PAFAH2 suppresses synchronized ferroptosis to ameliorate acute kidney injury.

Synchronized ferroptosis contributes to nephron loss in acute kidney injury (AKI). However, the propagation signals and the underlying mechanisms of the synchronized ferroptosis for renal tubular injury remain unresolved. Here we report that platelet-activating factor (PAF) and PAF-like phospholipids (PAF-LPLs) mediated synchronized ferroptosis and contributed to AKI. The emergence of PAF and PAF-LPLs in ferroptosis caused the instability of biomembranes and signaled the cell death of neighboring cells. This cascade could be suppressed by PAF-acetylhydrolase (II) (PAFAH2) or by addition of antibodies against PAF. Genetic knockout or pharmacological inhibition of PAFAH2 increased PAF production, augmented synchronized ferroptosis and exacerbated ischemia/reperfusion (I/R)-induced AKI. Notably, intravenous administration of wild-type PAFAH2 protein, but not its enzymatically inactive mutants, prevented synchronized tubular cell death, nephron loss and AKI. Our findings offer an insight into the mechanisms of synchronized ferroptosis and suggest a possibility for the preventive intervention of AKI.

Targeted activation of ferroptosis in colorectal cancer via LGR4 targeting overcomes acquired drug resistance.

Acquired drug resistance is a major challenge for cancer therapy and is the leading cause of cancer mortality; however, the mechanisms of drug resistance are diverse and the strategy to specifically target drug-resistant cancer cells remains an unmet clinical issue. Here, we established a colorectal cancer-derived organoid biobank and induced acquired drug resistance by repeated low-level exposures of chemo-agents. Chemosensitivity profiling and transcriptomic analysis studies revealed that chemoresistant cancer-derived organoids exhibited elevated expression of LGR4 and activation of the Wnt signaling pathway. Further, we generated a monoclonal antibody (LGR4-mAb) that potently inhibited LGR4-Wnt signaling and found that treatment with LGR4-mAb notably sensitized drug-induced ferroptosis. Mechanistically, LGR4-dependent Wnt signaling transcriptionally upregulated SLC7A11, a key inhibitor of ferroptosis, to confer acquired drug resistance. Our findings reveal that targeting of Wnt signaling by LGR4-mAb augments ferroptosis when co-administrated with chemotherapeutic agents, demonstrating a potential opportunity to fight refractory and recurrent cancers.

IL-6 is a predictor and potential therapeutic target for coronavirus disease 2019-related heart failure: A single-center retrospective study.

BACKGROUND: Inflammation is linked to coronavirus disease 2019 (COVID-19)-related heart failure (HF), but the specific mechanisms are unclear. This study aimed to assess the relationship between specific inflammatory factors, such as interleukin (IL)-1ß, IL-2, IL-4, IL-5, IL-6, IL-8, IL-10, IL-12, IL-17, interferon (IFN)-α, and IFN-γ, and COVID-19-related HF. METHODS: We retrospectively identified 212 adult patients with COVID-19 who were hospitalized at Shanghai Public Health Center from March 1 to May 30, 2022 (including 80 patients with HF and 132 without HF). High-sensitivity C-reactive protein (hs-CRP), procalcitonin (PCT), and inflammatory factors, including IL-1ß, IL-2, IL-4, IL-5, IL-6, IL-8, IL-10, IL-12, IL-17, IFN-α, and IFN-γ, were compared between patients with COVID-19 with and without HF. RESULTS: Patients with COVID-19 having and not having HF differed with regard to sex, age, hs-CRP, PCT, and IL-6 levels (p < 0.05). Logistic regression analysis indicated a significant positive association between IL and 6 and HF (odds ratio = 1.055; 95 % confidence interval: 1.019-1.093, p < 0.005). Sex, age, and hs-CRP were also associated with HF. Women had a greater risk of HF than men. Older age, higher levels of hs-CRP, and IL-6 were associated with a greater risk of HF. CONCLUSIONS: In patients with COVID-19, increased IL-6 levels are significantly associated with COVID-19-related HF.

Integrin ß Expression as a New Diagnostic Marker for Arteriovenous Thrombosis: A Single-Center Prospective Study.

Integrin ß plays an important role in the pathogenesis of thrombosis and inflammation, and it may be a shared pathogenic mechanism between arterial and venous thromboses. With the goal of identifying new treatment targets for thrombotic diseases and specific diagnostic markers for venous thromboembolism (VTE), this prospective clinical study was performed to clarify the relationship between integrin and thrombosis. The levels of integrin ß1-3, interleukin-6 (IL-6), and C-reactive protein were significantly higher in patients with acute myocardial infarction (AMI; n = 44) and acute VTE (n = 43) compared to healthy controls (n = 33). The IL-6 and integrin ß1-3 levels were also significantly higher in the AMI group compared to the VTE and control groups. Logistic regression analysis identified IL-6 and integrin ß1-3 levels as independent risk factors for thrombotic disease. Based on the receiver-operating characteristic curve, Youden index, sensitivity, and specificity, the diagnostic accuracy value for VTE was greater than 0.8 when integrins ß1, ß2, and ß3 were combined. Overall, these results suggest that integrin ß levels can contribute to improving the diagnosis and treatment of arteriovenous thrombosis.

Early-life exposure to lead changes cardiac development and compromises long-term cardiac function.

Lead (Pb) is widely used in industrial and daily-use consumer products. Early-life exposure may increase the risk of lead-related heart problems in childhood. However, the effects of early-life lead exposure on fetal heart development and long-term cardiac outcomes are unknown. In this study, pregnant ICR mice were exposed to lead acetate trihydrate (50 mg/kg/d) via oral gavage from gestation day 1.5 until offspring weaning. Thereafter, the second hit model was established, two groups of offspring (4 weeks old) were either administered sterile saline or Angiotensin II (Ang II) for 4 weeks until euthanasia. We investigated lead-induced offspring heart damage from embryonic period to adulthood by echocardiographic analysis, pathological H&E staining, and ultrastructural examination, as well as mitochondrial function detection. The results showed early-life lead exposure predisposed offspring mice to decreased ejection fraction, increased left ventricular volume, accompanied by hypertrophy and dilation, cardiomyocyte sarcomere dysplasia, abnormal mitochondrial structure, mitochondrial dysfunction, and decreased expression of key sarcomeric and mitochondrial genes, rendering them more susceptible to cardiac hypertrophy, vascular wall thickening, cardiac fibrosis, apoptosis, and heart failure induced by Ang II infusion. This study elucidates early-life low dose lead exposure compromises cardiac development and exacerbates second hit-induced cardiac pathological responses in adulthood, which furnishes crucial scientific evidence pertaining to the cardiac toxicity and risk evaluation associated with early-life exposure to lead.

NRF1-mediated mitochondrial biogenesis antagonizes innate antiviral immunity.

Mitochondrial biogenesis is the process of generating new mitochondria to maintain cellular homeostasis. Here, we report that viruses exploit mitochondrial biogenesis to antagonize innate antiviral immunity. We found that nuclear respiratory factor-1 (NRF1), a vital transcriptional factor involved in nuclear-mitochondrial interactions, is essential for RNA (VSV) or DNA (HSV-1) virus-induced mitochondrial biogenesis. NRF1 deficiency resulted in enhanced innate immunity, a diminished viral load, and morbidity in mice. Mechanistically, the inhibition of NRF1-mediated mitochondrial biogenesis aggravated virus-induced mitochondrial damage, promoted the release of mitochondrial DNA (mtDNA), increased the production of mitochondrial reactive oxygen species (mtROS), and activated the innate immune response. Notably, virus-activated kinase TBK1 phosphorylated NRF1 at Ser318 and thereby triggered the inactivation of the NRF1-TFAM axis during HSV-1 infection. A knock-in (KI) strategy that mimicked TBK1-NRF1 signaling revealed that interrupting the TBK1-NRF1 connection ablated mtDNA release and thereby attenuated the HSV-1-induced innate antiviral response. Our study reveals a previously unidentified antiviral mechanism that utilizes a NRF1-mediated negative feedback loop to modulate mitochondrial biogenesis and antagonize innate immune response.

Krüppel-Like Factors Orchestrate Endothelial Gene Expression Through Redundant and Non-Redundant Enhancer Networks.

Background Proper function of endothelial cells is critical for vascular integrity and organismal survival. Studies over the past 2 decades have identified 2 members of the KLF (Krüppel-like factor) family of proteins, KLF2 and KLF4, as nodal regulators of endothelial function. Strikingly, inducible postnatal deletion of both KLF2 and KLF4 resulted in widespread vascular leak, coagulopathy, and rapid death. Importantly, while transcriptomic studies revealed profound alterations in gene expression, the molecular mechanisms underlying these changes remain poorly understood. Here, we seek to determine mechanisms of KLF2 and KLF4 transcriptional control in multiple vascular beds to further understand their roles as critical endothelial regulators. Methods and Results We integrate chromatin occupancy and transcription studies from multiple transgenic mouse models to demonstrate that KLF2 and KLF4 have overlapping yet distinct binding patterns and transcriptional targets in heart and lung endothelium. Mechanistically, KLFs use open chromatin regions in promoters and enhancers and bind in context-specific patterns that govern transcription in microvasculature. Importantly, this occurs during homeostasis in vivo without additional exogenous stimuli. Conclusions Together, this work provides mechanistic insight behind the well-described transcriptional and functional heterogeneity seen in vascular populations, while also establishing tools into exploring microvascular endothelial dynamics in vivo.

A targetable pathway in neutrophils mitigates both arterial and venous thrombosis.

Arterial and venous thrombosis constitutes a major source of morbidity and mortality worldwide. Long considered as distinct entities, accumulating evidence indicates that arterial and venous thrombosis can occur in the same populations, suggesting that common mechanisms are likely operative. Although hyperactivation of the immune system is a common forerunner to the genesis of thrombotic events in both vascular systems, the key molecular control points remain poorly understood. Consequently, antithrombotic therapies targeting the immune system for therapeutics gain are lacking. Here, we show that neutrophils are key effectors of both arterial and venous thrombosis and can be targeted through immunoregulatory nanoparticles. Using antiphospholipid antibody syndrome (APS) as a model for arterial and venous thrombosis, we identified the transcription factor Krüppel-like factor 2 (KLF2) as a key regulator of neutrophil activation. Upon activation through genetic loss of KLF2 or administration of antiphospholipid antibodies, neutrophils clustered P-selectin glycoprotein ligand 1 (PSGL-1) by cortical actin remodeling, thereby increasing adhesion potential at sites of thrombosis. Targeting clustered PSGL-1 using nanoparticles attenuated neutrophil-mediated thrombosis in APS and KLF2 knockout models, illustrating the importance and feasibility of targeting activated neutrophils to prevent pathological thrombosis. Together, our results demonstrate a role for activated neutrophils in both arterial and venous thrombosis and identify key molecular events that serve as potential targets for therapeutics against diverse causes of immunothrombosis.

Hypoxia-induced proteasomal degradation of DBC1 by SIAH2 in breast cancer progression.

DBC1 has been characterized as a key regulator of physiological and pathophysiological activities, such as DNA damage, senescence, and tumorigenesis. However, the mechanism by which the functional stability of DBC1 is regulated has yet to be elucidated. Here, we report that the ubiquitination-mediated degradation of DBC1 is regulated by the E3 ubiquitin ligase SIAH2 and deubiquitinase OTUD5 under hypoxic stress. Mechanistically, hypoxia promoted DBC1 to interact with SIAH2 but not OTUD5, resulting in the ubiquitination and subsequent degradation of DBC1 through the ubiquitin-proteasome pathway. SIAH2 knockout inhibited tumor cell proliferation and migration, which could be rescued by double knockout of SIAH2/CCAR2. Human tissue microarray analysis further revealed that the SIAH2/DBC1 axis was responsible for tumor progression under hypoxic stress. These findings define a key role of the hypoxia-mediated SIAH2-DBC1 pathway in the progression of human breast cancer and provide novel insights into the metastatic mechanism of breast cancer.

Changes in transcriptomic landscape in human end-stage heart failure with distinct etiology.

Heart failure (HF) is the ultimate outcome of a variety of heart diseases, including restrictive cardiomyopathy (RCM), ischemic heart disease (IHD), and valvular heart disease (VHD). To date, accumulating evidence has suggested an important role of noncoding RNAs (ncRNAs) in HF. We performed RNA-sequencing studies with myocardial mRNAs/lncRNAs/circRNAs/miRNAs from non-failing hearts (donor heart tissue from heart transplantation) and three groups of patients with HF (RCM, IHD, and VHD). HF-related gene regulatory networks and gene co-expression networks were constructed based on the interaction relationship and expression profiles of differentially expressed mRNAs/ncRNAs. Our results indicated that HF with different etiologies is regulated by complex lncRNA/circRNA/miRNA/mRNA regulatory networks, comprising common pathways that are shared by all HF types as well as distinct pathways that are enriched in specific HF types. In addition, the HF biomarkers identified in our study have an important clinical application value in HF staging and HF type diagnosis.

Cardiac macrophages regulate isoproterenol-induced Takotsubo-like cardiomyopathy.

Takotsubo syndrome (TTS) is an acute, stress-induced cardiomyopathy that occurs predominantly in women after extreme physical and/or emotional stress. To date, our understanding of the molecular basis for TTS remains unknown and, consequently, specific therapies are lacking. Myocardial infiltration of monocytes and macrophages in TTS has been documented in clinical studies. However, the functional importance of these findings remains poorly understood. Here, we show that a single high dose of isoproterenol (ISO) in mice induced a TTS-like cardiomyopathy phenotype characterized by female predominance, severe cardiac dysfunction, and robust myocardial infiltration of macrophages. Single-cell RNA-Seq studies of myocardial immune cells revealed that TTS-like cardiomyopathy is associated with complex activation of innate and adaptive immune cells in the heart, and macrophages were identified as the dominant immune cells. Global macrophage depletion (via clodronate liposome administration) or blockade of macrophage infiltration (via a CCR2 antagonist or in CCR2-KO mice) resulted in recovery of cardiac dysfunction in ISO-challenged mice. In addition, damping myeloid cell activation by HIF1α deficiency or exposure to the immunomodulatory agent bortezomib ameliorated ISO-induced cardiac dysfunction. Collectively, our findings identify macrophages as a critical regulator of TTS pathogenesis that can be targeted for therapeutic gain.

KLF2 regulates neutrophil activation and thrombosis in cardiac hypertrophy and heart failure progression.

It is widely recognized that inflammation plays a critical role in cardiac hypertrophy and heart failure. However, clinical trials targeting cytokines have shown equivocal effects, indicating the need for a deeper understanding of the precise role of inflammation and inflammatory cells in heart failure. Leukocytes from human subjects and a rodent model of heart failure were characterized by a marked reduction in expression of Klf2 mRNA. Using a mouse model of angiotensin II-induced nonischemic cardiac dysfunction, we showed that neutrophils played an essential role in the pathogenesis and progression of heart failure. Mechanistically, chronic angiotensin II infusion activated a neutrophil KLF2/NETosis pathway that triggered sporadic thrombosis in small myocardial vessels, leading to myocardial hypoxia, cell death, and hypertrophy. Conversely, targeting neutrophils, neutrophil extracellular traps (NETs), or thrombosis ameliorated these pathological changes and preserved cardiac dysfunction. KLF2 regulated neutrophil activation in response to angiotensin II at the molecular level, partly through crosstalk with HIF1 signaling. Taken together, our data implicate neutrophil-mediated immunothrombotic dysregulation as a critical pathogenic mechanism leading to cardiac hypertrophy and heart failure. This neutrophil KLF2-NETosis-thrombosis mechanism underlying chronic heart failure can be exploited for therapeutic gain by therapies targeting neutrophils, NETosis, or thrombosis.

Hypoxic vasodilatory defect and pulmonary hypertension in mice lacking hemoglobin ß-cysteine93 S-nitrosylation.

Systemic hypoxia is characterized by peripheral vasodilation and pulmonary vasoconstriction. However, the system-wide mechanism for signaling hypoxia remains unknown. Accumulating evidence suggests that hemoglobin (Hb) in RBCs may serve as an O2 sensor and O2-responsive NO signal transducer to regulate systemic and pulmonary vascular tone, but this remains unexamined at the integrated system level. One residue invariant in mammalian Hbs, ß-globin cysteine93 (ßCys93), carries NO as vasorelaxant S-nitrosothiol (SNO) to autoregulate blood flow during O2 delivery. ßCys93Ala mutant mice thus exhibit systemic hypoxia despite transporting O2 normally. Here, we show that ßCys93Ala mutant mice had reduced S-nitrosohemoglobin (SNO-Hb) at baseline and upon targeted SNO repletion and that hypoxic vasodilation by RBCs was impaired in vitro and in vivo, recapitulating hypoxic pathophysiology. Notably, ßCys93Ala mutant mice showed marked impairment of hypoxic peripheral vasodilation and developed signs of pulmonary hypertension with age. Mutant mice also died prematurely with cor pulmonale (pulmonary hypertension with right ventricular dysfunction) when living under low O2. Altogether, we identify a major role for RBC SNO in clinically relevant vasodilatory responses attributed previously to endothelial NO. We conclude that SNO-Hb transduces the integrated, system-wide response to hypoxia in the mammalian respiratory cycle, expanding a core physiological principle.

Correction: The thromboprotective effect of traditional Chinese medicine Tongji 2 granules is dependent on anti-inflammatory activity by suppression of NF-κB pathways.

[This corrects the article DOI: 10.1371/journal.pone.0241607.].

Mitophagy receptor FUNDC1 is regulated by PGC-1α/NRF1 to fine tune mitochondrial homeostasis.

Mitophagy is an essential cellular autophagic process that selectively removes superfluous and damaged mitochondria, and it is coordinated with mitochondrial biogenesis to fine tune the quantity and quality of mitochondria. Coordination between these two opposing processes to maintain the functional mitochondrial network is of paramount importance for normal cellular and organismal metabolism. However, the underlying mechanism is not completely understood. Here we report that PGC-1α and nuclear respiratory factor 1 (NRF1), master regulators of mitochondrial biogenesis and metabolic adaptation, also transcriptionally upregulate the gene encoding FUNDC1, a previously characterized mitophagy receptor, in response to cold stress in brown fat tissue. NRF1 binds to the classic consensus site in the promoter of Fundc1 to upregulate its expression and to enhance mitophagy through its interaction with LC3. Specific knockout of Fundc1 in BAT results in reduced mitochondrial turnover and accumulation of functionally compromised mitochondria, leading to impaired adaptive thermogenesis. Our results demonstrate that FUNDC1-dependent mitophagy is directly coupled with mitochondrial biogenesis through the PGC-1α/NRF1 pathway, which dictates mitochondrial quantity, quality, and turnover and contributes to adaptive thermogenesis.

The thromboprotective effect of traditional Chinese medicine Tongji 2 granules is dependent on anti-inflammatory activity by suppression of NF-κB pathways.

Inflammation is a vital physiological response of the immune system meant to protect against the invasion of pathogens. However, accumulating evidence describes an intimate link between inflammation and thrombosis and cellular elements of the immune system of the immune system such as neutrophils and monocytes/macrophages are emerging as key players in the generation of a prothrombotic milieu suggesting that anti-inflammatory therapy may have a role in the management of thrombosis that is driven by inflammation. Tongji 2 (TJ2) is a traditional Chinese medication manufactured as granules by Tongji hospital of Tongji University (Shanghai, China) with known anti-inflammatory properties. In this study, we examine the effects of TJ2 on inflammation and thrombosis. Our study shows that TJ2 modulates NF-κB activation and thus generates a prominent anti-inflammatory effect. Further, we use mouse models of thrombosis to demonstrate that TJ2 has a beneficial effect in both arterial and venous thrombosis that occurs in the absence of alterations in platelet activation or coagulation.

Myeloid Krüppel-like factor 2 is a critical regulator of metabolic inflammation.

Substantial evidence implicates crosstalk between metabolic tissues and the immune system in the inception and progression of obesity. However, molecular regulators that orchestrate metaflammation both centrally and peripherally remains incompletely understood. Here, we identify myeloid Krüppel-like factor 2 (KLF2) as an essential regulator of obesity and its sequelae. In mice and humans, consumption of a fatty diet downregulates myeloid KLF2 levels. Under basal conditions, myeloid-specific KLF2 knockout mice (K2KO) exhibit increased feeding and weight gain. High-fat diet (HFD) feeding further exacerbates the K2KO metabolic disease phenotype. Mechanistically, loss of myeloid KLF2 increases metaflammation in peripheral and central tissues. A combination of pair-feeding, bone marrow-transplant, and microglial ablation implicate central and peripheral contributions to K2KO-induced metabolic dysfunction observed. Finally, overexpression of myeloid KLF2 protects mice from HFD-induced obesity and insulin resistance. Together, these data establish myeloid KLF2 as a nodal regulator of central and peripheral metabolic inflammation in homeostasis and disease.

P7C3-A20 treatment one year after TBI in mice repairs the blood-brain barrier, arrests chronic neurodegeneration, and restores cognition.

Chronic neurodegeneration in survivors of traumatic brain injury (TBI) is a major cause of morbidity, with no effective therapies to mitigate this progressive and debilitating form of nerve cell death. Here, we report that pharmacologic restoration of the blood-brain barrier (BBB), 12 mo after murine TBI, is associated with arrested axonal neurodegeneration and cognitive recovery, benefits that persisted for months after treatment cessation. Recovery was achieved by 30 d of once-daily administration of P7C3-A20, a compound that stabilizes cellular energy levels. Four months after P7C3-A20, electron microscopy revealed full repair of TBI-induced breaks in cortical and hippocampal BBB endothelium. Immunohistochemical staining identified additional benefits of P7C3-A20, including restoration of normal BBB endothelium length, increased brain capillary pericyte density, increased expression of BBB tight junction proteins, reduced brain infiltration of immunoglobulin, and attenuated neuroinflammation. These changes were accompanied by cessation of TBI-induced chronic axonal degeneration. Specificity for P7C3-A20 action on the endothelium was confirmed by protection of cultured human brain microvascular endothelial cells from hydrogen peroxide-induced cell death, as well as preservation of BBB integrity in mice after exposure to toxic levels of lipopolysaccharide. P7C3-A20 also protected mice from BBB degradation after acute TBI. Collectively, our results provide insights into the pathophysiologic mechanisms behind chronic neurodegeneration after TBI, along with a putative treatment strategy. Because TBI increases the risks of other forms of neurodegeneration involving BBB deterioration (e.g., Alzheimer's disease, Parkinson's disease, vascular dementia, chronic traumatic encephalopathy), P7C3-A20 may have widespread clinical utility in the setting of neurodegenerative conditions.

Corin plays a protective role via upregulating MAPK and downregulating eNOS in diabetic nephropathy endothelial dysfunction.

Diabetic nephropathy (DN) is one of the leading causes of mortality in diabetic patients, but its pathogenesis is unclear. We aimed to study the role of the pro-ANP convertase Corin in the pathogenesis of DN. Corin and ANP expression in DN rat kidneys and high-glucose-treated HK-2 cells was analyzed by real-time PCR, western blotting, and immunohistochemical staining. The effect of Corin-siRNA or ANP-siRNA HK-2 cells on EA.hy926 cell migration was determined by scratch-wound healing assay. The expression of mitogen-activated protein kinase (MAPK) and endothelial NO synthase (eNOS) in EA.hy926 cells treated with conditioned medium from Corin-siRNA- or ANP-siRNA-transfected HK-2 cells was determined by western blotting. We found a significant reduction in Corin and ANP expression in DN rat kidneys. These results were recapitulated in HK-2 cells treated with high glucose. EA.hy926 cells treated with conditioned medium from Corin-deficient HK-2 cells had inhibited migration, increased MAPK activity, and decreased eNOS activity. Similar effects were observed with ANP-siRNA transfection. Finally, adding ANP to the Corin-deficient HK-2 conditioned medium rescued the above defects, indicating that Corin mediates its effects through ANP. In conclusion, Corin plays a renoprotective role through pro-ANP processing, and defects in Corin cause endothelial dysfunction through MAPK and eNOS signaling in DN.