HIF-1alpha Research Articles

A Multi-Responsive Hydrogel Combined With Mild Heat Stimulation Promotes Diabetic Wound Healing by Regulating Inflammatory and Enhancing Angiogenesis.

The repair of diabetic wound still encounters huge challenges, such as disordered inflammatory regulation and impaired neovascularization. Here, a pH/ROS/glucose responsive and photothermal hydrogel is developed for diabetic wound healing. The hydrogel is formed through cross-linkage between phenylboronic acid-modified carboxymethyl chitosan (CMCS-PBA) and oxide dextran (OXD), utilizing Schiff base and phenylboronate ester bonds. Additionally, insulin-like growth factor 1 C domain (IGF-1C) and deferoxamine-loaded polydopamine nanoparticles (D@P) are incorporated into the hydrogel. The hydrogel demonstrates sustained drug release, excellent photo thermal effect, prominent antioxidant, antibacterial and anti-inflammatory activities, desirable mechanical and tissue adhesive properties, enhanced tube formation, and cell migration. Furthermore, the hydrogel combined with mild heat treatment can regulate chronic inflammation by promoting the transformation of macrophages from M1 phenotype to M2 phenotype and enhance angiogenesis by up-regulating the expression levels of angiogenesis-related factors such as hypoxia-inducible factor-1 alpha (HIF-1α), vascular endothelial growth factor (VEGF), CD31, and α-SMA, thus greatly accelerates the wound healing in streptozotocin (STZ)-induced diabetic mice. Therefore, this multi-responsive and multifunctional hydrogel holds potential as a therapeutic strategy for diabetic wounds.

Alleviating Tumor Hypoxia and Immunosuppression via Sononeoperfusion: A New Ally for potentiating anti-PD-L1 blockade of solid Tumor

The hypoxic and immunosuppressive tumor microenvironment (TME) remains a major obstacle to impede cancer immunotherapy. Here, we found that sononeoperfusion—a new effect of tumor perfusion enhancement induced by low mechanical index ultrasound stimulated microbubble cavitation (USMC)—ameliorated tumor tissue oxygenation and induced tumor vascular normalization (TVN). This TVN might be associated with the down-regulation of hypoxia-inducible factor 1-alpha (HIF-1α) and vascular endothelial growth factor (VEGF) within tumors. Moreover, the sononeoperfusion effect reduced the accumulation of immunosuppressive cells, such as regulatory T cells (Tregs), myeloid-derived suppressor cells (MDSCs) and M2-like tumor-associated macrophages (M2-TAMs), and decreased the production of immune inhibitory factors like transforming growth factor-β1 (TGF-β1), interleukin 10 (IL-10), chemoattractant chemokines CC-chemokine ligand 22 (CCL22), CCL28, adenosine and lactate within tumors. Notably, flow cytometry analysis revealed that sononeoperfusion not only increased the percentage of tumor infiltrating-CD8+ T cells, but also promoted the generation of interferon-γ (IFN-γ) and tumor necrosis factor-α (TNF-α) by these cells. Furthermore, the improved immune TME by sononeoperfusion effect sensitized anti-PD-L1 treatment both in MC38 colon cancer and Lewis lung carcinoma mice, resulting in tumor regression and prolonged survival. Mechanically, the enhanced efficacy of combination therapy was mainly based on promoting the infiltration and function of CD8+ T cells within tumors. Together, sononeoperfusion could ameliorate hypoxia and immunosuppression in the TME, thereby potentiating anti-PD-L1 therapy for solid tumors. This novel method of USMC generating sononeoperfusion effect may provide a new therapeutic modality for facilitating cancer immunotherapy.

A novel quinazoline derivative exhibits potent anticancer cytotoxicity via apoptosis and inhibition of angiogenesis in DMBA-induced mammary gland carcinoma.

Mammary gland carcinoma is one of the most prevalent and deadly diseases among women globally. It is a type of solid malignant tumor. In this malignant tumor, the microenvironment becomes hypoxic in rapidly proliferating cancer cells. These cells undergo adaptive changes through the expression of hypoxia-inducible factor-1alpha (HIF-1α) which is regulated by factor inhibiting HIF-1α (FIH-1). Considering this, we hypothesized that the chemical activation of FIH-1 would inhibit the hypoxic activity of HIF-1α in mammary gland carcinoma. A library of 67,609 chemical compounds was virtually screened against FIH-1 based on Lipinski's rule from the ZINC database. The BBAP-8 has been selected based on an excellent docking score (-8.352 Kcal/mol), favorable ADMET, and potential FIH-1 activator profile. Further, its in-vitro cytotoxicity and apoptotic activity were scrutinized against MCF-7 cells and in-vivo activity against 7,12-dimethylbenz[a]anthracene (DMBA) induced mammary gland carcinoma in Wistar rats. It exhibited significant cytotoxicity (IC50 = 16.59 ± 0.49 μM) and activated apoptosis when scrutinized through DAPI, AO/EB, and JC-1 staining. Also, oral administration of BBAP-8 restored hemodynamic changes, normalized tissue architecture, and corrected metabolic abnormalities. The western blot analysis and mRNA expression analysis validated that BBAP-8 has the potential to activate FIH-1 with the downregulation of GLUT-1, VEGF, and Twist-1. Moreover, BBAP-8 fostered apoptosis, when evaluated through BCL-2, BAX, Caspase-8, and Caspase-3. Based on research findings, this implies that BBAP-8 activates FIH-1 and can be effective in chemotherapeutic treatment of mammary gland carcinoma.

Genistein inhibits HIF-1α and attenuates high glucose-induced peritoneal mesothelial-mesenchymal transition and fibrosis via the mTOR/OGT pathway

Peritoneal fibrosis has been linked to hypoxia-inducible factor 1-alpha (HIF-1α) as well as O-linked-N-acetylglucosaminylation (O-GlcNAcylation) in peritoneal dialysis (PD). Genistein, recognized for its HIF-1α inhibitory and antifibrotic effects, presents a potential intervention against peritoneal mesothelial-mesenchymal transition (MMT) as well as fibrosis in PD. This study employed human peritoneal mesothelial cells (HPMCs) together with adenine-induced chronic kidney disease (CKD) rats undergoing peritoneal dialysis to explore Genistein’s role in high glucose-induced peritoneal MMT and fibrosis. Our findings reveal that Genistein exerts anti-MMT and anti-fibrotic effects by inhibiting HIF-1α in HPMCs under high glucose conditions. Genistein inhibited O-GlcNAcylation status of HIF-1α through the mTOR/O-GlcNAc transferase (OGT) pathway, promoting its ubiquitination as well as the subsequent proteasomal degradation. In adenine-induced CKD rats undergoing peritoneal dialysis, Genistein suppressed the mTOR/OGT expression and reduced the abundance of O-GlcNAcylation along with HIF-1α in the peritoneum. Additionally, Genistein protected against increased peritoneal thickness, fibrosis, and angiogenesis, while improving peritoneal function. Based on our results, it could be inferred that Genistein might inhibit the abundance of HIF-1α via the mTOR/OGT pathway, thereby ameliorating MMT as well as fibrosis in PD.

3D biological scaffold delivers Bergenin to reduce neuroinflammation in rats with cerebral hemorrhage

BackgroundIntracerebral hemorrhage (ICH) is a severe form of stroke characterized by high incidence and mortality rates. Currently, there is a significant lack of effective treatments aimed at improving clinical outcomes. Our research team has developed a three-dimensional (3D) biological scaffold that incorporates Bergenin, allowing for the sustained release of the compound.MethodsThis 3D biological scaffold was fabricated using a combination of photoinitiator, GEMA, silk fibroin, and decellularized brain matrix (dECM) to encapsulate Bergenin through advanced 3D bioprinting techniques. The kinetics of drug release were evaluated through both in vivo and in vitro studies. A cerebral hemorrhage model was established, and a 3D biological scaffold containing Bergenin was transplanted in situ. Levels of inflammatory response, oxidative stress, and apoptosis were quantified. The neurological function of rats with cerebral hemorrhage was assessed on days 1, 3, and 5 using the turning test, forelimb placement test, Longa score, and Bederson score.ResultsThe 3D biological scaffold incorporating Bergenin significantly enhances the maintenance of drug concentration in the bloodstream, leading to a marked reduction in inflammatory markers such as IL-6, iNOS, and COX-2 levels in a cerebral hemorrhage model, primarily through the inhibition of the NF-κB pathway. Additionally, the scaffold effectively reduces the expression of hypoxia-inducible factor 1-alpha (HIF-1α) in primary cultured astrocytes, which in turn decreases the production of reactive oxygen species (ROS) and inhibits IL-6 production induced by hemin. Subsequent experiments reveal that the 3D biological scaffold containing Bergenin promotes the activation of the Nrf-2/HO-1 signaling pathway, both in vivo and in vitro, thereby preventing cell death. Moreover, the application of this 3D biological scaffold has been demonstrated to improve drug retention in the bloodstream.ConclusionThis strategy effectively mitigates inflammation, oxidative stress, and cell death in rats with cerebral hemorrhage by inhibiting the NF-κB pathway while concurrently activating the Nrf-2/HO-1 pathway.

Clinical significance of HIF-1α, ET-1, and NO as biomarkers in chronic obstructive pulmonary disease patients with pulmonary hypertension.

Pulmonary hypertension (PH) is a common complication of chronic obstructive pulmonary disease (COPD), with repeated infections and airflow limitation leading to endothelial dysfunction. The objective of this study was to retrospectively investigate the clinical significance of serum hypoxia-inducible factor 1-alpha (HIF-1α), plasma endothelin-1 (ET-1), and nitric oxide (NO) as non-invasive blood biomarkers for diagnosing patients with acute exacerbation of chronic obstructive pulmonary disease (AECOPD). A total of 230 AECOPD patients were assessed for serum HIF-1α, plasma ET-1, and NO levels using Doppler echocardiography and blood tests. Clinical characteristics, including age, COPD duration, and comorbidities, were compared between patients with and without PH. The diagnostic value was determined through ROC analysis. Pearson correlation and multivariate logistic analyses explored the correlation of biomarkers in AECOPD with PH. Serum HIF-1α, plasma ET-1, and NO levels showed significant differences between COPD patients with and without PH. The combination model (0.027 * HIF-1α + 0.028 * ET-1 - 0.049 * NO) demonstrated an AUC of 0.89, indicating superior diagnostic value compared to individual biomarkers. Multivariate logistic analysis identified smoking, higher GOLD grade, hypertension, and elevated levels of HIF-1α, ET-1, and NO as independent risk factors for AECOPD. Positive correlations were observed between serum HIF-1α and plasma ET-1 levels with pulmonary artery systolic pressure (PASP), while NO demonstrated a negative correlation. Serum HIF-1α, plasma ET-1, and NO were associated with AECOPD and PH, and detecting individual or combined levels of these biomarkers in the blood can predict COPD-related PH.

Biofabricated adipose-derived mesenchymal cell sheets recover cryo-injured kidneys in rats.

This study aimed to develop a treatment for chronic kidney disease (CKD) by investigating whether transplantation of biofabricated adipose-derived mesenchymal cell (AMC) sheets could improve renal tissue and function. Thirty-nine 10-week-old male Sprague-Dawley rats underwent the harvesting of adipose tissues and right nephrectomy. AMCs that were collected from adipose tissues were labeled and cultured on temperature-responsive dishes, and applied to a gelatin hydrogel sheet. Subsequently, two identical AMC-gelatin sheets were attached together to biofabricate a bilayered AMC-gelatin sheet. Further, 3 weeks after nephrectomy, the renal artery and vein of the left kidney were clamped, and the kidney was sprayed with liquid nitrogen for 60 seconds. The biofabricated AMC sheet was autologously transplanted into the renal capsule of the cryo-injured region (n = 14). Control rats were given acellular sheet (n = 25). One day before and four weeks after transplantation, blood and 24-hour urinary specimens were collected. Histological analysis of the experimental kidneys was performed four weeks after transplantation. Four weeks after transplantation, in the acellular control-transplanted rats, creatinine clearance levels tended to increase, while serum creatinine levels significantly increased. However, in the biofabricated AMC sheet-transplanted rats, creatinine clearance levels significantly increased, and serum creatinine levels remained unchanged and were significantly lower than that of the control rats. The ratio of damaged to undamaged renal tubules in the AMC sheet-transplanted rats was lower than that in the control rats. In addition, the occupancy rate of fibrotic areas in the renal cortex under the AMC sheet-transplanted regions was significantly lower than that in the control regions. After transplantation, while the expressions of transforming growth factor-beta 1 and hypoxia-inducible factor-1 alpha were observed in both the control- and AMC sheet-transplanted regions, these expressions tended to be lower in the AMC sheet-transplanted rats than in the control rats. The labeled transplanted AMCs were detected in the transplanted regions, with some of them also showing positive staining for the vascular endothelial growth factor antibody. In conclusion, the biofabricated AMC sheets improved renal functions by ameliorating renal tubule disorders and renal fibrosis. Therefore, biofabricated AMC sheets would serve as a potential treatment for CKD.

Fetal hypoxia exposure induced Hif1a activation and autophagy in adult ovary granulosa cells.

Environmental hypoxia adversely impacts the reproduction of humans and animals. Previously, we showed that fetal hypoxia exposure led to granulosa cell (GC) autophagic cell death via the Foxo1/Pi3k/Akt pathway. However, the upstream regulatory mechanisms underlying GC dysfunction remain largely unexplored. Here, we tested the hypothesis that fetal hypoxia exposure altered gene expression programs in adult GCs and impaired ovarian function. We established a fetal hypoxia model in which pregnant mice were maintained in a high-plateau hypoxic environment from gestation day (E) 0--16.5 to study the impact of hypoxia exposure on the ovarian development and subsequent fertility of offspring. Compared with the unexposed control, fetal hypoxia impaired fertility by disordering ovarian function. Specifically, fetal hypoxia caused mitochondrial dysfunction, oxidant stress and autophagy in GCs in the adult ovary. RNA-seq analysis revealed that 437 genes were differentially expressed in the adult GCs of exposed animals. Western blotting results also revealed that fetal exposure induced high levels of hypoxia-inducible factor 1-alpha (Hif1a) expression in adult GCs. We then treated GCs isolated from exposed mice with PX-478, a specific pharmacological inhibitor of Hif-1a, and found that autophagy and apoptosis were effectively alleviated. Finally, by using a human ovarian granulosa-like tumor cell line (KGN) to simulate hypoxia in vitro, we showed that Hif1a regulated autophagic cell death in GCs through the Pi3k/Akt pathway. Together, these findings suggest that fetal hypoxia exposure induced persistent Hif1a expression, which impaired mitochondrial function and led to autophagic cell death in the GCs of the adult ovary.

Hypoxia-Inducible Factor 1-Alpha (HIF-1α) and Cancer: Mechanisms of Tumor Hypoxia and Therapeutic Targeting.

Hypoxia-inducible factor 1-alpha (HIF-1α) is necessary for cells to adapt to low oxygen levels often present in the tumor microenvironment. HIF-1α triggers a transcriptional program that promotes invasion, angiogenesis, metabolic reprogramming, and cell survival when it is active in hypoxic environments. These processes together lead to the growth and spread of tumors. This review article examines the molecular mechanisms by which HIF-1α contributes to tumor progression, including its regulation by oxygen-dependent and independent pathways, interactions with oncogenic signaling networks, and impact on the tumor microenvironment. Additionally, we explore current therapeutic strategies targeting HIF-1α, such as small molecule inhibitors, RNA interference, and immunotherapy approaches. Understanding the multifaceted roles of HIF-1α in cancer biology not only elucidates the complexities of tumor hypoxia but also opens avenues for developing novel and more effective cancer therapies.

Macrophage migration inhibitory factor facilitates replication of Senecavirus A by enhancing the glycolysis via hypoxia inducible factor 1 alpha

Senecavirus A (SVA) induced porcine idiopathic vesicular disease (PIVD) has been spread worldwide due to persistent infection, causing economic losses in swine industry. Host factors play an important role in replication of SVA, while, the interaction of migration inhibitory factor (MIF) and the virus has not been verified. Here, MIF facilitates the replication of SVA by enhancing the glycolysis via hypoxia-inducible factor alpha (HIF-1α) was reported. SVA infection up-regulates the expression of MIF in 3D4/21 cells, and infection experiment of cells with overexpression and interference expression of MIF showed that MIF facilitates the replication of SVA. MIF promoted the glycolysis in SVA infection to facilitate its replication by enhancing the accumulation of lactate and decreasing the production of adenosine triphosphate (ATP) and inhibiting the expression of retinoic acid-inducible gene I (RIG-I), mitochondrial antiviral-signaling protein (MAVS), interferon regulatory factor 3 (IRF3), interferon-beta (IFN-β), IFN-α, interferon-stimulating gene 15 (ISG15), and ISG56. Meanwhile, specific inhibitor verified MIF facilitates the replication of SVA by enhancing glycolysis. Further results showed MIF induces the increased expression of HIF-1α, which enhances MIF-induced glycolysis. These results provide new data on host factors in replication of SVA, as well as better understanding the role of MIF in virus infection.

PYGL regulation of glycolysis and apoptosis in glioma cells under hypoxic conditions via HIF1α-dependent mechanisms.

Gliomas are highly aggressive brain tumors with complex metabolic and molecular alterations. The role of glycolysis in glioma progression and its regulation by hypoxia remain poorly understood. This study investigated the function of glycogen phosphorylase L (PYGL) in glioma and its interaction with glycolytic pathways under hypoxic conditions. Differential expression analysis was conducted using The Cancer Genome Atlas (TCGA) glioma and GSE67089 datasets, revealing significant changes in the expression of genes. A prognostic risk model incorporating PYGL was built by univariate and multivariate Cox regression analyses. The impacts of PYGL on glioma cell proliferation, glycolysis, apoptosis, and metabolic activities were evaluated by in vitro assays. Additionally, the influences of hypoxia and hypoxia-inducible factor 1-alpha (HIF1α) on PYGL expression were evaluated. Our prognostic prediction model showed a C-index of 0.76 [95% confidence interval (CI): 0.70-0.82], indicating a good predictive accuracy of the model. In addition, genetic predictors included in the nomogram included PYGL, HIF1α, and other genes associated with the glycolytic pathway. Differential expression analysis identified PYGL as a key gene associated with glioma survival. PYGL expression was significantly upregulated in glioma cells. PYGL knockdown inhibited cell invasion, proliferation, migration, and colony formation and enhanced apoptosis via modulation of Bcl-2, caspase-3, and Bax. Glycolysis was impaired in PYGL-knockdown cells, as indicated by increased glycogen levels and a reduced extracellular acidification rate (ECAR), adenosine triphosphate (ATP) levels, lactate levels, and PKM2 and LDHA expression. PYGL overexpression promoted glycolysis and cell viability, which was counteracted by 2-deoxy-D-glucose (2-DG). Hypoxia-induced PYGL expression was regulated by HIF1α, underscoring the interplay between the hypoxia and glycolysis pathways. PYGL is a crucial regulator of glycolysis in gliomas and contributes to tumor progression under hypoxic conditions. Targeting PYGL and its associated metabolic pathways may offer new therapeutic strategies for glioma treatment.

Exercise preconditioning mitigates brain injury after cerebral ischemia-reperfusion injury in rats by restraining TIMP1.

Cerebral ischemic disease is a common cerebrovascular disease, especially ischemic stroke. Exercise has protective functions on brain tissues following cerebral ischemia-reperfusion injury (CIRI), but its preventive effects and mechanisms in CIRI remain unclear. We aimed to investigate the effects and mechanisms of exercise preconditioning on CIRI. The middle cerebral artery occlusion (MCAO) operation was prepared to establish CIRI rats. All rats were randomized into the MCAO, exercise (exercise preconditioning plus MCAO operation), vector (exercise preconditioning, MCAO operation plus intraventricular injection of empty vector), and tissue inhibitor of metalloprotease 1 overexpression (OE-TIMP1, exercise preconditioning, MCAO operation plus intraventricular injection of OE-TIMP1) groups. The results indicated that exercise preconditioning suppressed approximately 66.67% of neurological deficit scores and 73.79% of TIMP1 mRNA expression in MCAO rats, which were partially offset by OE-TIMP1. The protective effects of exercise against neuron death status and cerebral infarction size in MCAO rats were reversed by OE-TIMP1. It also confirmed that exercise weakened apoptosis and oxidative stress damage, with notable increases of B-cell lymphoma-2, superoxide dismutase, and glutathione peroxidase production, and evident decreases of BCL2-associated X, caspase 3, and malondialdehyde in MCAO rats, while these effects were partially reversed by OE-TIMP1. Additionally, the inhibitory effects of exercise on the protein levels of TIMP1, hypoxia-inducible factor-alpha, vascular endothelial growth factor receptor 2, vascular endothelial growth factor, and neurogenic locus notch homolog protein 1 in MCAO rats were partially reversed by OE-TIMP1. Altogether, exercise preconditioning had protective effects on CIRI by restraining TIMP1, which provided new therapeutic strategies for preventing CIRI.

Pre-treatment with notoginsenoside R1 from Panax notoginseng protects against high-altitude-induced pulmonary edema by inhibiting pyroptosis through the NLRP3/caspase-1/GSDMD pathway

High-altitude pulmonary edema (HAPE) is a potentially fatal condition that occurs when exposed to high-altitude hypoxia environments. Currently, there is no effective treatment for HAPE, and available interventions focus on providing relief. Notoginsenoside R1 (NGR1), a major active constituent of Panax notoginseng (Burkill) F.H.Chen (sānqī), has demonstrated heart and lung-protective effects under hypobaric hypoxia. However, there is a lack of clarity regarding the precise mechanisms that underlie the protective effects of NGR1 against inflammation. In this study, a rat model of HAPE was developed to assess the effect of NGR1 on this pathology. High-altitude hypoxia corresponding to 6000 m altitude was simulated with a hypobaric chamber. We found that NGR1 dose-dependently alleviated pulmonary oxidative stress damage and inflammatory response, and prevented acid-base balance disruption. In addition, NGR1 restored the expression levels of hypoxia-inducible factor-1 alpha, vascular endothelial growth factor, and aquaporin protein-5, correlated with the development of pulmonary edema induced by hypobaric hypoxia. Furthermore, NGR1 pre-treatment remarkably mitigated NOD-like receptor family pyrin domain containing 3 (NLRP3) inflammasome-induced pyroptosis, and this effect was partially counteracted by the use of an NLRP3 agonist. Thus, NGR1 may exert a lung-protective effect against HAPE by ameliorating hypoxia-induced lung edema, oxidative damage, and inflammation through inhibition of the NLRP3/Caspase-1/ GSDMD signaling pathway.

HIF-1α-HPRT1 axis promotes tumorigenesis and gefitinib resistance by enhancing purine metabolism in EGFR-mutant lung adenocarcinoma

BackgroundThe mutations of oncogenic epidermal growth factor receptor (EGFR) is an important cause of lung adenocarcinoma (LUAD) malignance. It has been knowm that metabolic reprogramming is an important hallmark of malignant tumors, and purine metabolism is a key metabolic pathway for tumor progression and drug resistance, but its relationship with the EGFR-mutant LUAD is unclear.MethodsMetabolic reprogramming was studied through capillary electrophoresis-time of flight mass spectrometry (CE-TOF/MS)-based metabolic profiling analysis. Cell proliferation in vitro was evaluated by EdU staining and cell cycle assay. Tumorigenicity in vivo was tested by subcutaneous tumor formation experiment in nude mice. The binding of hypoxia-inducible factor-1 alpha (HIF-1α) and hypoxanthine phosphoribosyltransferase 1 (HPRT1) was detected by DNA pull‑down assay and Chromatin immunoprecipitation (ChIP) assays. HIF-1α, HPRT1, DNA damage and cell apoptosis related genes were examined by western blot. In addition, RNA sequencing, mass spectrometry and bioinformatics analysis were performed.ResultsWe found that mutated EGFR (muEGFR) upregulates HPRT1 to promote purine metabolism and tumorigenesis of EGFR-mutant LUAD. Mechanistically, muEGFR increases HIF-1α expression through protein stability. Meanwhile, up-regulated HIF-1α bound to the promoter of HPRT1 and transcriptionally activates HPRT1 expression, enhancing purine metabolism to maintain rapid tumor cell proliferation in EGFR-mutant LUAD. Further, gefitinib inhibited the synthesis of purine nucleotides, and HPRT1 inhibition increased the sensitivity of gefitinib to EGFR-mutant LUAD.ConclusionsOur study reveals that muEGFR-HIF-1α-HPRT1 axis plays a key role in EGFR-mutant LUAD and provides a new strategy-inhibiting purine metabolism for treating EGFR-mutant LUAD.

Claudin-9 (CLDN9) promotes gastric cancer progression by enhancing the glycolysis pathway and facilitating PD-L1 lactylation to suppress CD8+ T cell anti-tumor immunity

BackgroundGastric cancer (GC) is a common malignancy characterized by the absence of reliable prognostic indicators and effective therapeutic targets. Claudin-9 (CLDN9) has been demonstrated to be upregulated in various cancers. However, its prognostic value, biological function, and regulatory mechanisms in GC remain unclear. Therefore, this study aimed to elucidate the role of CLDN9 in GC progression and its underlying mechanisms. MethodsWe utilized consensus cluster, random survival forest, and multivariate Cox regression analyses to identify CLDN9 in GC. Subsequently, we evaluated the mRNA and protein levels of CLDN9 in GC using quantitative real-time polymerase chain reaction (PCR) (qRT-PCR), western blotting (WB), and immunohistochemistry (IHC). Furthermore, the role of CLDN9 in GC progression was investigated using a series of functional in vivo and in vitro experiments. Finally, we elucidated the molecular mechanisms of CLDN9 using bioinformatics, molecular biology, animal models, and patient tissue specimens. ResultsTwo GC subtypes with survival and functional differences were identified based on glycolytic metabolic genes in the Cancer Genome Atlas (TCGA)- Stomach adenocarcinoma (STAD) dataset. A prognostic risk score was calculated using seven genes to assess the overall survival (OS) in GC. Using random survival forest and multivariate Cox analyses, we identified CLDN9 as the key gene linked to the glycolytic subtype and prognosis of GC. CLDN9 expression was significantly upregulated in patients with GC as well as in GC cells. CLDN9 knockdown inhibited tumor proliferation, invasion, and metastasis both in vivo and in vitro. Mechanistically, CLDN9 was found to regulate lactate dehydrogenase A (LDHA) expression and promote glycolytic metabolism by activating the phosphatidylinositol 3-kinase (PI3K)/protein kinase B (AKT)/hypoxia-inducible factor 1-alpha (HIF1α) signaling pathway. Additionally, lactate, a glycolytic metabolite, enhanced programmed cell death ligand 1 (PD-L1) lactylation and stability, which suppressed anti-tumor immunity in CD8+ T cells, thereby contributing to GC progression. ConclusionsCLDN9 expression is associated with GC development and progression. Mechanistically, CLDN9 enhances the glycolysis pathway and facilitates PD-L1 lactylation through the PI3K/AKT/HIF1α signaling pathway, thereby suppressing anti-tumor immunity in CD8+ T cells. CLDN9 has the potential to serve as a novel prognostic marker and therapeutic target for GC.

RNA binding protein ELAVL1-mediated USP33 stabilizes HIF1A to promote pathological proliferation, migration and angiogenesis of RECs.

Dysfunction of retinal vascularization plays pathogenic roles in retinopathy of prematurity (ROP). Hypoxia-inducible factor 1 alpha (HIF1A) is activated by hypoxia and contributes to ROP progression. Herein, we clarified the mechanism underlying HIF1A activation in human retinal vascular endothelial cells (HRECs) under hypoxia. Protein expression was assayed by immunoblot analysis. Cell migration, microtubule formation, invasion, proliferation, and viability were detected by wound-healing, tube formation, transwell, EdU, and CCK-8 assays, respectively. Bioinformatics was used to predict the deubiquitinase-HIF1A interactions and RNA binding proteins (RBPs) bound to USP33. The impact of USP33 on HIF1A deubiquitination was validated by immunoprecipitation (IP) assay. RNA stability analysis was performed with actinomycin D (Act D) treatment. The ELAVL1/USP33 interaction was assessed by RNA immunoprecipitation experiment. In hypoxia-exposed HRECs, HIF1A and USP33 protein levels were upregulated. Deficiency of HIF1A or USP33 suppressed cell migration, proliferation and microtubule formation of hypoxia-exposed HRECs. Mechanistically, USP33 deficiency led to an elevation in HIF1A ubiquitination and degradation. USP33 deficiency reduced HIF1A protein levels to suppress the proliferation and microtubule formation of hypoxia-induced HRECs. Moreover, the RBP ELAVL1 stabilized USP33 mRNA to increase USP33 protein levels. ELAVL1 decrease repressed the proliferation and microtubule formation of hypoxia-induced HRECs by reducing USP33. Our study identifies a novel ELAVL1/USP33/HIF1A regulatory cascade with the ability to affect hypoxia-induced pathological proliferation, angiogenesis, and migration in HRECs.

A New Combination of Bifidobacterium bifidum and Lactococcus lactis Strains with Synergistic Effects Alleviates Colitis-Associated Colorectal Cancer.

Chronic inflammation is a factor in the development of cancer, and probiotics play a role in preventing or treating inflammation as an adjuvant therapy. To investigate potential probiotics for the prevention of colitis-associated colorectal cancer (CAC), Bifidobacterium bifidum H3-R2 and Lactococcus lactis KLDS4.0325 were used to examine the effects on colon cancer cells and in an inflammation-related cancer animal model. The results revealed that B. bifidum H3-R2 in combination with L. lactis KLDS4.0325 caused apoptosis in colon cancer cells by increasing caspase-3 and caspase-9 protein levels, enhancing Bax expression, and lowering Bcl-2 expression. In addition, the combination of the two strains relieved the tissue damage; reduced proinflammatory cytokines, myeloperoxidase (MPO) activity, and hypoxia-inducible factor 1-alpha (HIF-1α) level; upregulated anti-inflammatory cytokines; increased colonic tight junction protein expression; regulated intestinal homeostasis by inhibiting NLRP3 inflammasome signaling pathway; and improved the imbalance of gut microbiota in animal models. Moreover, the combination of the two strains had a greater preventive impact than each strain alone. These findings are supportive of clinical studies and product development of multi-strain probiotic preparations for diseases associated with colitis.

Proteoglycan 4 (Lubricin) and Regulation of Xanthine Oxidase in Synovial Macrophage as A Mechanism of Controlling Synovitis.

Synovial macrophages (SMs) are important effectors of joint health and disease. A novel Cx3CR1 + TREM2 + SM population expressing the tight junction protein claudin-5, was recently discovered in synovial lining. Ablation of these SMs was associated with onset of arthritis. Proteoglycan 4 (PRG4) is a mucinous glycoprotein that fulfills lubricating and homeostatic roles in the joint. The aim of this work is to study the role of PRG4 in modulating synovitis in the context of SM homeostasis and assess the contribution of xanthine oxidase (XO)-hypoxia inducible factor alpha (HIF-1α) axis to this regulation. We used Prg4 FrlioxP/FrtloxP ;R26 FlPoER/+ , a novel transgenic mouse, where the Prg4 Frt allele normally expresses the PRG4 protein and was designed to flank the first two exons of Prg4 with a flippase recognition target and "LOXP" sites. Inducing flippase activity with tamoxifen (TAM) inactivates the Frt allele and thus creates a conditional knockout state. We studied anti-inflammatory SMs and XO by quantitative immunohistochemistry, isolated RNA and studied immune pathway activations by multiplexed assays and isolated SMs and studied PRG4 signaling dysfunction in relation to glycolytic switching due to pro-inflammatory activation. Prg4 inactivated mice were treated with oral febuxostat, a specific XO inhibitor, and quantification of Cx3CR1 + TREM2 + SMs, XO immunostaining and synovitis assessment were conducted. Prg4 inactivation induced Cx3CR1 + TREM2 + SM loss (p < 0.001) and upregulated glycolysis and innate immune pathways in the synovium. In isolated SMs, Xdh (p < 0.01) and Hif1a (p < 0.05) were upregulated. Pro-inflammatory activation of SMs was evident by enhanced glycolytic flux and XO-generated reactive oxygen species (ROS). Febuxostat reduced glycolytic flux (p < 0.001) and HIF-1α levels (p < 0.0001) in SMs. Febuxostat also reduced systemic inflammation (p < 0.001), synovial hyperplasia (p < 0.001) and preserved Cx3CR1 + TREM2 + SMs (p < 0.0001) in synovia of Prg4 inactivated mice. PRG4 is a biologically significant modulator of synovial homeostasis via inhibition of XO expression and downstream HIF-1a activation. PRG4 signaling is anti-inflammatory and promotes synovial homeostasis in chronic synovitis, where direct XO inhibition is potentially therapeutic in chronic synovitis.

HIF-1α knockdown suppresses breast cancer metastasis via epithelial mesenchymal transition Abrogation

Lung metastasis, a leading cause of breast cancer mortality, lacks effective therapeutic options. Hypoxia-inducible factor 1-alpha (HIF-1α) plays important roles in breast cancer progression, but its direct impact on lung metastasis remains unclear. Herein, in this study, we investigated the role of HIF-1α in breast cancer lung metastasis and the potential of targeting it for therapeutic benefit. HIF-1α expression was knocked down in the 4T1 mouse mammary carcinoma cell line using a lentiviral vector. HIF-1α knockdown significantly reduced the migratory ability of 4T1 cells in vitro and lung metastasis in a mouse model. Mechanistically, HIF-1α knockdown decreased the expression of matrix metalloproteinases (MMP-2 and MMP-9) that degrade the extracellular matrix and suppressed the epithelial-to-mesenchymal transition (EMT) by increasing E-cadherin and decreasing vimentin expression. The findings of this study demonstrate that HIF-1α knockdown effectively inhibits lung metastasis of 4T1 cells both in vitro and in vivo by suppressing EMT. These results underscore a promising new approach for managing breast cancer metastasis.

Limiting pre-menstrual endometrial Hypoxia Inducible Factor 2 Alpha may fine-tune endometrial function at menstruation.

Heavy menstrual bleeding (HMB) is common and debilitating, but the precise endometrial mechanisms causing increased menstrual blood loss (MBL) remain undefined. We have previously identified a role for hypoxia in endometrial repair following progesterone withdrawal. As hypoxia inducible factor 2 alpha (HIF2A) is known to alter vascular function in other tissues, we hypothesised that endometrial HIF2A is involved in pre-menstrual optimisation of endometrial function during the secretory phase to limit MBL. Women with objective HMB had higher endometrial HIF2A during the mid-secretory phase when compared to those with normal MBL (p=0.0269). In a mouse model of simulated menses, genetic or pharmacological manipulation of HIF2A did not significantly affect endometrial breakdown/repair, volume of MBL or endometrial hypoxia. However, 88% of Hif2a heterozygote mice reached early-full repair by 24h versus only 65% of wild-type mice. Mean MBL was 0.39 μl (±0.67) in Hif2a heterozygote mice versus 0.98 μl (±0.79) in wild-type mice. Conversely, when we increased HIF2A pre-menstrually, 11% reached early repair at by 8h versus 30% of vehicle-treated mice. Mean MBL was 2.61 μl (±1.10) in mice with HIF2A stabilisation and 2.24 μl (±1.14) in vehicle-treated mice. These non-significant but consistent trends indicate that increased endometrial HIF2A may contribute to delayed endometrial repair and HMB. Increased HIF2A in the secretory endometrium is unlikely to be sufficient to account for the phenotype of HMB, but limitation of HIF2 levels may optimise endometrial function at menstruation.