Hypoxia-mediated repression of pyruvate carboxylase drives immunosuppression.

BACKGROUND: Metabolic plasticity mediates breast cancer survival, growth, and immune evasion during metastasis. However, how tumor cell metabolism is influenced by and feeds back to regulate breast cancer progression are not fully understood. We identify hypoxia-mediated suppression of pyruvate carboxylase (PC), and subsequent induction of lactate production, as a metabolic regulator of immunosuppression. METHODS: We used qPCR, immunoblot, and reporter assays to characterize repression of PC in hypoxic primary tumors. Steady state metabolomics were used to identify changes in metabolite pools upon PC depletion. In vivo tumor growth and metastasis assays were used to evaluate the impact of PC manipulation and pharmacologic inhibition of lactate transporters. Immunohistochemistry, flow cytometry, and global gene expression analyzes of tumor tissue were employed to characterize the impact of PC depletion on tumor immunity. RESULTS: PC is essential for metastatic colonization of the lungs. In contrast, depletion of PC in tumor cells promotes primary tumor growth. This effect was only observed in immune competent animals, supporting the hypothesis that repression of PC can suppress anti-tumor immunity. Exploring key differences between the pulmonary and mammary environments, we demonstrate that hypoxia potently downregulated PC. In the absence of PC, tumor cells produce more lactate and undergo less oxidative phosphorylation. Inhibition of lactate metabolism was sufficient to restore T cell populations to PC-depleted mammary tumors. CONCLUSIONS: We present a dimorphic role for PC in primary mammary tumors vs. pulmonary metastases. These findings highlight a key contextual role for PC-directed lactate production as a metabolic nexus connecting hypoxia and antitumor immunity.

Hypoxia-responsive lncRNA MIR155HG promotes PD-L1 expression in hepatocellular carcinoma cells by enhancing HIF-1α mRNA stability.

The microenvironment of hepatocellular carcinoma (HCC) is characterized by hypoxia, which leads to immune evasion of HCC. Therefore, gaining a comprehensive understanding of the mechanism underlying the impact of hypoxia on HCC cells may provide valuable insights into immune checkpoint therapy. Based on analysis of databases and clinical samples, we observed that expression level of programmed cell death ligand 1 (PD-L1) and long non-coding RNA (lncRNA) MIR155HG in patients in the hypoxia group were higher than those in the non-hypoxia group. Furthermore, there was a positive correlation between the expression of PD-L1 and MIR155HG with that of HIF-1α. In vitro experiments using hypoxic treatment demonstrated an increase in PD-L1 and MIR155HG expression levels in HCC cells. While the hypoxia-induced upregulation of PD-L1 could be reversed by knocking down MIR155HG. Mechanistically, as a transcription factor, HIF-1α binds to the promoter region of MIR155HG to enhance its transcriptional activity under hypoxic conditions. Hypoxia acts as a stressor promoting nuclear output of ILF3 leading to increased binding of ILF3 to MIR155HG, thereby enhancing stability for HIF-1α mRNA. In vivo, knocking down MIR155HG inhibit subcutaneous tumor growth, reduce the expression of HIF-1α and PD-L1 within tumors; additionally, it enhances anti-tumor immunity response. These findings suggested that through inducing MIR155HG to interact with ILF3, hypoxia increases HIF-1α mRNA stability resulting in elevated PD-L1 expression in HCC and thus promoting immune escape. In summary, this study provides new insights into the effects of hypoxia on HCC immunosuppression.

The impact of hypoxia preconditioning on mesenchymal stem cells performance in hypertensive kidney disease.

BACKGROUND: Autologous mesenchymal stem cells (MSCs) have emerged as a therapeutic option for many diseases. Hypertensive kidney disease (HKD) might impair MSCs' reparative ability by altering the biomolecular properties, but the characteristics of this impairment are unclear. In our previous pre-clinical studies, we found hypoxic preconditioning (HPC) enhanced angiogenesis and suppressed senescence gene expression. Thus, we hypothesize that HPC would improve human MSCs by enhancing their functionality and angiogenesis, creating an anti-inflammatory and anti-senescence environment. METHODS: MSC samples (n = 12 each) were collected from the abdominal fat of healthy kidney donors (HC), hypertensive patients (HTN), and patients with hypertensive kidney disease (HKD). MSCs were harvested and cultured in Normoxic (20% O2) or Hypoxic (1% O2) conditions. MSC functionality was measured by proliferation assays and cytokine released in conditioned media. Senescence was evaluated by senescence-associated beta-galactosidase (SA-beta-gal) activity. Additionally, transcriptome analysis using RNA-sequencing and quantitative PCR (qPCR) were performed. RESULTS: At baseline, normoxic HTN-MSCs had higher proliferation capacity compared to HC. However, HPC augmented proliferation in HC. HPC did not affect the release of pro-angiogenic protein VEGF, but increased EGF in HC-MSC, and decreased HGF in HC and HKD MSCs. Under HPC, SA-ß-gal activity tended to decrease, particularly in HC group. HPC upregulated mostly the pro-angiogenic and inflammatory genes in HC and HKD and a few senescence genes in HKD. CONCLUSIONS: HPC has a more favorable functional effect on HC- than on HKD-MSC, reflected in increased proliferation and EGF release, and modest decrease in senescence, whereas it has little effect on HTN or HKD MSCs.

An activatable azophenyl fluorescent probe for hypoxic fluorescence imaging in living cells.

Cellular hypoxia is a common pathological process in various diseases. Detecting cellular hypoxia is of great scientific significance for early diagnosis of tumors. The hypoxia fluorescence probe analysis method can efficiently and conveniently evaluate the hypoxia status in tumor cells. These probes are covalently linked by hypoxic recognition groups and organic fluorescent molecules. Currently, the fluorescent molecules used in these probes often exhibit the aggregation-caused quenching effect, which is not conducive to fluorescence imaging in water. Herein, an activatable hypoxia fluorescence probe was constructed by covalently linking aggregation-induced emission luminogens to the hypoxic recognition group azobenzene. It does not emit fluorescence in solution and in solid state under light excitation due to the presence of photosensitive azo bonds. It can be cleaved by intracellular azoreductase into fluorescent amino derivatives with aggregation-induced emission characteristic. As the concentration of oxygen in cells decreases, its fluorescence intensity increases, making it suitable for fluorescence imaging to detect hypoxic environment in live cancer cells. This work broadens the molecular design approach for activatable hypoxia fluorescent probes.

MiR-18a affects hypoxia induced glucose metabolism transition in HT22 hippocampal neuronal cell line through the Hif1a gene.

Hypoxia can cause a variety of diseases, including ischemic stroke and neurodegenerative diseases. Within a certain range of partial pressure of oxygen, cells can respond to changes in oxygen. Changes in oxygen concentration beyond a threshold will cause damage or even necrosis of tissues and organs, especially for the central nervous system. Therefore, it is very important to find appropriate measures to alleviate damage. MiRNAs can participate in the regulation of hypoxic responses in various types of cells. MiRNAs are involved in regulating hypoxic responses in many types of tissues by activating the hypoxia-inducible factor (HIF) to affect angiogenesis, glycolysis and other biological processes. By analyzing differentially expressed miRNAs in hypoxia and hypoxia-related studies, as well as the HT22 neuronal cell line under hypoxic stress, we found that the expression of miR-18a was changed in these models. MiR-18a could regulate glucose metabolism in HT22 cells under hypoxic stress by directly regulating the 3'UTR of the Hif1a gene. As a small molecule, miRNAs are easy to be designed into small nucleic acid drugs, so this study can provide a theoretical basis for the research and treatment of nervous system diseases caused by hypoxia.

Endoplasmic reticulum stress-dependent regulation of the expression of serine hydroxymethyltransferase 2 in glioblastoma cells.

Objective. Serine hydroxymethyltransferase (SHMT2) plays a multifunctional role in mitochondria (folate-dependent tRNA methylation, translation, and thymidylate synthesis). The endoplasmic reticulum stress, hypoxia, and glucose and glutamine supply are significant factors of malignant tumor growth including glioblastoma. Previous studies have shown that the knockdown of the endoplasmic reticulum to nucleus signaling 1 (ERN1) pathway of endoplasmic reticulum stress strongly suppressed glioblastoma cell proliferation and modified the sensitivity of these cells to hypoxia and glucose or glutamine deprivations. The present study aimed to investigate the regulation of the SHMT2 gene in U87MG glioblastoma cells by ERN1 knockdown, hypoxia, and glucose or glutamine deprivations with the intent to reveal the role of ERN1 signaling in sensitivity of this gene expression to hypoxia and nutrient supply. Methods. The control U87MG glioblastoma cells (transfected by an empty vector) and ERN1 knockdown cells with inhibited ERN1 endoribonuclease and protein kinase (dnERN1) or only ERN1 endoribonuclease (dnrERN1) were used. Hypoxia was introduced by dimethyloxalylglycine (500 ng/ml for 4 h). For glucose and glutamine deprivations, cells were exposed in DMEM without glucose and glutamine, respectively for 16 h. RNA was extracted from cells and reverse transcribed. The expression level of the SHMT2 gene was studied by real-time qPCR and normalized to ACTB. Results. It was found that inhibition of ERN1 endoribonuclease and protein kinase in glioblastoma cells led to a down-regulation of SHMT2 gene expression in U87MG cells. At the same time, the expression of this gene did not significantly change in cells with inhibited ERN1 endoribonuclease, but tunicamycin strongly increased its expression. Moreover, the expression of the SHMT2 gene was not affected in U87MG cells after silencing of XBP1. Hypoxia up-regulated the expression level of the SHMT2 gene in both control and ERN1 knockdown U87MG cells. The expression of this gene was significantly up-regulated in glioblastoma cells under glucose and glutamine deprivations and ERN1 knockdown significantly increased the sensitivity of the SHMT2 gene to these nutrient deprivation conditions. Conclusion. The results of the present study demonstrate that the expression of the SHMT2 gene responsible for serine metabolism and formation of folate one-carbon is controlled by ERN1 protein kinase and induced by hypoxia as well as glutamine and glucose deprivation conditions in glioblastoma cells and reflects the ERN1-mediated reprogramming of sensitivity this gene expression to nutrient deprivation.

Hypoxia-related THBD+ macrophages as a prognostic factor in glioma: Construction of a powerful risk model.

Glioma is a prevalent malignant tumour characterized by hypoxia as a pivotal factor in its progression. This study aims to investigate the impact of the most severely hypoxic cell subpopulation in glioma. Our findings reveal that the THBD+ macrophage subpopulation is closely associated with hypoxia in glioma, exhibiting significantly higher infiltration in tumours compared to non-tumour tissues. Moreover, a high proportion of THBD+ cells correlates with poor prognosis in glioblastoma (GBM) patients. Notably, THBD+ macrophages exhibit hypoxic characteristics and epithelial-mesenchymal transition features. Silencing THBD expression leads to a notable reduction in the proliferation and metastasis of glioma cells. Furthermore, we developed a THBD+ macrophage-related risk signature (THBDMRS) through machine learning techniques. THBDMRS emerges as an independent prognostic factor for GBM patients with a substantial prognostic impact. By comparing THBDMRS with 119 established prognostic features, we demonstrate the superior prognostic performance of THBDMRS. Additionally, THBDMRS is associated with glioma metastasis and extracellular matrix remodelling. In conclusion, hypoxia-related THBD+ macrophages play a pivotal role in glioma pathogenesis, and THBDMRS emerges as a potent and promising prognostic tool for GBM, contributing to enhanced patient survival outcomes.

Newsights of endoplasmic reticulum in hypoxia.

The endoplasmic reticulum (ER) is important to cells because of its essential functions, including synthesizing three major nutrients and ion transport. When cellular homeostasis is disrupted, ER quality control (ERQC) system is activated effectively to remove misfolded and unfolded proteins through ER-phagy, ER-related degradation (ERAD), and molecular chaperones. When unfolded protein response (UPR) and ER stress are activated, the cell may be suffering a huge blow, and the most probable consequence is apoptosis. The membrane contact points between the ER and sub-organelles contribute to communication between the organelles. The decrease in oxygen concentration affects the morphology and structure of the ER, thereby affecting its function and further disrupting the stable state of cells, leading to the occurrence of disease. In this study, we describe the functions of ER-, ERQC-, and ER-related membrane contact points and their changes under hypoxia, which will help us further understand ER and treat ER-related diseases.

Intervention of exogenous VEGF protect brain microvascular endothelial cells from hypoxia-induced injury by regulating PLCγ/RAS/ERK and PI3K/AKT pathways.

Ischemic stroke rapidly increases the expression level of vascular endothelial growth factor (VEGF), which promotes neovascularization during hypoxia. However, the effect and mechanism of VEGF intervention on cerebrovascular formation remain unclear. Therefore, our research discussed the protective effect of exogenous VEGF on cells in hypoxia environment in cerebral microvascular endothelial cells, simulating ischemic stroke in hypoxic environment. Firstly, we detected the proliferation and apoptosis of cerebral microvascular endothelial cells under hypoxia environment, as well the expression levels of VEGF-E, vascular endothelial growth factor re-ceptor-2 (VEGFR-2), BCL2, PRKCE and PINK1. Moreover, immunofluorescence and western blotting were used to verify the regulation of exogenous VEGF-E on VEGFR-2 expression in hypoxic or normal oxygen environment. Lastly, we manipulated the concentration of VEGF-E in the culture medium to investigate its impact on phospholipase Cγ1 (PLCγ1)/extracellular signaling regulatory protein kinase (ERK) -1/2 and protein kinase B (AKT) pathways. Additionally, we employed a PLCγ1 inhibitor (U73122) to investigate its impact on proliferation and PLCγ1/ERK pathways. The results show that hypoxia inhibited the proliferation of cerebral microvascular endothelial cells, promoted cell apoptosis, significantly up-regulated the expression of VEGF-E, VEGFR-2, PRKCE and PINK1, but down-regulated the expression of BCL2. Interference from exogenous VEGF-E activated PLCγ1/ERK-1/2 and AKT pathways, promoting cell proliferation and inhibiting apoptosis of hypoxic brain microvascular endothelial cells. In summary, exogenous VEGF-E prevents hypoxia-induced damage to cerebral microvascular endothelial cells by activating the PLCγ1/ERK and AKT pathways. This action inhibits the apoptosis pathway in hypoxic cerebral microvascular endothelial cells, thereby safeguarding the blood-brain barrier and the nervous system.

MicroRNA-135b-5p Is a Pathologic Biomarker in the Endothelial Cells of Arteriovenous Malformations.

Arteriovenous malformations (AVMs) are congenital vascular anomalies with a poor prognosis. AVMs are considered intractable diseases, as there is no established approach for early diagnosis and treatment. Therefore, this study aimed to provide new evidence by analyzing microRNAs (miRNAs) associated with AVM. We present fundamental evidence for the early diagnosis and treatment of AVM by analyzing miRNAs in the endothelial cells of AVMs. This study performed sequencing and validation of miRNAs in endothelial cells from normal and AVM tissues. Five upregulated and two downregulated miRNAs were subsequently analyzed under hypoxia and vascular endothelial growth factor (VEGF) treatment by one-way analysis of variance (ANOVA). Under hypoxic conditions, miR-135b-5p was significantly upregulated in the AVM compared to that under normal conditions, corresponding to increased endothelial activity (p-value = 0.0238). VEGF treatment showed no significant increase in miR-135b-5p under normal conditions, however, a surge in AVM was observed. Under both hypoxia and VEGF treatment, comparison indicated a downregulation of miR-135b-5p in AVM. Therefore, miR-135b-5p was assumed to affect the pathophysiological process of AVM and might play a vital role as a potential biomarker of AVMs for application related to diagnosis and treatment.

Sevoflurane postconditioning attenuates cardiomyocytes hypoxia/reoxygenation injury via PI3K/AKT pathway mediated HIF-1α to regulate the mitochondrial dynamic balance.

BACKGROUND: Myocardial ischemia-reperfusion injury (I/RI) is a major cause of perioperative cardiac-related adverse events and death. Studies have shown that sevoflurane postconditioning (SpostC), which attenuates I/R injury and exerts cardioprotective effects, regulates mitochondrial dynamic balance via HIF-1α, but the exact mechanism is unknown. This study investigates whether the PI3K/AKT pathway in SpostC regulates mitochondrial dynamic balance by mediating HIF-1α, thereby exerting myocardial protective effects. METHODS: The H9C2 cardiomyocytes were cultured to establish the hypoxia-reoxygenation (H/R) model and randomly divided into 4 groups: Control group, H/R group, sevoflurane postconditioning (H/R + SpostC) group and PI3K/AKT blocker (H/R + SpostC + LY) group. Cell survival rate was determined by CCK-8; Apoptosis rate was determined by flow cytometry; mitochondrial membrane potential was evaluated by Mito Tracker™ Red; mRNA expression levels of AKT, HIF-1α, Opa1and Drp1 were detected by quantitative real-time polymerase chain reaction (qRT-PCR); Western Blot assay was used to detect the protein expression levels of AKT, phosphorylated AKT (p-AKT), HIF-1α, Opa1 and Drp1. RESULTS: Compared with the H/R group, the survival rate of cardiomyocytes in the H/R + SpostC group increased, the apoptosis rate decreased and the mitochondrial membrane potential increased. qRT-PCR showed that the mRNA expression of HIF-1α and Opa1 were higher in the H/R + SpostC group compared with the H/R group, whereas the transcription level of Drp1 was lower in the H/R + SpostC group. In the H/R + SpostC + LY group, the mRNA expression of HIF-1α was lower than the H/R + SpostC group. There was no difference in the expression of Opa1 mRNA between the H/R group and the H/R + SpostC + LY group. WB assay results showed that compared with the H/R group, the protein expression levels of HIF-1α, Opa1, P-AKT were increased and Drp1 protein expression levels were decreased in the H/R + SpostC group. HIF-1α, P-AKT protein expression levels were decreased in the H/R + SpostC + LY group compared to the H/R + SpostC group. CONCLUSION: SpostC mediates HIF-1α-regulated mitochondrial fission and fusion-related protein expression to maintain mitochondrial dynamic balance by activating the PI3K/AKT pathway and increasing AKT phosphorylation, thereby attenuating myocardial I/R injury.

3-Hydroxytanshinone Inhibits the Activity of Hypoxia-Inducible Factor 1-α by Interfering with the Function of α-Enolase in the Glycolytic Pathway.

Tumor cells in hypoxic conditions control cancer metabolism and angiogenesis by expressing HIF-1α. Tanshinone is a traditional Chinese medicine that has been shown to possess antitumor properties and exerts a therapeutic impact on angiogenesis. However, the precise molecular mechanism responsible for the antitumor activity of 3-Hydroxytanshinone (3-HT), a type of tanshinone, has not been fully understood. Therefore, our study aimed to investigate the mechanism by which 3-HT regulates the expression of HIF-1α. Our findings demonstrate that 3-HT inhibits HIF-1α activity and expression under hypoxic conditions. Additionally, 3-HT inhibits hypoxia-induced angiogenesis by suppressing the expression of VEGF. Moreover, 3-HT was found to directly bind to α-enolase, an enzyme associated with glycolysis, resulting in the suppression of its activity. This inhibition of α-enolase activity by 3-HT leads to the blockade of the glycolytic pathway and a decrease in glycolysis products, ultimately altering HIF1-α expression. Furthermore, 3-HT negatively regulates the expression of HIF-1α by altering the phosphorylation of AMP-activated protein kinase (AMPK). Our study's findings elucidate the mechanism by which 3-HT regulates HIF-1α through the inhibition of the glycolytic enzyme α-enolase and the phosphorylation of AMPK. These results suggest that 3-HT holds promise as a potential therapeutic agent for hypoxia-related angiogenesis and tumorigenesis.

Activation of the nonneuronal cholinergic cardiac system by hypoxic preconditioning protects isolated adult cardiomyocytes from hypoxia/reoxygenation injury.

Activation of the vagus nerve mediates cardioprotection and attenuates myocardial ischemia/reperfusion (I/R) injury. In response to vagal activation, acetylcholine (ACh) is released from the intracardiac nervous system (ICNS) and activates intracellular cardioprotective signaling cascades. Recently, however, a nonneuronal cholinergic cardiac system (NNCCS) in cardiomyocytes has been described as an additional source of ACh. To investigate whether the NNCCS mediates cardioprotection in the absence of vagal and ICNS activation, we used a reductionist approach of isolated adult rat ventricular cardiomyocytes without neuronal cells, using hypoxic preconditioning (HPC) as a protective stimulus. Adult rat ventricular cardiomyocytes were isolated, the absence of neuronal cells was confirmed, and HPC was induced by 10/20 min hypoxia/reoxygenation (H/R) before subjection to 30/5 min H/R to simulate I/R injury. Cardiomyocyte viability was assessed by trypan blue staining at baseline and after HPC+H/R or H/R. Intra- and extracellular ACh was quantified using liquid chromatography-coupled mass spectrometry at baseline, after HPC, after hypoxia, and after reoxygenation, respectively. In a subset of experiments, muscarinic and nicotinic ACh receptor (m- and nAChR) antagonists were added during HPC or during H/R. Cardiomyocyte viability at baseline (69 ± 4%) was reduced by H/R (10 ± 3%). With HPC, cardiomyocyte viability was preserved after H/R (25 ± 6%). Intra- and extracellular ACh increased during hypoxia; HPC further increased both intra- and extracellular ACh (from 0.9 ± 0.7 to 1.5 ± 1.0 nmol/mg; from 0.7 ± 0.6 to 1.1 ± 0.7 nmol/mg, respectively). The addition of mAChR and nAChR antagonists during HPC had no impact on HPC's protection; however, protection was abrogated when antagonists were added during H/R (cardiomyocyte viability after H/R: 23 ± 5%; 13 ± 4%). In conclusion, activation of the NNCCS is involved in cardiomyocyte protection; HPC increases intra- and extracellular ACh during H/R, and m- and nAChRs are causally involved in HPC's cardiomyocyte protection during H/R. The interplay between upstream ICNS activation and NNCCS activation in myocardial cholinergic metabolism and cardioprotection needs to be investigated in future studies.NEW & NOTEWORTHY The intracardiac nervous system is considered to be involved in ischemic conditioning's cardioprotection through the release of acetylcholine (ACh). However, we demonstrate that hypoxic preconditioning (HPC) protects from hypoxia/reoxygenation injury and increases intra- and extracellular ACh during hypoxia in isolated adult ventricular rat cardiomyocytes. HPC's protection involves cardiomyocyte muscarinic and nicotinic ACh receptor activation. Thus, besides the intracardiac nervous system, a nonneuronal cholinergic cardiac system may also be causally involved in cardiomyocyte protection by ischemic conditioning.

Mitochondrial outer membrane protein Samm50 protects against hypoxia-induced cardiac injury by interacting with Shmt2.

Cardiac remodeling is a critical process following myocardial infarction (MI), potentially leading to heart failure if untreated. The significance of mitochondrial homeostasis in MI remains insufficiently understood. Samm50 is an essential component of mitochondria. Our study aimed to investigate its role in hypoxia-induced cardiac injury and the underlying mechanisms. First, we observed that Samm50 was dynamically downregulated in mice with MI compared to the control mice. In vitro, Samm50 was also downregulated in oxygen-glucose-deprived neonatal rat cardiomyocytes and fibroblasts. Overexpression and knockdown of Samm50 mitigated and exacerbated cardiac apoptosis and fibrosis, while also improving and worsening mitochondrial homeostasis, respectively. Protein interactions with Samm50 during the protective process were identified via immune-coprecipitation/mass spectroscopy. Mechanistically, serine hydroxymethyltransferase 2 (Shmt2) interacted with Samm50, acting as a crucial element in the protective process by hindering the transfer of Bax from the cytoplasm to the mitochondria and subsequent activation of caspase-3. Inhibition of Shmt2 diminished the protective effect of Samm50 overexpression against cardiac injury. Finally, Samm50 overexpression in vivo mitigated cardiac remodeling and enhanced cardiac function in both acute and chronic MI. In conclusion, Samm50 overexpression mitigated hypoxia-induced cardiac remodeling by inhibiting apoptosis and fibrosis, with Shmt2 acting as a key regulator in this protective process. The Samm50/Shmt2 axis represents a newly discovered mitochondria-related pathway for mitigating hypoxia-induced cardiac injury.

Lactylome analyses suggest systematic lysine-lactylated substrates in oral squamous cell carcinoma under normoxia and hypoxia.

Cancer cells tend to live in hypoxic environment characterized by enhanced glycolysis and accumulation of lactate. Intracellular lactate is shown to drive a novel type of post-translational modification (PTM), lysine lactylation (Kla). Kla has been confirmed to affect the malignant progression of tumors such as hepatocellular carcinoma (HCC) and colon cancer, whereas the global lactylomic profiling of oral squamous cell carcinoma (OSCC) is unclear. Here, the integrative lactylome and proteome analyses by using liquid chromatography-tandem mass spectrometry (LC-MS/MS) identified 1011 Kla sites within 532 proteins and 1197 Kla sites within 608 proteins in SCC25 cells under normoxic and hypoxic environments, respectively. Among these lactylated proteins, histones accounted for only a small fraction, suggesting the presence of Kla modification of OSCC in a large number of non-histone proteins. Notably, Kla preferred to enrich in spliceosome, ribosome and glycolysis/gluconeogenesis pathway in both normoxic and hypoxic cultures. Compared with normoxia, 589 differential proteins with 898 differentially lactylated sites were detected under hypoxia, which were mainly associated with the glycolysis/gluconeogenesis pathway by KEGG analysis. Importantly, we verified the presence of lactylation modification in the spliceosomal proteins hnRNPA1, SF3A1, hnRNPU and SLU7, as well as in glycolytic enzyme PFKP. In addition, the differential alternative splicing analysis described the divergence of pre-mRNA splicing patterns in the presence or absence of sodium lactate and at different oxygen concentrations. Finally, a negative correlation between tissue Kla levels and the prognosis of OSCC patients was revealed by immunohistochemistry. Our study is the first report to elucidate the lactylome and its biological function in OSCC, which deepens our understanding of the mechanisms underlying OSCC progression and provides a novel strategy for targeted therapy for OSCC.

S100A16 is a potential target for reshaping the tumor microenvironment in the hypoxic context of liver cancer.

BACKGROUND: The research on the S100 family has garnered significant attention; however, there remains a dearth of understanding regarding the precise role of S100A16 in the tumor microenvironment of liver cancer. METHOD: Comprehensive analysis was conducted on the expression of S100A16 in tumor tissues and its correlation with hypoxia genes. Furthermore, an investigation was carried out to examine the association between S100A16 and infiltration of immune cells in tumors as well as immunotherapy. Relevant findings were derived from the analysis of single cell sequencing data, focusing on the involvement of S100A16 in both cellular differentiation and intercellular communication. Finally, we validated the expression of S100A16 in liver cancer by Wuhan cohort and multiplexed immunofluorescence to investigate the correlation between S100A16 and hypoxia. RESULT: Tumor tissues displayed a notable increase in the expression of S100A16. A significant correlation was observed between S100A16 and genes associated with hypoxic genes. Examination of immune cell infiltration revealed an inverse association between T cell infiltration and the level of S100A16 expression. The high expression group of S100A16 exhibited a decrease in the expression of genes related to immune cell function. Single-cell sequencing data analysis revealed that non-immune cells predominantly expressed S100A16, and its expression levels increased along with the trajectory of cell differentiation. Additionally, there were significant variations observed in hypoxia genes as cells underwent differentiation. Cellular communication identified non-immune cells interacting with immune cells through multiple signaling pathways. The Wuhan cohort verified that S100A16 expression was increased in liver cancer. The expression of S100A16 and HIF was simultaneously elevated in endothelial cells. CONCLUSION: The strong association between S100A16 and immune cell infiltration is observed in the context of hypoxia, indicating its regulatory role in shaping the hypoxic tumor microenvironment in liver cancer.

Extracellular vesicles deviced from hypoxia-3D-GMSCs rescue the mitochondrial dysfunction of aging-GMSCs.

Mesenchymal stem cells (MSCs) are ubiquitous multipotent cells exhibiting significant therapeutic potential for various diseases. It is generally accepted that clinical application requires massive expansion of MSCs, which is often accompanied by the occurrence of replicative senescence. Additionally, senescent MSCs exhibit significantly reduced proliferation, differentiation, and therapeutic potential. The scale-up of MSCs production and cellular senescence are major challenges for translational applications. This study first collected extracellular vesicles (EVs) from gingival MSCs (GMSCs) under hypoxia preconditioning combined with 3D dynamic culture (obtained EVs designed as H-3D-EVs). Subsequently, we further explored the effects and mechanisms of H-3D-EVs on aging-GMSCs. The results showed that H-3D-EVs improved the proliferation ability and cell activity of aging-GMSCs, and ameliorated their senescence. mRNA sequencing reveals transcriptomic changes in aging-GMSCs. It was found that H-3D-EVs up-regulated genes related to mitochondrial dynamics, cell cycle, and DNA repair, while down-regulated aging-related genes. Furthermore, we verified that H-3D-EVs corrected the mitochondrial dysfunction of aging-GMSCs by improving mitochondrial dynamics. In summary, this study provides a promising strategy for improving the culture methods of GMSCs and avoiding its senescence in large-scale production.

Synthesis, antioxidant and anti-hypoxia activities of 6-hydroxygenistein and its methylated derivatives. / 6-ç¾ŸåŸºæŸ“æ–™æœ¨ç´ åŠå ¶ç”²åŸºåŒ–è¡ç”Ÿç‰©çš„åˆæˆå’ŒæŠ—æ°§åŒ–ä¸ŽæŠ—ç¼ºæ°§æ´»æ€§.

OBJECTIVES: Hypoxia is a common pathological phenomenon, usually caused by insufficient oxygen supply or inability to use oxygen effectively. Hydroxylated and methoxylated flavonoids have significant anti-hypoxia activity. This study aims to explore the synthesis, antioxidant and anti-hypoxia activities of 6-hydroxygenistein (6-OHG) and its methoxylated derivatives. METHODS: The 6-OHG and its methoxylated derivatives, including 4',6,7-trimethoxy-5-hydroxyisoflavone (compound 3), 4',5,6,7-tetramethoxyisoflavone (compound 4), 4',6-imethoxy-5,7-dihydroxyisoflavone (compound 6), and 4'-methoxy-5,6,7-trihydroxyisoflavone (compound 7), were synthesized by methylation, bromination, methoxylation, and demethylation using biochanin A as raw material. The structure of these products were characterized by 1hydrogen-nuclear magnetic resonance spectroscopy (1H-NMR) and mass spectrometry (MS). The purity of these compounds was detected by high pressure chromatography (HPLC). The antioxidant activity in vitro was investigated by 1,1-diphenyl-2-picrylhydrazyl radical (DPPH) free radical scavenging assay. PC12 cells were divided into a normal group, a hypoxia model group, rutin (1×10-9-1×10-5 mol/L) groups, and target compounds (1×10-9-1×10-5 mol/L) groups under normal and hypoxic conditions. Cell viability was detected by cell counting kit-8 (CCK-8) assay, the target compounds with excellent anti-hypoxia activity and the drug concentration at the maximum anti-hypoxia activity were screened. PC12 cells were treated with the optimal concentration of the target compound or rutin with excellent anti-hypoxia activity, and the cell morphology was observed under light microscope. The apoptotic rate was determined by flow cytometry, and the expressions of hypoxia inducible factor-1α (HIF-1α) and vascular endothelial growth factor (VEGF) were detected by Western blotting. RESULTS: The structure of 6-OHG and its 4 methylated derivatives were correct, and the purity was all more than 97%. When the concentration was 4 mmol/L, the DPPH free radical removal rates of chemical compounds 7 and 6-OHG were 81.16% and 86.94%, respectively, which were higher than those of rutin, the positive control. The removal rates of chemical compounds 3, 4, and 6 were all lower than 20%. Compared with the normal group, the cell viability of the hypoxia model group was significantly decreased (P<0.01). Compared with the hypoxia model group, compounds 3, 4, and 6 had no significant effect on cell viability under hypoxic conditions. At all experimental concentrations, the cell viability of the 6-OHG group was significantly higher than that of the hypoxia model group (all P<0.05). The cell viability of compound 7 group at 1×10-7 and 1×10-6 mol/L was significantly higher than that of the hypoxia model group (both P<0.05). The anti-hypoxia activity of 6-OHG and compound 7 was excellent, and the optimal drug concentration was 1×10-6 and 1×10-7 mol/L. After PC12 cells was treated with 6-OHG (1×10-6 mol/L) and compound 7 (1×10-7 mol/L), the cell damage was reduced, the apoptotic rate was significantly decreased (P<0.01), and the protein expression levels of HIF-1α and VEGF were significantly decreased in comparison with the hypoxia model group (both P<0.01). CONCLUSIONS: The optimized synthesis route can increase the yield of 6-OHG and obtain 4 derivatives by methylation and selective demethylation. 6-OHG and compound 7 have excellent antioxidant and anti-hypoxia activities, which are related to the structure of the A-ring ortho-triphenol hydroxyl group in the molecule.

GSK-3α-BNIP3 axis promotes mitophagy in human cardiomyocytes under hypoxia.

Dysregulated autophagy/mitophagy is one of the major causes of cardiac injury in ischemic conditions. Glycogen synthase kinase-3alpha (GSK-3α) has been shown to play a crucial role in the pathophysiology of cardiac diseases. However, the precise role of GSK-3α in cardiac mitophagy remains unknown. Herein, we investigated the role of GSK-3α in cardiac mitophagy by employing AC16 human cardiomyocytes under the condition of acute hypoxia. We observed that the gain-of-GSK-3α function profoundly induced mitophagy in the AC16 cardiomyocytes post-hypoxia. Moreover, GSK-3α overexpression led to increased ROS generation and mitochondrial dysfunction in cardiomyocytes, accompanied by enhanced mitophagy displayed by increased mt-mKeima intensity under hypoxia. Mechanistically, we identified that GSK-3α promotes mitophagy through upregulation of BNIP3, caused by GSK-3α-mediated increase in expression of HIF-1α and FOXO3a in cardiomyocytes post-hypoxia. Moreover, GSK-3α displayed a physical interaction with BNIP3 and, inhibited PINK1 and Parkin recruitment to mitochondria was observed specifically under hypoxia. Taken together, we identified a novel mechanism of mitophagy in human cardiomyocytes. GSK-3α promotes mitochondrial dysfunction and regulates FOXO3a -mediated BNIP3 overexpression in cardiomyocytes to facilitate mitophagy following hypoxia. An interaction between GSK-3α and BNIP3 suggests a role of GSK-3α in BNIP3 recruitment to the mitochondrial membrane where it enhances mitophagy in stressed cardiomyocytes independent of the PINK1/Parkin.