardiac myocyte-specific Hmbox1 knockout and AAV9-mediated Hmbox1 knockdown protected against cardiac I/R injury and heart failure. Therapeutic effects were observed when sh-Hmbox1 AAV9 was administered after I/R injury. Inhibition of Hmbox1 activated the Akt/mTOR/P70S6K pathway and transcriptionally upregulated Gck, leading to reduced apoptosis and improved mitochondrial respiration and glycolysis in cardiomyocytes. ETS1 functioned as an upstream negative regulator of Hmbox1 transcription, and its overexpression was protective against cardiac I/R injury. CONCLUSIONS: Our studies unravel a new role for the transcriptional repressor Hmbox1 in exercise-induced physiological cardiac growth. They also highlight the therapeutic potential of targeting Hmbox1 to improve myocardial survival and glucose metabolism after I/R injury.

Mitochondrial Calcium Regulation of Cardiac Metabolism in Health and Disease.

Oxidative phosphorylation is regulated by mitochondrial calcium (Ca2+) in health and disease. In physiological states, Ca2+ enters via the mitochondrial Ca2+ uniporter and rapidly enhances NADH and ATP production. However, maintaining Ca2+ homeostasis is critical: insufficient Ca2+ impairs stress adaptation, and Ca2+ overload can trigger cell death. In this review, we delve into recent insights further defining the relationship between mitochondrial Ca2+ dynamics and oxidative phosphorylation. Our focus is on how such regulation affects cardiac function in health and disease, including heart failure, ischemia-reperfusion, arrhythmias, catecholaminergic polymorphic ventricular tachycardia, mitochondrial cardiomyopathies, Barth syndrome, and Friedreich's ataxia. Several themes emerge from recent data. First, mitochondrial Ca2+ regulation is critical for fuel substrate selection, metabolite import, and matching of ATP supply to demand. Second, mitochondrial Ca2+ regulates both the production and response to reactive oxygen species (ROS), and the balance between its pro- and antioxidant effects is key to how it contributes to physiological and pathological states. Third, Ca2+ exerts localized effects on the electron transport chain (ETC), not through traditional allosteric mechanisms but rather indirectly. These effects hinge on specific transporters, such as the uniporter or the Na+/Ca2+ exchanger, and may not be noticeable acutely, contributing differently to phenotypes depending on whether Ca2+ transporters are acutely or chronically modified. Perturbations in these novel relationships during disease states may either serve as compensatory mechanisms or exacerbate impairments in oxidative phosphorylation. Consequently, targeting mitochondrial Ca2+ holds promise as a therapeutic strategy for a variety of cardiac diseases characterized by contractile failure or arrhythmias.

Liver Transplantation: A Test of Cellular Physiology, Preservation, and Injury.

Liver transplantation has evolved into a mature clinical field, but scarcity of usable organs poses a unique challenge. Expanding the donor pool requires novel approaches for protecting hepatic physiology and cellular homeostasis. Here we define hepatocellular injury during transplantation, with an emphasis on modifiable cell death pathways as future therapeutics.

MSC-Derived Small Extracellular Vesicles Exert Cardioprotective Effect Through Reducing VLCFAs and Apoptosis in Human Cardiac Organoid IRI Model.

Cardiovascular diseases (CVDs) are the leading cause of death worldwide, accounting for 31% of all deaths globally. Myocardial ischemia-reperfusion injury (IRI), a common complication of CVDs, is a major cause of mortality and morbidity. Studies have shown efficacious use of mesenchymal stem cells-derived small extracellular vesicles (MSCs-EVs) to mitigate IRI in animals, but few research has been done on human-related models. In this study, human embryonic stem cell-derived chambered cardiac organoid (CCO) was used as a model system to study the effects of MSC-EVs on myocardial IRI. The results revealed that MSC-EVs treatment reduced apoptosis and improved contraction resumption of the CCOs. Metabolomics analysis showed that this effect could be attributed to EVs' ability to prevent the accumulation of unsaturated very long-chain fatty acids (VLCFAs). This was corroborated when inhibition of fatty acid synthase, which was reported to reduce VLCFAs, produced a similar protective effect to EVs. Overall, this study uncovered the mechanistic role of MSC-EVs in mitigating IRI that involves preventing the accumulation of unsaturated VLCFA, decreasing cell death, and improving contraction resumption in CCOs.

Enforced HCELL Expression Enhances Bone Marrow Stromal Cells Homing and Amelioration of Cerebral Ischemia-Reperfusion Injury via induction of PGE2.

Ischemic stroke (IS) is a significant and potentially life-threatening disease with limited treatment options, often resulting in severe disability. Bone marrow stromal cells (BMSCs) transplantation has exhibited promising neuroprotection following cerebral ischemia-reperfusion injury (CIRI). However, the effectiveness is hindered by their low homing rate when administered through the vein. In this study, we aimed to enhance the homing ability of BMSCs through lentivirus transfection to express fucosyltransferase 7. This glycosylation engineered CD44 on BMSCs to express hematopoietic cell E-selectin/L-selectin ligand (HCELL), which is the most potent E-selectin ligand. Following enforced HCELL expression, the transplantation of BMSCs was then evaluated in a middle cerebral artery occlusion (MCAO) model. Results showed that HCELL+BMSCs significantly ameliorated neurological deficits and reduced the volume of cerebral infarction. Furthermore, the transplantation led to a decrease in apoptosis by up-regulating BCL-2 and down-regulating BAX, also reduced the mRNA levels of inflammatory factors, such as interleukin-1ß (IL-1ß), IL-2, IL-6 and tumor necrosis factor-alpha (TNF-α) in the ischemic brain tissue. Notably, enforced HCELL expression facilitated the migration of BMSCs towards cerebral ischemic lesions and their subsequent transendothelial migration through the up-regulation of PTGS-2, increased production of PGE2 and activation of VLA-4. In summary, our study demonstrates that transplantation of HCELL+BMSCs effectively alleviates CIRI, and that enforced HCELL expression enhances the homing of BMSCs to cerebral ischemic lesions and their transendothelial migration via PTGS-2/PGE2/VLA-4. These findings indicate that enforced expression of HCELL on BMSCs could serve as a promising therapeutic strategy for the treatment of ischemic stroke.

Mid1 aggravates hepatic ischemia-reperfusion injury by inducing immune cell infiltration.

Hepatic ischemia-reperfusion injury (HIRI) represents a major risk factor in liver transplantation and resection surgeries. Kupffer cells (KCs) produce proinflammatory cytokines and lead to hepatic neutrophil infiltration in the liver, which is one of the leading causes of HIRI. Mid1 is involved in immune infiltration, but the role of Mid1 remains poorly understood. Herin, our study aimed to investigate the effect of Mid1 on HIRI progression. Male C57BL/6 mice aged 6 weeks were used for the HIRI model established. The function of Mid1 on liver injury and hepatic inflammation was evaluated. In vitro, KCs were used to investigate the function and mechanism of Mid1 in modulating KC inflammation upon lipopolysaccharide (LPS) stimulation. We found that Mid1 expression was up-regulated upon HIRI. Mid1 inhibition alleviated liver damage, as evidenced by neutrophil infiltration, intrahepatic inflammation, and hepatocyte apoptosis. In vitro experiments further revealed that Mid1 knockdown reduced the secretion of proinflammatory cytokines and chemokines in KCs. Moreover, silenced-Mid1 suppressed proinflammatory responses by the inhibition of NF-κB, JNK, and p38 signaling pathways. Taken together, Mid1 contributes to HIRI via regulating the proinflammatory response of KCs and inducing neutrophil infiltration. Targeting Mid1 may be a promising strategy to protect against HIRI.

Scutellarin alleviates renal ischemia-reperfusion injury by inhibiting the MAPK pathway and pro-inflammatory macrophage polarization.

Renal ischemia-reperfusion injury (IRI) is an integral process in renal transplantation, which results in compromised graft survival. Macrophages play an important role in both the early inflammatory period and late fibrotic period in response to IRI. In this study, we investigated whether scutellarin (SCU) could protect against renal IRI by regulating macrophage polarization. Mice were given SCU (5-50 mg/kg) by gavage 1 h earlier, followed by a unilateral renal IRI. Renal function and pathological injury were assessed 24 h after reperfusion. The results showed that administration of 50 mg/kg SCU significantly improved renal function and renal pathology in IRI mice. In addition, SCU alleviated IRI-induced apoptosis. Meanwhile, it reduced macrophage infiltration and inhibited pro-inflammatory macrophage polarization. Moreover, in RAW 264.7 cells and primary bone marrow-derived macrophages (BMDMs) exposed to SCU, we found that 150 µM SCU inhibited these cells to polarize to an inflammatory phenotype induced by lipopolysaccharide (LPS) and interferon-γ (IFN-γ). However, SCU has no influence on anti-inflammatory macrophage polarization in vivo and in vitro induced by in interleukin-4 (IL-4). Finally, we explored the effect of SCU on the activation of the mitogen-activated protein kinase (MAPK) pathway both in vivo and in vitro. We found that SCU suppressed the activation of the MAPK pathway, including the extracellular signal-regulated kinase (ERK), Jun N-terminal kinase (JNK), and p38. Our results demonstrated that SCU protects the kidney against IRI by inhibiting macrophage infiltration and polarization toward pro-inflammatory phenotype via the MAPK pathway, suggesting that SCU may be therapeutically important in treatment of IRI.

Histone deacetylase 6 deficiency protects the liver against ischemia/reperfusion injury by activating PI3K/AKT/mTOR signaling.

Liver transplantation (LT) is the only effective method to treat end-stage liver disease. Hepatic ischemia-reperfusion injury (IRI) continues to limit the prognosis of patients receiving LT. Histone deacetylase 6 (HDAC6) is a unique HDAC member involved in inflammation and apoptosis. However, its role and mechanism in hepatic IRI have not yet been reported. We examined HDAC6 levels in liver tissue from LT patients, mice challenged with liver IRI, and hepatocytes subjected to hypoxia/reoxygenation (H/R). In addition, HDAC6 global-knockout (HDAC6-KO) mice, adeno-associated virus-mediated liver-specific HDAC6 overexpressing (HDAC6-LTG) mice, and their corresponding controls were used to construct hepatic IRI models. Hepatic histology, inflammatory responses, and apoptosis were detected to assess liver injury. The molecular mechanisms of HDAC6 in hepatic IRI were explored in vivo and in vitro. Moreover, the HDAC6-selective inhibitor tubastatin A was used to detect the therapeutic effect of HDAC6 on liver IRI. Together, our results showed that HDAC6 expression was significantly upregulated in liver tissue from LT patients, mice subjected to hepatic I/R surgery, and hepatocytes challenged by hypoxia/reoxygenation (H/R) treatment. Compared with control mice, HDAC6 deficiency mitigated liver IRI by inhibiting inflammatory responses and apoptosis, whereas HDAC6-LTG mice displayed the opposite phenotype. Further molecular experiments show that HDAC6 bound to and deacetylated AKT and HDAC6 deficiency improved liver IRI by activating PI3K/AKT/mTOR signaling. In conclusion, HDAC6 is a key mediator of hepatic IRI that functions to promote inflammation and apoptosis via PI3K/AKT/mTOR signaling. Targeting hepatic HDAC6 inhibition may be a promising approach to attenuate liver IRI.

Preconditioning with ß-hydroxybutyrate attenuates lung ischemia-reperfusion injury by suppressing alveolar macrophage pyroptosis through the SIRT1-FOXO3 signaling pathway.

The complex pathogenesis of lung ischemia-reperfusion injury (LIRI) was examined in a murine model, focusing on the role of pyroptosis and its exacerbation of lung injury. We specifically examined the levels and cellular localization of pyroptosis within the lung, which revealed alveolar macrophages as the primary site. The inhibition of pyroptosis by VX-765 reduced the severity of lung injury, underscoring its significant role in LIRI. Furthermore, the therapeutic potential of ß-hydroxybutyrate (ß-OHB) in ameliorating LIRI was examined. Modulation of ß-OHB levels was evaluated by ketone ester supplementation and 3-hydroxybutyrate dehydrogenase 1 (BDH-1) gene knockout, along with the manipulation of the SIRT1-FOXO3 signaling pathway using EX-527 and pCMV-SIRT1 plasmid transfection. This revealed that ß-OHB exerts lung-protective and anti-pyroptotic effects, which were mediated through the upregulation of SIRT1 and the enhancement of FOXO3 deacetylation, leading to decreased pyroptosis markers and lung injury. In addition, ß-OHB treatment of MH-S cells in vitro showed a concentration-dependent improvement in pyroptosis, linking its therapeutic benefits to specific cell mechanisms. Overall, this study highlights the significance of alveolar macrophage pyroptosis in the exacerbation of LIRI and indicates the potential of ß-OHB in mitigating injury by modulating the SIRT1-FOXO3 signaling pathway.

Interdependent Nuclear Co-Trafficking of ASPP1 and p53 Aggravates Cardiac Ischemia/Reperfusion Injury.

OBJECTIVE: ASPP1 (apoptosis stimulating of p53 protein 1) is critical in regulating cell apoptosis as a cofactor of p53 to promote its transcriptional activity in the nucleus. However, whether cytoplasmic ASPP1 affects p53 nuclear trafficking and its role in cardiac diseases remains unknown. This study aims to explore the mechanism by which ASPP1 modulates p53 nuclear trafficking and the subsequent contribution to cardiac ischemia/reperfusion (I/R) injury. METHODS AND RESULTS: The immunofluorescent staining showed that under normal condition ASPP1 and p53 colocalized in the cytoplasm of neonatal mouse ventricular cardiomyocytes, while they were both upregulated and translocated to the nuclei upon hypoxia/reoxygenation treatment. The nuclear translocation of ASPP1 and p53 was interdependent, as knockdown of either ASPP1 or p53 attenuated nuclear translocation of the other one. Inhibition of importin-ß1 resulted in the cytoplasmic sequestration of both p53 and ASPP1 in neonatal mouse ventricular cardiomyocytes with hypoxia/reoxygenation stimulation. Overexpression of ASPP1 potentiated, whereas knockdown of ASPP1 inhibited the expression of Bax (Bcl2-associated X), PUMA (p53 upregulated modulator of apoptosis), and Noxa, direct apoptosis-associated targets of p53. ASPP1 was also increased in the I/R myocardium. Cardiomyocyte-specific transgenic overexpression of ASPP1 aggravated I/R injury as indicated by increased infarct size and impaired cardiac function. Conversely, knockout of ASPP1 mitigated cardiac I/R injury. The same qualitative data were observed in neonatal mouse ventricular cardiomyocytes exposed to hypoxia/reoxygenation injury. Furthermore, inhibition of p53 significantly blunted the proapoptotic activity and detrimental effects of ASPP1 both in vitro and in vivo. CONCLUSIONS: Binding of ASPP1 to p53 triggers their nuclear cotranslocation via importin-ß1 that eventually exacerbates cardiac I/R injury. The findings imply that interfering the expression of ASPP1 or the interaction between ASPP1 and p53 to block their nuclear trafficking represents an important therapeutic strategy for cardiac I/R injury.

Platelet-Derived MicroRNAs Regulate Cardiac Remodeling After Myocardial Ischemia.

BACKGROUND: Platelets can infiltrate ischemic myocardium and are increasingly recognized as critical regulators of inflammatory processes during myocardial ischemia and reperfusion (I/R). Platelets contain a broad repertoire of microRNAs (miRNAs), which, under certain conditions such as myocardial ischemia, may be transferred to surrounding cells or released into the microenvironment. Recent studies could demonstrate that platelets contribute substantially to the circulating miRNA pool holding the potential for so far undiscovered regulatory functions. The present study aimed to determine the role of platelet-derived miRNAs in myocardial injury and repair following myocardial I/R. METHODS: In vivo model of myocardial I/R, multimodal in vivo and ex vivo imaging approaches (light-sheet fluorescence microscopy, positron emission tomography and magnetic resonance imaging, speckle-tracking echocardiography) of myocardial inflammation and remodeling, and next-generation deep sequencing analysis of platelet miRNA expression. RESULTS: In mice with a megakaryocyte/platelet-specific knockout of pre-miRNA processing ribonuclease Dicer, the present study discloses a key role of platelet-derived miRNAs in the tightly regulated cellular processes orchestrating left ventricular remodeling after myocardial I/R following transient left coronary artery ligation. Disruption of the miRNA processing machinery in platelets by deletion of Dicer resulted in increased myocardial inflammation, impaired angiogenesis, and accelerated development of cardiac fibrosis, culminating in an increased infarct size by d7 that persisted through d28 of myocardial I/R. Worsened cardiac remodeling after myocardial infarction in mice with a platelet-specific Dicer deletion resulted in an increased fibrotic scar formation and distinguishably increased perfusion defect of the apical and anterolateral wall at day 28 post-myocardial infarction. Altogether, these observations culminated in an impaired left ventricular function and hampered long-term cardiac recovery after experimental myocardial infarction and reperfusion therapy. Treatment with the P2Y12 (P2Y purinoceptor 12) antagonist ticagrelor completely reversed increased myocardial damage and adverse cardiac remodeling observed in DicerPf4∆/Pf4∆ mice. CONCLUSIONS: The present study discloses a critical role of platelet-derived miRNA in myocardial inflammation and structural remodeling processes following myocardial I/R.

Air pollution aggravates renal ischaemia-reperfusion-induced acute kidney injury.

Chronic kidney disease (CKD) has emerged as a significant global public health concern. Recent epidemiological studies have highlighted the link between exposure to fine particulate matter (PM2.5) and a decline in renal function. PM2.5 exerts harmful effects on various organs through oxidative stress and inflammation. Acute kidney injury (AKI) resulting from ischaemia-reperfusion injury (IRI) involves biological processes similar to those involved in PM2.5 toxicity and is a known risk factor for CKD. The objective of this study was to investigate the impact of PM2.5 exposure on IRI-induced AKI. Through a unique environmentally controlled setup, mice were exposed to urban PM2.5 or filtered air for 12 weeks before IRI followed by euthanasia 48 h after surgery. Animals exposed to PM2.5 and IRI exhibited reduced glomerular filtration, impaired urine concentration ability, and significant tubular damage. Further, PM2.5 aggravated local innate immune responses and mitochondrial dysfunction, as well as enhancing cyclic GMP-AMP synthase-stimulator of interferon genes (cGAS-STING) pathway activation. This increased renal senescence and suppressed the anti-ageing protein klotho, leading to early fibrotic changes. In vitro studies using proximal tubular epithelial cells exposed to PM2.5 and hypoxia/reoxygenation revealed heightened activation of the STING pathway triggered by cytoplasmic mitochondrial DNA, resulting in increased tubular damage and a pro-inflammatory phenotype. In summary, our findings imply a role for PM2.5 in sensitising proximal tubular epithelial cells to IRI-induced damage, suggesting a plausible association between PM2.5 exposure and heightened susceptibility to CKD in individuals experiencing AKI. Strategies aimed at reducing PM2.5 concentrations and implementing preventive measures may improve outcomes for AKI patients and mitigate the progression from AKI to CKD. © 2024 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland.

Glycyrrhizin attenuates myocardial ischemia reperfusion injury by suppressing Inflammation, oxidative stress, and ferroptosis via the HMGB1-TLR4-GPX4 pathway.

Ferroptosis, a form of regulated cell death process, play an important role in myocardial ischemia‒reperfusion (I/R) injury. Glycyrrhizin (GL), a natural glycoconjugate triterpene, has the property to improve growth rate, immune regulation, antioxidant, anti-inflammatory. However, whether GL can attenuate myocardial I/R injury by modulating ferroptosis or other mechanisms are still unclear. In this study, SD rats underwent in vivo myocardial ischemia/reperfusion (I/R) surgery, while H9C2 cells were subjected to the hypoxia/reoxygenation (H/R) model for in vitro experiments. In addition, TAK-242, a TLR4-specific antagonist, and GL were also used to evaluate the effect and mechanisms of GL on the cardiac function and expression of ferroptosis-related gene and protein in vivo and vitro. The results show that GL decreased not only the expression of the inflammation-related factors (HMGB1, TNF-α, IL-6, IL-18 and IL-1ß), but also reduced the number of TUNEL-positive cardiomyocytes, and mitigated pathological alterations in I/R injury. In addition, GL decreased the levels of MDA, promoted antioxidant capacity such as GSH, CAT, Cu/Zn-SOD, Mn-SOD, and SOD in vivo and vitro. More importantly, GL and TAK-242 regulate ferroptosis-related protein and gene expression in I/R and H/R model. Surprisingly, GL may ameliorate cardiomyocyte ferroptosis and ultimately improves cardiac function induced by H/R via the HMGB1-TLR4-GPX4 axis. Therefore, we have highlighted a novel mechanism by which GL regulates inflammation, oxidative stress, and ferroptosis via the HMGB1-TLR4-GPX4 pathway to prevent myocardial I/R injury. GL appears to be a potentially applicable drug for the treatment of myocardial I/R injury.

MSR1-dependent efferocytosis improved ischemia-reperfusion injury following aged-donor liver transplantation in mice by regulating the pro-resolving polarisation of macrophages.

Compared with young liver donors, aged liver donors are more susceptible to ischemia-reperfusion injury (IRI) following transplantation, which may be related to excessive inflammatory response and macrophage dysfunction, but the specific mechanism is unclear. Macrophage scavenger receptor 1 (MSR1) is a member of the scavenger receptor family, and plays an important regulatory role in inflammation response and macrophage function regulation. But its role in IRI following aged-donor liver transplantation is still unclear. This study demonstrates that MSR1 expression is decreased in macrophages from aged donor livers, inhibiting their efferocytosis and pro-resolving polarisation. Decreased MSR1 is responsible for the more severe IRI suffered by aged donor livers. Overexpression of MSR1 using F4/80-labelled AAV9 improved intrahepatic macrophage efferocytosis and promoted pro-resolving polarisation, ultimately ameliorating IRI following aged-donor liver transplantation. In vitro co-culture experiments further showed that overexpression of MSR1 promoted an increase in calcium concentration, which further activated the PI3K-AKT-GSK3ß pathway, and induced the upregulation of ß-catenin. Overall, MSR1-dependent efferocytosis promoted the pro-resolving polarisation of macrophages through the PI3K-AKT-GSK3ß pathway-induced up-regulating of ß-catenin leading to improved IRI following aged-donor liver transplantation.

In vitro modeling of skeletal muscle ischemia-reperfusion injury based on sphere differentiation culture from human pluripotent stem cells.

Skeletal muscle ischemia-reperfusion (IR) injury poses significant challenges due to its local and systemic complications. Traditional studies relying on two-dimensional (2D) cell culture or animal models often fall short of faithfully replicating the human in vivo environment, thereby impeding the translational process from animal research to clinical applications. Three-dimensional (3D) constructs, such as skeletal muscle spheroids with enhanced cell-cell interactions from human pluripotent stem cells (hPSCs) offer a promising alternative by partially mimicking human physiological cellular environment in vivo processes. This study aims to establish an innovative in vitro model, human skeletal muscle spheroids based on sphere differentiation from hPSCs, to investigate human skeletal muscle developmental processes and IR mechanisms within a controlled laboratory setting. By eticulously recapitulating embryonic myogenesis through paraxial mesodermal differentiation of neuro-mesodermal progenitors, we successfully established 3D skeletal muscle spheroids that mirror the dynamic colonization observed during human skeletal muscle development. Co-culturing human skeletal muscle spheroids with spinal cord spheroids facilitated the formation of neuromuscular junctions, providing functional relevance to skeletal muscle spheroids. Furthermore, through oxygen-glucose deprivation/re-oxygenation treatment, 3D skeletal muscle spheroids provide insights into the molecular events and pathogenesis of IR injury. The findings presented in this study significantly contribute to our understanding of skeletal muscle development and offer a robust platform for in vitro studies on skeletal muscle IR injury, holding potential applications in drug testing, therapeutic development, and personalized medicine within the realm of skeletal muscle-related pathologies.

Proximal tubular FHL2, a novel downstream target of hypoxia inducible factor 1, is a protector against ischemic acute kidney injury.

Four-and-a-half LIM domains protein 2 (FHL2) is an adaptor protein that may interact with hypoxia inducible factor 1α (HIF-1α) or ß-catenin, two pivotal protective signaling in acute kidney injury (AKI). However, little is known about the regulation and function of FHL2 during AKI. We found that FHL2 was induced in renal tubular cells in patients with acute tubular necrosis and mice model of ischemia-reperfusion injury (IRI). In cultured renal proximal tubular cells (PTCs), hypoxia induced FHL2 expression and promoted the binding of HIF-1 to FHL2 promoter. Compared with control littermates, mice with PTC-specific deletion of FHL2 gene displayed worse renal function, more severe morphologic lesion, more tubular cell death and less cell proliferation, accompanying by downregulation of AQP1 and Na, K-ATPase after IRI. Consistently, loss of FHL2 in PTCs restricted activation of HIF-1 and ß-catenin signaling simultaneously, leading to attenuation of glycolysis, upregulation of apoptosis-related proteins and downregulation of proliferation-related proteins during IRI. In vitro, knockdown of FHL2 suppressed hypoxia-induced activation of HIF-1α and ß-catenin signaling pathways. Overexpression of FHL2 induced physical interactions between FHL2 and HIF-1α, ß-catenin, GSK-3ß or p300, and the combination of these interactions favored the stabilization and nuclear translocation of HIF-1α and ß-catenin, enhancing their mediated gene transcription. Collectively, these findings identify FHL2 as a direct downstream target gene of HIF-1 signaling and demonstrate that FHL2 could play a critical role in protecting against ischemic AKI by promoting the activation of HIF-1 and ß-catenin signaling through the interactions with its multiple protein partners.

Necroptosis triggers spatially restricted neutrophil-mediated vascular damage during lung ischemia reperfusion injury.

Ischemia reperfusion injury represents a common pathological condition that is triggered by the release of endogenous ligands. While neutrophils are known to play a critical role in its pathogenesis, the tissue-specific spatiotemporal regulation of ischemia-reperfusion injury is not understood. Here, using oxidative lipidomics and intravital imaging of transplanted mouse lungs that are subjected to severe ischemia reperfusion injury, we discovered that necroptosis, a nonapoptotic form of cell death, triggers the recruitment of neutrophils. During the initial stages of inflammation, neutrophils traffic predominantly to subpleural vessels, where their aggregation is directed by chemoattractants produced by nonclassical monocytes that are spatially restricted in this vascular compartment. Subsequent neutrophilic disruption of capillaries resulting in vascular leakage is associated with impaired graft function. We found that TLR4 signaling in vascular endothelial cells and downstream NADPH oxidase 4 expression mediate the arrest of neutrophils, a step upstream of their extravasation. Neutrophil extracellular traps formed in injured lungs and their disruption with DNase prevented vascular leakage and ameliorated primary graft dysfunction. Thus, we have uncovered mechanisms that regulate the initial recruitment of neutrophils to injured lungs, which result in selective damage to subpleural pulmonary vessels and primary graft dysfunction. Our findings could lead to the development of new therapeutics that protect lungs from ischemia reperfusion injury.