Deubiquitinase UCHL1 promotes angiogenesis and blood-spinal cord barrier function recovery after spinal cord injury by stabilizing Sox17.

Improving the function of the blood-spinal cord barrier (BSCB) benefits the functional recovery of mice following spinal cord injury (SCI). The death of endothelial cells and disruption of the BSCB at the injury site contribute to secondary damage, and the ubiquitin-proteasome system is involved in regulating protein function. However, little is known about the regulation of deubiquitinated enzymes in endothelial cells and their effect on BSCB function after SCI. We observed that Sox17 is predominantly localized in endothelial cells and is significantly upregulated after SCI and in LPS-treated brain microvascular endothelial cells. In vitro Sox17 knockdown attenuated endothelial cell proliferation, migration, and tube formation, while in vivo Sox17 knockdown inhibited endothelial regeneration and barrier recovery, leading to poor functional recovery after SCI. Conversely, in vivo overexpression of Sox17 promoted angiogenesis and functional recovery after injury. Additionally, immunoprecipitation-mass spectrometry revealed the interaction between the deubiquitinase UCHL1 and Sox17, which stabilized Sox17 and influenced angiogenesis and BSCB repair following injury. By generating UCHL1 conditional knockout mice and conducting rescue experiments, we further validated that the deubiquitinase UCHL1 promotes angiogenesis and restoration of BSCB function after injury by stabilizing Sox17. Collectively, our findings present a novel therapeutic target for treating SCI by revealing a potential mechanism for endothelial cell regeneration and BSCB repair after SCI.

Expression of Tissue Factor and Platelet/Leukocyte Markers on Extracellular Vesicles Reflect Platelet-Leukocyte Interaction in Severe COVID-19.

Severe COVID-19 is frequently associated with thromboembolic complications. Increased platelet activation and platelet-leukocyte aggregate formation can amplify thrombotic responses by inducing tissue factor (TF) expression on leukocytes. Here, we characterized TF-positive extracellular vesicles (EVs) and their cellular origin in 12 patients suffering from severe COVID-19 (time course, 134 samples overall) and 25 healthy controls. EVs exposing phosphatidylserine (PS) were characterized by flow cytometry. Their cellular origin was determined by staining with anti-CD41, anti-CD45, anti-CD235a, and anti-CD105 as platelet, leukocyte, red blood cell, and endothelial markers. We further investigated the association of EVs with TF, platelet factor 4 (PF4), C-reactive protein (CRP), and high mobility group box-1 protein (HMGB-1). COVID-19 patients showed higher levels of PS-exposing EVs compared to controls. The majority of these EVs originated from platelets. A higher amount of EVs in patient samples was associated with CRP, HMGB-1, PF4, and TF as compared to EVs from healthy donors. In COVID-19 samples, 16.5% of all CD41+ EVs displayed the leukocyte marker CD45, and 55.5% of all EV aggregates (CD41+CD45+) co-expressed TF, which reflects the interaction of platelets and leukocytes in COVID-19 on an EV level.

Two mitochondrial HMG-box proteins, Cim1 and Abf2, antagonistically regulate mtDNA copy number in Saccharomyces cerevisiae.

The mitochondrial genome, mtDNA, is present in multiple copies in cells and encodes essential subunits of oxidative phosphorylation complexes. mtDNA levels have to change in response to metabolic demands and copy number alterations are implicated in various diseases. The mitochondrial HMG-box proteins Abf2 in yeast and TFAM in mammals are critical for mtDNA maintenance and packaging and have been linked to mtDNA copy number control. Here, we discover the previously unrecognized mitochondrial HMG-box protein Cim1 (copy number influence on mtDNA) in Saccharomyces cerevisiae, which exhibits metabolic state dependent mtDNA association. Surprisingly, in contrast to Abf2's supportive role in mtDNA maintenance, Cim1 negatively regulates mtDNA copy number. Cells lacking Cim1 display increased mtDNA levels and enhanced mitochondrial function, while Cim1 overexpression results in mtDNA loss. Intriguingly, Cim1 deletion alleviates mtDNA maintenance defects associated with loss of Abf2, while defects caused by Cim1 overexpression are mitigated by simultaneous overexpression of Abf2. Moreover, we find that the conserved LON protease Pim1 is essential to maintain low Cim1 levels, thereby preventing its accumulation and concomitant repressive effects on mtDNA. We propose a model in which the protein ratio of antagonistically acting Cim1 and Abf2 determines mtDNA copy number.

Evaluating the protective role of trimetazidine versus nano-trimetazidine in amelioration of bilateral renal ischemia/reperfusion induced neuro-degeneration: Implications of ERK1/2, JNK and Galectin-3 /NF-κB/TNF-α/HMGB-1 signaling.

BACKGROUND: Renal ischemia/reperfusion (I/R) is a primary culprit of acute kidney injury. Neurodegeneration can result from I/R, but the mechanisms are still challenging. We studied the implications of bilateral renal I/R on brain and potential involvement of the oxidative stress (OS) driven extracellular signal-regulated kinase1/2, c-Jun N-terminal kinase (ERK1/2, JNK) and Galectin-3 (Gal-3)/nuclear factor Kappa B (NF-қB)/tumor necrosis factor-alpha (TNF-α), high mobility group box-1 (HMGB-1), and caspase-3 paths upregulation. We tested the impact of Nano-trimetazidine (Nano-TMZ) on these pathways being a target of its neuroprotective effects. METHODS: Study groups; Sham, I/R, TMZ+I/R, and Nano-TMZ+I/R. Kidney functions, cognition, hippocampal OS markers, Gal-3, NF-қB, p65 and HMGB-1 gene expression, TNF-α level, t-JNK/p-JNK and t-ERK/p-ERK proteins, caspase-3, glial fibrillary acidic protein (GFAP) and ionized calcium binding protein-1 (Iba-1) were assessed. RESULTS: Nano-TMZ averted renal I/R-induced hippocampal impairment by virtue of its anti: oxidative, inflammatory, and apoptotic properties. CONCLUSION: Nano-TMZ is more than anti-ischemic.

Early and Dose-Dependent Xenogeneic Mesenchymal Stem Cell Therapy Improved Outcomes in Acute Respiratory Distress Syndrome Rodent Through Ameliorating Inflammation, Oxidative Stress, and Immune Reaction.

This study tested whether human umbilical cord-derived mesenchymal stem cells (HUCDMSCs) treatment effectively protected the rat lung against acute respiratory distress syndrome (ARDS) injury, and benefits of early and dose-dependent treatment. Rat pulmonary epithelial cell line L2 (PECL2) were categorized into G1 (PECL2), G2 (PECL2 + healthy rat lung-derived extraction/50 mg/ml co-cultured for 24 h), G3 (PECL2 + ARDS rat lung-derived extraction/50 mg/ml co-cultured for 24 h), and G4 (condition as G3 + HUCDMSCs/1 × 105/co-cultured for 24 h). The result showed that the protein expressions of inflammatory (HMGB-1/TLR-2/TLR-4/MAL/TRAM/MyD88/TRIF/TRAF6/IkB/NF-κB/IL-1ß/TNF-α), oxidative-stress/mitochondrial-damaged (NOX-1/NOX-2/ASK1/p-MKK4/p-MKK7/JNKs/JUN/cytosolic-cytochrome-C/cyclophilin-D/DRP1), and cell-apoptotic/fibrotic (cleaved-caspase 3/cleaved-PARP/TGF-ß/p-Smad3) biomarkers were significantly increased in G3 than in G1/G2 and were significantly reversed in G4 (all P < 0.001), but they were similar between G1/G2. Adult male rats (n = 42) were equally categorized into group 1 (normal control), group 2 (ARDS only), group 3 [ARDS + HUCDMSCs/1.2 × 106 cells intravenous administration at 3 h after 48 h ARDS induction (i.e., early treatment)], group 4 [ARDS + HUCDMSCs/1.2 × 106 cells intravenous administration at 24 h after 48 h ARDS induction (late treatment)], and group 5 [ARDS + HUCDMSCs/1.2 × 106 cells intravenous administration at 3 h/24 h after-48 h ARDS induction (dose-dependent treatment)]. By day 5 after ARDS induction, the SaO2%/immune regulatory T cells were highest in group 1, lowest in group 2, significantly lower in group 4 than in groups 3/5, and significantly lower in group 3 than in group 5, whereas the circulatory/bronchioalveolar lavage fluid inflammatory cells (CD11b-c+/LyG6+/MPO+)/circulatory immune cells (CD3-C4+/CD3-CD8+)/lung-leakage-albumin level/lung injury score/lung protein expressions of inflammatory (HMGB-1/TLR-2/TLR-4/MAL/TRAM/MyD88/TRIF/TRAF6/IκB-ß/p-NF-κB/IL-1ß/TNF-α)/fibrotic (p-SMad3/TGF-ß), apoptosis (mitochondrial-Bax/cleaved-caspase-3)/oxidative-cell-stress (NOX-1/NOX-2/ASK1/p-MKK4/p-MKK7/p-JNKs/p-cJUN)/mitochondrial damaged (cyclophilin-D/DRP1/cytosolic-cytochrome-C) biomarkers displayed an opposite pattern of SaO2% among the groups (all P < 0.0001). Early administration was superior to and two-dose counterpart was even more superior to late HUCDMSCs treatment for protecting the lung against ARDS injury.

Inhibition of BRD4 expression attenuates the inflammatory response and apoptosis by downregulating the HMGB-1/NF-κB signaling pathway following traumatic brain injury in rats.

Neuroinflammation plays an important part in secondary traumatic brain injury (TBI). Bromodomain-4 (BRD4) exerts specific proinflammatory effects in various neuropathological conditions. However, the underlying mechanism of action of BRD4 after TBI is not known. We measured BRD4 expression after TBI and investigated its possible mechanism of action. We established a model of craniocerebral injury in rats. After different intervention measures, we used western blotting, immunofluorescence, real-time reverse transcription-quantitative polymerase chain reaction, neuronal apoptosis, and behavioral tests to evaluate the effect of BRD4 on brain injury. At 72 h after brain injury, BRD4 overexpression aggravated the neuroinflammatory response, neuronal apoptosis, neurological dysfunction, and blood-brain-barrier damage, whereas upregulating expression of HMGB-1 and NF-κB had the opposite effect. Glycyrrhizic acid could reverse the proinflammatory effect of BRD4 overexpression upon TBI. Our results suggest that: (i) BRD4 may have a proinflammatory role in secondary brain injury through the HMGB-1/NF-κB signaling pathway; (ii) inhibition of BRD4 expression may play a part in secondary brain injury. BRD4 could be targeted therapy strategy for brain injury.

Sesquiterpene nootkatone counteracted the melamine-induced neurotoxicity via repressing of oxidative stress, inflammatory, and apoptotic trajectories.

Melamine (ML), a chemical substance of high nitrogen content, is used as a food adulterant. Former evidences implied that ML could induce a variety of toxic effects including neurotoxicity and cognitive impairment. Therefore, the aim of this study was to delineate the protective effect of the nootkatone (NK) against ML-induced neural adverse effects. Rats were orally pretreated with NK (5 and 10 mg/kg) prior to the oral administration of ML (700 mg/kg) for a period of 28 days. Our findings unveiled remarkable alleviating effect of NK on MK-induced neurobehavioral disturbance in open field test. Furthermore, NK lessened ML-caused increases in the acetylcholine esterase level in the brain tissue of exposed rats. NK also decreased the neural oxidative stress as represented by elevated levels of SOD, CAT, and GSH along with decreased MDA and NO levels. Upregulated mRNA expression levels of neural NRF-2 and HO-1 were noticed after NK administration. Remarkable anti-inflammatory impact was prominent by decreased neural IL-1ß, and TNF-α along with downregulated NF-κB and TLR-4 gene expression levels in NK-treated rats. Noteworthily, pre-treatment with NK decreased the immune reaction of RAGE and HMGB-1 induced by oral ML exposure. Brain histological examination validated the obtained biochemical and molecular results. To sum up, these outcomes reveal that NK successfully alleviated the neural damage induced by ML via blocking of oxidative stress, and inflammatory signaling pathways. Consequently, our study may suggest NK as a new effective therapeutic supplement for treatment of ML-mediated neurotoxicity in rats via inhibition of HMGB-1-RAGE/TLR-4/NF-κB.

HMGB family proteins: Potential biomarkers and mechanistic factors in cardiovascular diseases.

Cardiovascular disease (CVD) is the most fatal disease that causes sudden death, and inflammation contributes substantially to its occurrence and progression. The prevalence of CVD increases as the population ages, and the pathophysiology is complex. Anti-inflammatory and immunological modulation are the potential methods for CVD prevention and treatment. High-Mobility Group (HMG) chromosomal proteins are one of the most abundant nuclear nonhistone proteins which act as inflammatory mediators in DNA replication, transcription, and repair by producing cytokines and serving as damage-associated molecular patterns in inflammatory responses. The most common and well-studied HMG proteins are those with an HMGB domain, which participate in a variety of biological processes. HMGB1 and HMGB2 were the first members of the HMGB family to be identified and are present in all investigated eukaryotes. Our review is primarily concerned with the involvement of HMGB1 and HMGB2 in CVD. The purpose of this review is to provide a theoretical framework for diagnosing and treating CVD by discussing the structure and function of HMGB1 and HMGB2.

Artemisinin attenuates type 2 diabetic cardiomyopathy in rats through modulation of AGE-RAGE/HMGB-1 signaling pathway.

Diabetes mellitus is a common metabolic disorder. About two-thirds of diabetic patients develop diabetic cardiomyopathy (DCM), which becomes a challenging issue as it severely threatens the patient's life. Hyperglycemia and the resulting advanced glycated end products (AGE) and their receptor (RAGE)/High Mobility Group Box-1 (HMGB-1) molecular pathway are thought to be key players. Recently, artemisinin (ART) has gained more attention owing to its potent biological activities beyond its antimalarial effect. Herein, we aim to evaluate the effect of ART on DCM and the possible underlying mechanisms. Twenty-four male Sprague-Dawley rats were divided into: control, ART, type 2 diabetic and type 2 diabetic treated with ART groups. At the end of the research, the ECG was recorded, then the heart weight to body weight (HW/BW) ratio, fasting blood glucose, serum insulin and HOMA-IR were evaluated. Cardiac biomarkers (CK-MB and LDH), oxidative stress markers, IL-1ß, AGE, RAGE and HMGB-1 expression were also measured. The heart specimens were stained for H&E as well as Masson's trichrome. DCM induced disturbances in all studied parameters; contrary to this, ART improved these insults. Our study concluded that ART could improve DCM through modulation of the AGE-RAGE/HMGB-1 signaling pathway, with subsequent impacts on oxidative stress, inflammation and fibrosis. ART could therefore be a promising therapy for the management of DCM.

Structure and Functions of HMGB2 Protein.

High-Mobility Group (HMG) chromosomal proteins are the most numerous nuclear non-histone proteins. HMGB domain proteins are the most abundant and well-studied HMG proteins. They are involved in variety of biological processes. HMGB1 and HMGB2 were the first members of HMGB-family to be discovered and are found in all studied eukaryotes. Despite the high degree of homology, HMGB1 and HMGB2 proteins differ from each other both in structure and functions. In contrast to HMGB2, there is a large pool of works devoted to the HMGB1 protein whose structure-function properties have been described in detail in our previous review in 2020. In this review, we attempted to bring together diverse data about the structure and functions of the HMGB2 protein. The review also describes post-translational modifications of the HMGB2 protein and its role in the development of a number of diseases. Particular attention is paid to its interaction with various targets, including DNA and protein partners. The influence of the level of HMGB2 expression on various processes associated with cell differentiation and aging and its ability to mediate the differentiation of embryonic and adult stem cells are also discussed.

Stem cells therapeutic effect in a reserpine-induced fibromyalgia rat model: A possible NLRP3 inflammasome modulation with neurogenesis promotion in the cerebral cortex.

Fibromyalgia is a chronic pain syndrome with a multifactorial pathophysiology affecting 2-8 % of the population. AIMS: To investigate the therapeutic effects of bone marrow mesenchymal stem cells (BMSCs) against fibromyalgia-related cerebral cortex damage and the possible underlying mechanisms of action. MATERIALS AND METHODS: Rats were randomly allocated into three groups; control, fibromyalgia and fibromyalgia treated with BMSCs groups. Physical and behavioural assessments were performed. Cerebral cortices were collected for biochemical and histological assessment. KEY FINDINGS: Fibromyalgia group showed behavioural changes indicating presence of pain, fatigue, depression, and sleep disturbances. Moreover, biochemical biomarkers alterations were demonstrated by a significant decrease in brain monoamines and GSH levels, but MDA, NO, TNF-alpha, HMGB-1, NLRP3, and caspase-1 levels significantly increased. Furthermore, histological assessment revealed structural and ultrastructural alterations indicating neuronal and neuroglial degeneration with microglia activation, an increase in mast cell number and IL-1ß immune-expression. Additionally, a significant decrease in Beclin-1 immune-expression, and blood brain barrier disruption were noticed. Interestingly, BMSCs administration significantly improved behavioural alterations, restored the reduced brain monoamines and oxidative stress markers, and reduced TNF-alpha, HMGB-1, NLRP3, and caspase-1 levels. Profoundly, cerebral cortices demonstrated improved histological structure, significant decrease in mast cell number and IL-1ß immune-expression, besides a significant increase in Beclin-1 and DCX immune-expression. SIGNIFICANCE: For the best of our knowledge, this is the first study showing ameliorative effects for BMSCs treatment in fibromyalgia-related cerebral cortical damage. The neurotherapeutic effects of BMSCs could be attributed to NLRP3 inflammasome signaling pathway inhibition, mast cell deactivation, and stimulation of neurogenesis and autophagy.

HMGB 1 acetylation mediates trichloroethylene-induced immune kidney injury by facilitating endothelial cell-podocyte communication.

More and more clinical evidence shows that occupational medicamentose-like dermatitis due to trichloroethylene (OMDT) patients often present immune kidney damage. However, the exact mechanisms of cell-to-cell transmission in TCE-induced immune kidney damage remain poorly understood. The present study aimed to explore the role of high mobility group box-1 (HMGB 1) in glomerular endothelial cell-podocyte transmission. 17 OMDT patients and 34 controls were enrolled in this study. We observed that OMDT patients had renal function injury, endothelial cell activation and podocyte injury, and these indicators were associated with serum HMGB 1. To gain mechanistic insight, a TCE-sensitized BALB/c mouse model was established under the interventions of sirtuin 1 (SIRT 1) activator SRT 1720 (0.1 ml, 5 mg/kg) and receptor for advanced glycation end products (RAGE) inhibitor FPS-ZM 1 (0.1 ml, 1.5 mg/kg). We identified HMGB 1 acetylation and its endothelial cytoplasmic translocation following TCE sensitization, but SRT 1720 abolished the process. RAGE was located on podocytes and co-precipitated with extracellular acetylated HMGB 1, promoting podocyte injury, while SRT 1720 and FPS-ZM 1 both alleviated podocyte injury. The results demonstrate that interventions to upstream and downstream pathways of HMGB 1 may weaken glomerular endothelial cell-podocyte transmission, thereby alleviating TCE-induced immune renal injury.

Inflachromene attenuates seizure severity in mouse epilepsy models via inhibiting HMGB1 translocation.

Epilepsy is not well controlled by current anti-seizure drugs (ASDs). High mobility group box 1 (HMGB1) is a DNA-binding protein in the nucleus regulating transcriptional activity and maintaining chromatin structure and DNA repair. In epileptic brains, HMGB1 is released by activated glia and neurons, interacting with various receptors like Toll-like receptor 4 (TLR4) and downstream glutamatergic NMDA receptor, thus enhancing neural excitability. But there is a lack of small-molecule drugs targeting the HMGB1-related pathways. In this study we evaluated the therapeutic potential of inflachromene (ICM), an HMGB-targeting small-molecule inhibitor, in mouse epilepsy models. Pentylenetetrazol-, kainic acid- and kindling-induced epilepsy models were established in mice. The mice were pre-treated with ICM (3, 10 mg/kg, i.p.). We showed that ICM pretreatment significantly reduced the severity of epileptic seizures in all the three epilepsy models. ICM (10 mg/kg) exerted the most apparent anti-seizure effect in kainic acid-induced epileptic status (SE) model. By immunohistochemical analysis of brain sections from kainic acid-induced SE mice, we found that kainic acid greatly enhanced HMGB1 translocation in the hippocampus, which was attenuated by ICM pretreatment in subregion- and cell type-dependent manners. Notably, in CA1 region, the seizure focus, ICM pretreatment mainly inhibited HMGB1 translocation in microglia. Furthermore, the anti-seizure effect of ICM was related to HMGB1 targeting, as pre-injection of anti-HMGB1 monoclonal antibody (5 mg/kg, i.p.) blocked the seizure-suppressing effect of ICM in kainic acid-induced SE model. In addition, ICM pretreatment significantly alleviated pyramidal neuronal loss and granule cell dispersion in kainic acid-induced SE model. These results demonstrate that ICM is an HMGB-targeting small molecule with anti-seizure potential, which may help develop a potential drug for treating epilepsy.

Single-Cell RNA Sequencing of Sox17-Expressing Lineages Reveals Distinct Gene Regulatory Networks and Dynamic Developmental Trajectories.

During early embryogenesis, the transcription factor SOX17 contributes to hepato-pancreato-biliary system formation and vascular-hematopoietic emergence. To better understand Sox17 function in the developing endoderm and endothelium, we developed a dual-color temporal lineage-tracing strategy in mice combined with single-cell RNA sequencing to analyze 6934 cells from Sox17-expressing lineages at embryonic days 9.0-9.5. Our analyses showed 19 distinct cellular clusters combined from all 3 germ layers. Differential gene expression, trajectory and RNA-velocity analyses of endothelial cells revealed a heterogenous population of uncommitted and specialized endothelial subtypes, including 2 hemogenic populations that arise from different origins. Similarly, analyses of posterior foregut endoderm revealed subsets of hepatic, pancreatic, and biliary progenitors with overlapping developmental potency. Calculated gene-regulatory networks predict gene regulons that are dominated by cell type-specific transcription factors unique to each lineage. Vastly different Sox17 regulons found in endoderm versus endothelial cells support the differential interactions of SOX17 with other regulatory factors thereby enabling lineage-specific regulatory actions.

Super-enhancer-driven TOX2 mediates oncogenesis in Natural Killer/T Cell Lymphoma.

BACKGROUND: Extranodal natural killer/T-cell lymphoma (NKTL) is an aggressive type of non-Hodgkin lymphoma with dismal outcome. A better understanding of disease biology and key oncogenic process is necessary for the development of targeted therapy. Super-enhancers (SEs) have been shown to drive pivotal oncogenes in various malignancies. However, the landscape of SEs and SE-associated oncogenes remain elusive in NKTL. METHODS: We used Nano-ChIP-seq of the active enhancer marker histone H3 lysine 27 acetylation (H3K27ac) to profile unique SEs NKTL primary tumor samples. Integrative analysis of RNA-seq and survival data further pinned down high value, novel SE oncogenes. We utilized shRNA knockdown, CRISPR-dCas9, luciferase reporter assay, ChIP-PCR to investigate the regulation of transcription factor (TF) on SE oncogenes. Multi-color immunofluorescence (mIF) staining was performed on an independent cohort of clinical samples. Various function experiments were performed to evaluate the effects of TOX2 on the malignancy of NKTL in vitro and in vivo. RESULTS: SE landscape was substantially different in NKTL samples in comparison with normal tonsils. Several SEs at key transcriptional factor (TF) genes, including TOX2, TBX21(T-bet), EOMES, RUNX2, and ID2, were identified. We confirmed that TOX2 was aberrantly overexpressed in NKTL relative to normal NK cells and high expression of TOX2 was associated with worse survival. Modulation of TOX2 expression by shRNA, CRISPR-dCas9 interference of SE function impacted on cell proliferation, survival and colony formation ability of NKTL cells. Mechanistically, we found that RUNX3 regulates TOX2 transcription by binding to the active elements of its SE. Silencing TOX2 also impaired tumor formation of NKTL cells in vivo. Metastasis-associated phosphatase PRL-3 has been identified and validated as a key downstream effector of TOX2-mediated oncogenesis. CONCLUSIONS: Our integrative SE profiling strategy revealed the landscape of SEs, novel targets and insights into molecular pathogenesis of NKTL. The RUNX3-TOX2-SE-TOX2-PRL-3 regulatory pathway may represent a hallmark of NKTL biology. Targeting TOX2 could be a valuable therapeutic intervene for NKTL patients and warrants further study in clinic.

SOX17 Enhancer Variants Disrupt Transcription Factor Binding And Enhancer Inactivity Drives Pulmonary Hypertension.

BACKGROUND: Pulmonary arterial hypertension (PAH) is a rare disease characterized by remodeling of the pulmonary arteries, increased vascular resistance, and right-sided heart failure. Genome-wide association studies of idiopathic/heritable PAH established novel genetic risk variants, including conserved enhancers upstream of transcription factor (TF) SOX17 containing 2 independent signals. SOX17 is an important TF in embryonic development and in the homeostasis of pulmonary artery endothelial cells (hPAEC) in the adult. Rare pathogenic mutations in SOX17 cause heritable PAH. We hypothesized that PAH risk alleles in an enhancer region impair TF-binding upstream of SOX17, which in turn reduces SOX17 expression and contributes to disturbed endothelial cell function and PAH development. METHODS: CRISPR manipulation and siRNA were used to modulate SOX17 expression. Electromobility shift assays were used to confirm in silico-predicted TF differential binding to the SOX17 variants. Functional assays in hPAECs were used to establish the biological consequences of SOX17 loss. In silico analysis with the connectivity map was used to predict compounds that rescue disturbed SOX17 signaling. Mice with deletion of the SOX17-signal 1 enhancer region (SOX17-4593/enhKO) were phenotyped in response to chronic hypoxia and SU5416/hypoxia. RESULTS: CRISPR inhibition of SOX17-signal 2 and deletion of SOX17-signal 1 specifically decreased SOX17 expression. Electromobility shift assays demonstrated differential binding of hPAEC nuclear proteins to the risk and nonrisk alleles from both SOX17 signals. Candidate TFs HOXA5 and ROR-α were identified through in silico analysis and antibody electromobility shift assays. Analysis of the hPAEC transcriptomes revealed alteration of PAH-relevant pathways on SOX17 silencing, including extracellular matrix regulation. SOX17 silencing in hPAECs resulted in increased apoptosis, proliferation, and disturbance of barrier function. With the use of the connectivity map, compounds were identified that reversed the SOX17-dysfunction transcriptomic signatures in hPAECs. SOX17 enhancer knockout in mice reduced lung SOX17 expression, resulting in more severe pulmonary vascular leak and hypoxia or SU5416/hypoxia-induced pulmonary hypertension. CONCLUSIONS: Common PAH risk variants upstream of the SOX17 promoter reduce endothelial SOX17 expression, at least in part, through differential binding of HOXA5 and ROR-α. Reduced SOX17 expression results in disturbed hPAEC function and PAH. Existing drug compounds can reverse the disturbed SOX17 pulmonary endothelial transcriptomic signature.

SOX17 is a Critical Factor in Maintaining Endothelial Function in Pulmonary Hypertension by an Exosome-Mediated Autocrine Manner.

Endothelial dysfunction is considered a predominant driver for pulmonary vascular remodeling in pulmonary hypertension (PH). SOX17, a key regulator of vascular homoeostasis, has been found to harbor mutations in PH patients, which are associated with PH susceptibility. Here, this study explores whether SOX17 mediates the autocrine activity of pulmonary artery ECs to maintain endothelial function and vascular homeostasis in PH and its underlying mechanism. It is found that SOX17 expression is downregulated in the endothelium of remodeled pulmonary arteries in IPH patients and SU5416/hypoxia (Su/hypo)-induced PH mice as well as dysfunctional HPAECs. Endothelial knockdown of SOX17 accelerates the progression of Su/hypo-induced PH in mice. SOX17 overexpression in the pulmonary endothelium of mice attenuates Su/hypo-induced PH. SOX17-associated exosomes block the proliferation, apoptosis, and inflammation of HPAECs, preventing pulmonary arterial remodeling and Su/hypo-induced PH. Mechanistic analyses demonstrates that overexpressing SOX17 promotes the exosome-mediated release of miR-224-5p and miR-361-3p, which are internalized by injured HPAECs in an autocrine manner, ultimately repressing the upregulation of NR4A3 and PCSK9 genes and improving endothelial function. These results suggest that SOX17 is a key gene in maintaining endothelial function and vascular homeostasis in PH through regulating exosomal miRNAs in an autocrine manner.

SOX17 Deficiency Mediates Pulmonary Hypertension: At the Crossroads of Sex, Metabolism, and Genetics.

Rationale: Genetic studies suggest that SOX17 (SRY-related HMG-box 17) deficiency increases pulmonary arterial hypertension (PAH) risk. Objectives: On the basis of pathological roles of estrogen and HIF2α (hypoxia-inducible factor 2α) signaling in pulmonary artery endothelial cells (PAECs), we hypothesized that SOX17 is a target of estrogen signaling that promotes mitochondrial function and attenuates PAH development via HIF2α inhibition. Methods: We used metabolic (Seahorse) and promoter luciferase assays in PAECs together with the chronic hypoxia murine model to test the hypothesis. Measurements and Main Results: Sox17 expression was reduced in PAH tissues (rodent models and from patients). Chronic hypoxic pulmonary hypertension was exacerbated by mice with conditional Tie2-Sox17 (Sox17EC-/-) deletion and attenuated by transgenic Tie2-Sox17 overexpression (Sox17Tg). On the basis of untargeted proteomics, metabolism was the top pathway altered by SOX17 deficiency in PAECs. Mechanistically, we found that HIF2α concentrations were increased in the lungs of Sox17EC-/- and reduced in those from Sox17Tg mice. Increased SOX17 promoted oxidative phosphorylation and mitochondrial function in PAECs, which were partly attenuated by HIF2α overexpression. Rat lungs in males displayed higher Sox17 expression versus females, suggesting repression by estrogen signaling. Supporting 16α-hydroxyestrone (16αOHE; a pathologic estrogen metabolite)-mediated repression of SOX17 promoter activity, Sox17Tg mice attenuated 16αOHE-mediated exacerbations of chronic hypoxic pulmonary hypertension. Finally, in adjusted analyses in patients with PAH, we report novel associations between a SOX17 risk variant, rs10103692, and reduced plasma citrate concentrations (n = 1,326). Conclusions: Cumulatively, SOX17 promotes mitochondrial bioenergetics and attenuates PAH, in part, via inhibition of HIF2α. 16αOHE mediates PAH development via downregulation of SOX17, linking sexual dimorphism and SOX17 genetics in PAH.

Lavender essential oil accelerates lipopolysaccharide-induced chronic wound healing by inhibiting caspase-11-mediated macrophage pyroptosis.

Chronic wounds seriously affect the quality of life of the elderly, obese people, and diabetic patients. The excessive inflammatory response is a key driver of delayed chronic wound healing. Although lavender essential oil (EO [lav]) has been proven to have anti-inflammatory and accelerate wound curative effects, the specific molecular mechanism involved is still ambiguous. The results showed that the wounds treated with lipopolysaccharide (LPS) not only had delayed healing, but also the expression levels of pro-inflammatory cytokines, such as tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), interleukin-1ß (IL-1ß), and the inflammatory mediator protein, high-mobility group box 1 protein (HMGB-1), in the wound tissues were significantly increased. However, treatment of LPS-induced chronic wounds with EO (lav) accelerated wound healing and decreased IL-1ß and HMGB-1 expression levels. It was further found that LPS induced macrophage pyroptosis to produce IL-1ß. After treatment with EO (lav), the expression level of macrophage pyroptosis marker Gasdermin D (GSDMD) and pyroptosis-related cytotoxic effects were significantly reduced. Immunofluorescence results also directly indicate that EO (lav) can protect macrophages from LPS-induced pyroptosis. Moreover, EO (lav) can down-regulate expression levels of IL-1ß, GSDMD, and nucleotide-binding oligomerization domain-like receptor protein 3 (NLRP3) in the caspase-11-related pyroptotic signaling pathway. This study demonstrates that EO (lav) can reduce proinflammatory factor production and ameliorate inflammatory response by inhibiting macrophage pyroptosis, which accelerates LPS-induced chronic wound healing.

The DNA binding high mobility group box protein family functionally binds RNA.

Nucleic acid binding proteins regulate transcription, splicing, RNA stability, RNA localization, and translation, together tailoring gene expression in response to stimuli. Upon discovery, these proteins are typically classified as either DNA or RNA binding as defined by their in vivo functions; however, recent evidence suggests dual DNA and RNA binding by many of these proteins. High mobility group box (HMGB) proteins have a DNA binding HMGB domain, act as transcription factors and chromatin remodeling proteins, and are increasingly understood to interact with RNA as means to regulate gene expression. Herein, multiple layers of evidence that the HMGB family are dual DNA and RNA binding proteins is comprehensively reviewed. For example, HMGB proteins directly interact with RNA in vitro and in vivo, are localized to RNP granules involved in RNA processing, and their protein interactors are enriched in RNA binding proteins involved in RNA metabolism. Importantly, in cell-based systems, HMGB-RNA interactions facilitate protein-protein interactions, impact splicing outcomes, and modify HMGB protein genomic or cellular localization. Misregulation of these HMGB-RNA interactions are also likely involved in human disease. This review brings to light that as a family, HMGB proteins are likely to bind RNA which is essential to HMGB protein biology. This article is categorized under: RNA Interactions with Proteins and Other Molecules > Protein-RNA Recognition RNA Interactions with Proteins and Other Molecules > RNA-Protein Complexes RNA Interactions with Proteins and Other Molecules > Protein-RNA Interactions: Functional Implications.