Hypoxia Model Research Articles

Fibronectin binds integrin α5β1 to regulate macular neovascularization through the Wnt/β-catenin signaling pathway

Age-related macular degeneration (AMD) is a progressive, degenerative disease of the macula. The formation of macular neovascularization (MNV) and subretinal fibrosis of AMD is the most classic cause of the loss of vision in older adults worldwide. While the underlying causes of MNV and subretinal fibrosis remain elusive, the common feature of many common retinal diseases is changes the proportions of protein deposition in extracellular matrix (ECM) when compared to normal tissue. In ECM, fibronectin (FN) is a crucial component and plays a pivotal part not only in fibrotic diseases but also in the process of angiogenesis. The study aims to understand the role of ligand FN and its common integrin receptor α5β1 on MNV, and to understand the molecular mechanism involved. To study this, the laser-induced MNV mouse model and the rhesus macaque choroid-retinal endothelial cell line (RF/6A) chemical hypoxia mode were established, and the FN-α5β1 expression levels were detected by immunohistochemistry (IHC) and quantitative real-time PCR analysis (qRT-PCR). Fibronectin expression was silenced using small interfering RNA (siRNA) targeting FN. The tube formation and vitro scratch assays were used to assess the ability to form blood vessels and cell migration. To measure the formation of MNV, immunofluorescence, and Western blot assays were used. These results revealed that the expressions of FN and integrin α5β1 were distinctly increased in the laser-induced MNV mouse model and in the RF/6A cytochemically induced hypoxia model, and the expression tendency was identical. After the use of FN siRNA, the tube formation and migration abilities of the RF/6A cells were lower, the ability of endothelial cells to proliferate was confined and the scope of damage caused by the laser in animal models was significantly cut down. In addition, FN gene knockdown dramatically inhibited the expression of Wnt/β-catenin signal. The interaction of FN with the integrin receptor α5β1 in the constructed model, which may act through the Wnt/β-catenin signaling pathway, was confirmed in this study. In conclusion, FN may be a potential new molecular target for the prevention and treatment of subretinal fibrosis and MNV.

Abstract 1241: Enhancing NK cell therapy for head and neck cancer within the solid tumor microenvironment using a B7H3-targeting tri-specific killer engager (TriKE)

Abstract With an annual incidence rate exceeding 660,000 cases, and a death toll surpassing 325,000 per year, head and neck cancer (HNC) ranks as the seventh most common cancer in the world. Surgery, radiation and chemotherapy are used to treat HNC patients with modest and variable clinical success. However, these treatments prove less effective for human papillomavirus negative (HPV-) HNC patients, a subset of HNC patients with markedly worse prognosis. While significant advancements have been made in cancer immunotherapy over the past decade, its success also remains elusive for HNC due to factors such as the hypoxic solid tumor microenvironment (TME). To address the critical need for an improved therapeutic intervention, we leveraged the ability of natural killer (NK) cells in killing cancer cells without prior sensitization by developing a novel tri-specific killer engager (TriKE) that can direct NK cell killing of tumor within the hypoxic solid TME. The TriKE is composed of three domains: a humanized nanobody arm binding the activating receptor CD16 on NK cells, an interleukin (IL)-15 moiety that can drive expansion of NK cells, and a nanobody arm binding B7H3, a protein which high expression can be negatively correlated with overall survival of HPV- HNC patients. B7H3 is a prime target candidate because it is highly expressed on HPV- HNC cells, but minimally expressed on healthy tissues. In vitro testing using HPV- HNC patient blood samples revealed that B7H3 TriKE enhances activation (measured by NK cell degranulation and interferon-gamma production) and expansion of NK cells from these patients at levels equivalent to those observed in healthy controls. Furthermore, B7H3 TriKE is efficacious in its ability to drive high NK cell cytotoxicity in both acute (<48-hours) and prolonged (7-days) hypoxic (1% oxygen) models of HNC. Under acute hypoxia, B7H3 TriKE induces significantly more killing of targets by NK cells compared to IL-15 treatment, where NK cell cytotoxicity is impaired. In addition, B7H3 TriKE can boost the killing efficacy of NK cells exposed to prolonged hypoxia, surpassing limitations seen with IL-15 treatment. These findings strongly suggest that the B7H3 TriKE can bypass hypoxic suppression of NK effector functions in the solid TME. In vivo studies using immunocompromised mice engrafted with HPV- HNC cells revealed that B7H3 TriKE treatment significantly extends the survival of mice, compared to IL-15 treatment. Moreover, NK cells persisted in the blood of B7H3 TriKE-treated mice 28 days post-NK cell injection, highlighting the promising clinical translation of this immunotherapy. More in-depth characterization of NK cells from HPV- HNC patients are underway but altogether, these robust pre-clinical data present a novel avenue for the management of HNC for these patients. We plan to translate results from these studies to clinical trials in fall 2024. Citation Format: Melissa Khaw, Nicholas A. Zorko, Carly Selleck, Laura Bendzick, Zachary Davis, Peter Hinderlie, Madison Shackelford, Ann Lu, James Lim, Naomi Fujioka, Margaret MacMillan, John Wagner, Martin Felices, Jeffrey S. Miller. Enhancing NK cell therapy for head and neck cancer within the solid tumor microenvironment using a B7H3-targeting tri-specific killer engager (TriKE) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1241.

Abstract 5413: In vitro modeling of the migration ability of lung adenocarcinoma cell lines under hypoxic conditions

Abstract Hypoxia is a common condition in the tumor microenvironment that contributes to tumor cell migration, invasion, and metastasis, partly through inducing epithelial-mesenchymal transition (EMT). One of the key proteins in this process is hypoxia-inducible factor 1 (HIF-1), which regulates the expression of various genes as transcription factor and is vital for the cellular response in tissue hypoxia. Several studies have investigated the causation between hypoxic conditions and increased in vitro tumor cell motility and the role of potential regulatory signaling pathways. Understanding the precise molecular mechanisms underlying hypoxia-induced responses is crucial for better targeting tumor progression. However, establishing sound and predictive models is urgently needed to achieve this.In this study, we investigated the migration ability of four non-small cell lung cancer cell lines carrying different mutations of the growth factor pathway (H1975: EGFR T790M, PF901: BRAF V600E, PF139: KRAS G12C mutant, and H838: triple WT) under normoxic, hypoxic, and hypoxia-mimicking CoCl2-treated conditions. The motility of the cells was investigated by 48 h video microscopy (single cell random migration) and 72 h scratch assay. We analyzed the expression of crucial genes and proteins, including HIF-1α, as well as molecules associated with epithelial-mesenchymal transition (vimentin, n-cadherin, and e-cadherin), proliferation (PCNA), and apoptosis (PARP), using Western blot and qPCR techniques. Our work showed no change in cellular motility between any of the cell lines following treatment with CoCl2 compared to normoxic conditions. However, PF139 and PF901 cell lines showed a significant decrease in single-cell motility upon hypoxia. Interestingly, immunoblot analysis revealed stabilization of HIF-1α in response to both CoCl2 and hypoxia in the meantime. Additionally, we observed cell line-dependent changes in EMT marker expression upon CoCl2 treatment and hypoxia. Our work complements previous results published by our group and others on the tumor-promoting role of hypoxia, demonstrating that short-term exposure to hypoxia may act as an acute stressor for tumor cell lines. Our work suggests that the tumor progression-promoting effects are tissue-specific and should be investigated using long-term hypoxia models. Acknowledgments: This work was supported by the Hungarian Thematic Excellence Program (TKP2021-EGA-44) and the National Laboratories Program - National Laboratory for Tumor Biology (NLP-17). Sára Eszter Surguta is a grantee of the ITM-NKFIA funded Cooperative Doctoral Program (1018567). Citation Format: Sára Eszter Surguta, Marcell Baranyi, Laura Svajda, Ivan Ranđelović, Mihály Cserepes, József Tóvári. In vitro modeling of the migration ability of lung adenocarcinoma cell lines under hypoxic conditions [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5413.

Effects of Hypoxia and Reoxygenation on Metabolic Profiles of Cardiomyocytes.

In vitro cellular models provide valuable insights into the adaptive biochemical mechanisms triggered by cells to cope with the stress situation induced by hypoxia and reoxygenation cycles. The first biological data generated in studies based on this micrometric life-scale has the potential to provide us a global overview about the main biochemical phenomena presented in some reported preconditioning therapies in life-scale of higher dimensions. Thus, in this study, a cell incubator was designed and manufactured to produce a cellular model of heart hypoxia followed by reoxygenation (HfR) through consecutive repetitions of hypoxia-normoxia gas exchange. Samples of cellular extracts and culture media were obtained from non-proliferative cardiomyocytes (CMs) cultivated under challenging HfR (stressed CMs) and regular cultivation (unstressed CMs) in rounds of four days for each case. Metabolomic based on proton magnetic resonance spectroscopy (1H-MRS) was used as an analytical approach to identify and quantify the metabolomes of these samples, the endo- and exo-metabolome. Despite the stressed CMs presented over 90% higher cellular death rate compared to the unstressed CMs, the metabolic profiles indicates that the surviving cells up-regulate their amino acid metabolism either by active protein degradation or by the consumption of culture media components to increase coenzyme A-dependent metabolic pathways. This cell auto-regulation mechanism could be well characterized in the first two days when the difference smears off under once the metabolomes become similar. The metabolic adaptations of stressed CMs identified the relevance of the cyclic oxidation/reduction reactions of nicotinamide adenine dinucleotide phosphate molecules, NADP+/NADPH, and the increased tricarboxylic acid cycle activity in an environment overloaded with such a powerful antioxidant agent to survive an extreme HfR challenge. Thus, the combination of cellular models based on CMs, investigative methods, such as metabolomic and 1H-MRS, and the instrumental development of hypoxia incubator shown in this work were able to provide the first biochemical evidences behind therapies of gaseous exchanges paving the way to future assays.

Blockage of αvβ3 integrin in 3D culture of triple-negative breast cancer and endothelial cells inhibits migration and discourages endothelial-to-mesenchymal plasticity

Breast cancer is a relevant cause of mortality in women and its triple-negative subtype (TNBC) is usually associated with poor prognosis. During tumor progression to metastasis, angiogenesis is triggered by the sprouting of endothelial cells from pre-existing vessels by a dynamic chain of events including VE-cadherin downregulation, actin protrusion, and integrin-mediated adhesion, allowing for migration and proliferation. The binding of tumoral and tumor-associated stromal cells with the extracellular matrix through integrins mediates angiogenic processes and certain integrin subtypes, such as the αvβ3 integrin, are upregulated in hypoxic TNBC models. Integrin αvβ3 inhibition by the high-affinity binding disintegrin DisBa-01 was previously demonstrated to induce anti-tumoral and anti-angiogenic responses in traditional 2D cell assays. Here, we investigate the effects of integrin αvβ3 blockage in endothelial and TNBC cells by DisBa-01 in 3D cultures under two oxygen conditions (1% and 20%). 3D cultures created using non-adhesive micromolds with Matrigel were submitted to migration assay in Boyden chambers and fluorescence analysis. DisBa-01 inhibited cell migration in normoxia and hypoxia in both MDA-MB-231 and HUVEC spheroids. Protein levels of integrin αvβ3 were overexpressed in HUVEC spheroids compared to MDA-MB-231 spheroids. In HUVEC 3D cultures, sprouting assays in collagen type I were decreased in normoxia upon DisBa-01 treatment, and VE-cadherin levels were diminished in HUVEC spheroids in hypoxia and upon DisBa-01 treatment. In conclusion, the blockage of integrin αvβ3 by DisBa-01 inhibits cell migration in 3D culture and interferes with tumor-derived responses in different oxygen settings, implicating its crucial role in angiogenesis and tumor progression.

Network pharmacology prediction, molecular docking and in vitro experiment explored the potential mechanism of Gaoyuan'an capsule in improving hypoxia tolerance.

Tibetan medicine Gaoyuan'an capsule (GYAC) is widely used to prevent pulmonary edema at high altitude, but the specific mechanism has not been explored. In this study, we analyzed the mechanism of GYAC in hypoxia tolerance, and provided a new idea for the prevention and treatment of altitude disease. The effective components and corresponding targets of GYAC were screened out by the Chinese herbal medicine network database, and the key targets of hypoxia tolerance were retrieved by Genecards, OMIM and PubMed database. Cytoscape 3.7.2 was used to construct GYAC ingredient-target-hypoxia tolerance-related target network. GO function annotation and KEGG enrichment analysis were performed to predict the pathways in which target genes may be involved, and molecular docking was used to verify the binding ability of the compound to target genes. In vitro, the above results were further verified by molecular experiment. We found that GYAC can improve hypoxia tolerance by regulating various target genes, including IL6, IFNG, etc. The main regulatory pathways were HIF-1 signaling pathway. Molecular docking showed that the affinity between luteolin and target genes (IL6, IFNG) were better. In vitro, we observed that hypoxia can inhibit cell viability and promote apoptosis of H9C2 cell. And hypoxia can promote the expression of LDH. After the addition of luteolin, the decrease of cell viability, the increase of cell apoptosis, LDH release and the decrease of mitochondrial membrane potential were inhibited. Besides, inflammatory related factors (IL-6, IL-10, IL-2, IFNG and VEGFA) expression were also inhibited hypoxic cell models. The results of network pharmacology and molecular docking showed that luteolin, a monomeric component of GYAC, played a role in hypoxia tolerance through a variety of target genes, such as IL6, IFNG. What's more, we have discovered that luteolin can reduce the inflammatory response in cardiac myocytes, thereby alleviating mitochondrial damage, and ultimately enhancing the hypoxia tolerance of H9C2 cardiomyocytes.

Hypoxia-induced circRNAs encoded by PPARA are highly expressed in human cardiomyocytes and are potential clinical biomarkers of acute myocardial infarction

BackgroundAcute myocardial infarction (AMI) is a serious cardiovascular disease that adversely affects human health. Circular RNAs (circRNAs) are involved in the pathological and physiological processes of AMI, but the biological mechanism of their involvement and their clinical significance remain unknown. We aimed to identify circRNAs that are significantly associated with morbidity in the peripheral blood of patients with AMI and evaluate their diagnostic utility.MethodsHigh-throughput sequencing was used to screen for differentially expressed circRNAs in peripheral blood samples obtained from five patients with AMI and five sex- and age-matched healthy controls. A series of bioinformatics tools and databases were used to determine the biological functional classification and pathway enrichment of the circRNAs based on data obtained from sequencing. A hypoxia model was established and used to evaluate the effect of hypoxia on circRNA expression in human cardiomyocytes. A cytoplasmic separation assay and enzyme resistance assay were employed to identify the biological characteristics of circRNA. Polymerase chain reaction validity testing and receiver operating characteristic (ROC) curve analysis were used to evaluate the utility of circRNA assessments in the diagnosis of AMI.ResultsA large number of circRNAs were found to be differentially expressed in the peripheral blood of patients with AMI, and significantly more of these circRNAs were highly expressed than lowly expressed. The genes encoding these circRNAs have a wide range of effects on various functions in the body. A hypoxic environment promoted the upregulation of circRNA expression in human cardiomyocytes, and hsa_circ_0116795 encoded by PPARA was highly expressed in the peripheral blood of the patients with AMI. In terms of biological characteristics, under physiological conditions, hsa_circ_0116795 (circ_PPARA) was mainly located in the cytoplasm of cardiomyocytes and found to be resistant to exonuclease. The ROC curve analysis showed that the expression levels of circ_PPARA in the peripheral blood of patients with AMI were significantly different from those in the peripheral blood of healthy controls.ConclusionA large number of abnormally expressed circRNAs are detectable in the peripheral blood of patients with AMI. In particular, circ_PPARA is highly expressed in human myocardial cells under hypoxic conditions, and its biological characteristics indicate that it could be employed as a biomarker for the early diagnosis of AMI.

Identifying key antioxidative stress factors regulating Nrf2 in the genioglossus with human umbilical cord mesenchymal stem-cell therapy

Intermittent hypoxia in patients with obstructive sleep apnea (OSA) hypopnea syndrome (OSAHS) is associated with pharyngeal cavity collapse during sleep. The effect of human umbilical cord mesenchymal stem cells (HUCMSCs) on OSA-induced oxidative damage in the genioglossus and whether nuclear factor erythroid 2-related factor 2 (Nrf2) or its upstream genes play a key role in this process remains unclear. This study aimed to identify the key factors responsible for oxidative damage during OSAHS through Nrf2 analysis and hypothesize the mechanism of HUCMSC therapy. We simulated OSA using an intermittent hypoxia model, observed the oxidative damage in the genioglossus and changes in Nrf2 expression during intermittent hypoxia, and administered HUCMSCs therapy. Nrf2 initially increased, then decreased, aggravating the oxidative damage in the genioglossus; Nrf2 protein content decreased during hypoxia. Using transcriptomics, we identified seven possible factors in HUCMSCs involved in ameliorating oxidative stress by Nrf2, of which DJ-1 and MEF2A, showing trends similar to Nrf2, were selected by polymerase chain reaction. HUCMSCs may reduce oxidative stress induced by intermittent hypoxia through Nrf2, and the possible upstream target genes in this process are MEF2A and DJ-1. Further studies are needed to verify these findings.

Abstract 1121 Identifying novel cytoplasmic mRNA capping enzyme-targeted, hypoxia-responsive RNAs in hypoxic osteosarcoma cellular model: A plausible therapeutic approach

Abstract 1121 Identifying novel cytoplasmic mRNA capping enzyme-targeted, hypoxia-responsive RNAs in hypoxic osteosarcoma cellular model: A plausible therapeutic approach

HIF-1 contributes to autophagy activation via BNIP3 to facilitate renal fibrosis in hypoxia in vitro and UUO in vivo.

The molecular basis of renal interstitial fibrosis, a major pathological feature of progressive kidney diseases, remains poorly understood. Autophagy has been implicated in renal fibrosis, but whether it promotes or inhibits fibrosis remains controversial. Moreover, it is unclear how autophagy is activated and sustained in renal fibrosis. The present study was designed to address these questions using the in vivo mouse model of unilateral ureteral obstruction and the in vitro model of hypoxia in renal tubular cells. Both models showed the activation of hypoxia-inducible factor-1 (HIF-1) and autophagy along with fibrotic changes. Inhibition of autophagy with chloroquine reduced renal fibrosis in unilateral ureteral obstruction model, whereas chloroquine and autophagy-related gene 7 knockdown decreased fibrotic changes in cultured renal proximal tubular cells, supporting a profibrotic role of autophagy. Notably, pharmacological and genetic inhibition of HIF-1 led to the suppression of autophagy and renal fibrosis in these models. Mechanistically, knock down of BCL2 and adenovirus E1B 19-kDa-interacting protein 3 (BNIP3), a downstream target gene of HIF, decreased autophagy and fibrotic changes during hypoxia in BUMPT cells. Together, these results suggest that HIF-1 may activate autophagy via BNIP3 in renal tubular cells to facilitate the development of renal interstitial fibrosis.NEW & NOTEWORTHY Autophagy has been reported to participate in renal fibrosis, but its role and underlying activation mechanism is unclear. In this study, we report the role of HIF-1 in autophagy activation in models of renal fibrosis and further investigate the underlying mechanism.

Hypoxia-inducible factor-2α promotes fibrosis in non-alcoholic fatty liver disease by enhancing glutamine catabolism and inhibiting yes-associated protein phosphorylation in hepatic stellate cells.

Non-alcoholic fatty liver disease (NAFLD) has a high global prevalence and affects approximately one-third of adults, owing to high-fat dietary habits and a sedentary lifestyle. The role of hypoxia-inducible factor 2α (HIF-2α) in NAFLD progression remains unknown. This study aimed to investigate the effects of chronic hypoxia on NAFLD progression by examining the role of hypoxia-inducible factor 2α (HIF-2α) activation and that of hepatic stellate cell (HSC)-derived myofibroblasts through glutaminolysis. We hypothesised that hypoxia exacerbates NAFLD by promoting HIF-2α upregulation and inhibiting phosphorylated yes-associated protein (YAP), and that increasing YAP expression enhances HSC-derived myofibroblasts. We studied patients with NAFLD living at high altitudes, as well as animal models and cultured cells. The results revealed significant increases in HSC-derived myofibroblasts and collagen accumulation caused by HIF-2α and YAP upregulation, both in patients and in a mouse model for hypoxia and NAFLD. HIF-2α and HIF-2α-dependent YAP downregulation reduced HSC activation and myofibroblast levels in persistent chronic hypoxia. Furthermore, hypoxia-induced HIF-2α upregulation promoted YAP and inhibited YAP phosphorylation, leading to glutaminase 1 (GLS1), SLC38A1, α-SMA, and Collagen-1 overexpression. Additionally, hypoxia restored mitochondrial adenosine triphosphate production and reactive oxygen species (ROS) overproduction. Thus, chronic hypoxia-induced HIF-2α activation enhances fibrosis and NAFLD progression by restoring mitochondrial ROS production and glutaminase-1-induced glutaminolysis, which is mediated through the inhibition of YAP phosphorylation and increased YAP nuclear translocation. In summary, HIF-2α plays a pivotal role in NAFLD progression during chronic hypoxia.

Synthesis, antioxidant and anti-hypoxia activities of 6-hydroxygenistein and its methylated derivatives.

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. 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. 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). 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.

Deciphering Oxygen‐Independent Augmented Photodynamic Oncotherapy by Facilitating the Separation of Electron‐Hole Pairs

AbstractDeveloping Type‐I photosensitizers provides an attractive approach to solve the dilemma of inadequate efficacy of photodynamic therapy (PDT) caused by the inherent oxygen consumption of traditional Type‐II PDT and anoxic tumor microenvironment. The challenge for the exploration of Type‐I PSs is to facilitate the electron transfer ability of photosensitization molecules for transforming oxygen or H2O to reactive oxygen species (ROS). Herein, we propose an electronic acceptor‐triggered photoinduced electron transfer (a‐PET) strategy promoting the separation of electron‐hole pairs by marriage of two organic semiconducting molecules of a non‐fullerene scaffold‐based photosensitizer and a perylene diimide that significantly boost the Type‐I PDT pathway to produce plentiful ROS, especially, inducing 3.5‐fold and 2.5‐fold amplification of hydroxyl (OH⋅) and superoxide (O2−⋅) generation. Systematic mechanism exploration reveals that intermolecular electron transfer and intramolecular charge separation after photoirradiation generate a competent production of radical ion pairs that promote the Type‐I PDT process by theoretical calculation and ultrafast femtosecond transient absorption (fs‐TA) spectroscopy. By complementary tumor diagnosis with photoacoustic imaging and second near‐infrared fluorescence imaging, this as‐prepared nanoplatform exhibits fabulous photocytotoxicity in harsh hypoxic conditions and terrific cancer revoked abilities in living mice. We envision that this work will broaden the insight into high‐efficiency Type‐I PDT for cancer phototheranostics.

Deciphering Oxygen-Independent Augmented Photodynamic Oncotherapy by Facilitating the Separation of Electron-Hole Pairs.

Developing Type-I photosensitizers provides an attractive approach to solve the dilemma of inadequate efficacy of photodynamic therapy (PDT) caused by the inherent oxygen consumption of traditional Type-II PDT and anoxic tumor microenvironment. The challenge for the exploration of Type-I PSs is to facilitate the electron transfer ability of photosensitization molecules for transforming oxygen or H2O to reactive oxygen species (ROS). Herein, we propose an electronic acceptor-triggered photoinduced electron transfer (a-PET) strategy promoting the separation of electron-hole pairs by marriage of two organic semiconducting molecules of a non-fullerene scaffold-based photosensitizer and a perylene diimide that significantly boost the Type-I PDT pathway to produce plentiful ROS, especially, inducing 3.5-fold and 2.5-fold amplification of hydroxyl (OH⋅) and superoxide (O2 -⋅) generation. Systematic mechanism exploration reveals that intermolecular electron transfer and intramolecular charge separation after photoirradiation generate a competent production of radical ion pairs that promote the Type-I PDT process by theoretical calculation and ultrafast femtosecond transient absorption (fs-TA) spectroscopy. By complementary tumor diagnosis with photoacoustic imaging and second near-infrared fluorescence imaging, this as-prepared nanoplatform exhibits fabulous photocytotoxicity in harsh hypoxic conditions and terrific cancer revoked abilities in living mice. We envision that this work will broaden the insight into high-efficiency Type-I PDT for cancer phototheranostics.

In vivo mitochondria-targeted protection against uterine artery vascular dysfunction and remodelling in rodent hypoxic pregnancy.

Gestational hypoxia adversely affects uterine artery function, increasing complications. However, an effective therapy remains unidentified. Here, we show in rodent uterine arteries that hypoxic pregnancy promotes hypertrophic remodelling, increases constrictor reactivity via protein kinase C signalling, and triggers compensatory dilatation via nitric oxide-dependent mechanisms and stimulation of large conductance Ca2+ -activated K+ -channels. Maternal in vivo oral treatment with the mitochondria-targeted antioxidant MitoQ in hypoxic pregnancy normalises uterine artery reactivity and prevents vascular remodelling. From days 6-20 of gestation (term ∼22 days), female Wistar rats were randomly assigned to normoxic or hypoxic (13-14% O2 ) pregnancy±daily maternal MitoQ treatment (500 µm in drinking water). At 20 days of gestation, maternal, placental and fetal tissue was frozen to determine MitoQ uptake. The uterine arteries were harvested and, in one segment, constrictor and dilator reactivity was determined by wire myography. Another segment was fixed for unbiased stereological analysis of vessel morphology. Maternal administration of MitoQ in both normoxic and hypoxic pregnancy crossed the placenta and was present in all tissues analysed. Hypoxia increased uterine artery constrictor responses to norepinephrine, angiotensin II and the protein kinase C activator, phorbol 12,13-dibutyrate. Hypoxia enhanced dilator reactivity to sodium nitroprusside, the large conductance Ca2+ -activated K+ -channel activator NS1619 and ACh via increased nitric oxide-dependent mechanisms. Uterine arteries from hypoxic pregnancy showed increased wall thickness and MitoQ treatment in hypoxic pregnancy prevented all effects on uterine artery reactivity and remodelling. The data support mitochondria-targeted therapy against adverse changes in uterine artery structure and function in high-risk pregnancy. KEY POINTS: Dysfunction and remodelling of the uterine artery are strongly implicated in many pregnancy complications, including advanced maternal age, maternal hypertension of pregnancy, maternal obesity, gestational diabetes and pregnancy at high altitude. Such complications not only have immediate adverse effects on the growth of the fetus, but also they can also increase the risk of cardiovascular disease in the mother and offspring. Despite this, there is a significant unmet clinical need for therapeutics that treat uterine artery vascular dysfunction in adverse pregnancy. Here, we show in a rodent model of gestational hypoxia that in vivo oral treatment of the mitochondria-targeted antioxidant MitoQ protects against uterine artery vascular dysfunction and remodelling, supporting the use of mitochondria-targeted therapy against adverse changes in uterine artery structure and function in high-risk pregnancy.

Shen-Shuai-II-Recipe inhibits tubular inflammation by PPARα-mediated fatty acid oxidation to attenuate fibroblast activation in fibrotic kidneys

BackgroundShen Shuai Ⅱ Recipe (SSR) is clinically used to treat chronic kidney diseases (CKDs) with remarkable efficacy and safety. In earlier research, we found the anti-inflammatory, antioxidant, and mitochondrial protective properties of SSR in hypoxic kidney injury model, which is closely related to its renal protection. Further work is needed to understand the underlying molecular mechanisms. PurposeFurther investigation of the mechanisms of action of SSR against renal interstitial fibrosis (RIF) building on previous research leads. MethodsRats receiving CKD model surgery were given with Fenofibrate or SSR once a day for eight weeks. In vitro, the NRK-52E cells were treated with SSR in the presence or absence of 10 μM Sc75741, 0.5 μM PMA, or 1 μM fenofibrate under 1% O2. The effects of SSR on NF-κB/NLRP3 inflammatory cascade, secretion of pro-inflammatory cytokines, fatty acid oxidation (FAO), and renal tubular injury were determined by immunoblotting, luminex liquid suspension chip assay, transmission electron microscopy, and Oil red O staining. Next, we delivered PPARα-interfering sequences to kidney tissue and NRK-52E cells by adeno-associated virus (AAV) injection and siRNA transfection methods. Finally, we evaluated the effect of renal tubular cells on fibroblast activation by co-culture method. ResultsSSR attenuated the release of IL-18, VEGF, and MCP1 cytokines, inhibited the activation of NF-κB/NLRP3 cascade, increased the PPARα, CPT-1α, CPT-2, ACADL, and MCAD protein expression, and improved the lipid accumulation. Further studies have demonstrated that one of the ways in which SSR suppresses the inflammatory response to protect renal tubular cells is through the restoration of PPARα-mediated FAO. In addition, by means of co-culture ways, the results demonstrated that SSR attenuated secretion of inflammatory mediators in NRK-52E cells by PPARα/NF-κB/NLRP3 pathway, thereby inhibiting renal fibroblast activation. ConclusionSSR inhibits RIF by suppressing inflammatory response of hypoxia-exposed RTECs through PPARα-mediated FAO.

Innovative in vivo rat model for global cerebral hypoxia: a new approach to investigate therapeutic and preventive drugs.

Introduction: Severe acute global cerebral hypoxia can lead to significant disability in humans. Although different animal models have been described to study hypoxia, there is no endogenous model that considers hypoxia and its effect on the brain as an independent factor. Thus, we developed a minimally invasive rat model, which is based on the non-depolarizing muscle blocking agent rocuronium in anesthetized animals. This drug causes respiratory insufficiency by paralysis of the striated muscles. Methods: In this study, 14 rats underwent 12min of hypoxemia with an oxygen saturation of approximately 60% measured by pulse oximetry; thereafter, animals obtained sugammadex to antagonize rocuronium immediately. Results: Compared to controls (14 rats, anesthesia only), hypoxic animals demonstrated significant morphological alterations in the hippocampus (cell decrease in the CA 1 region) and the cerebellum (Purkinje cell decrease), as well as significant changes in hypoxia markers in blood (Hif2α, Il1β, Tgf1β, Tnfα, S100b, cspg2, neuron-specific enolase), hippocampus (Il1β, Tnfα, S100b, cspg2, NSE), and cerebellum (Hif1α, Tnfα, S100b, cspg2, NSE). Effects were more pronounced in females than in males. Discussion: Consequently, this model is suitable to induce hypoxemia with consecutive global cerebral hypoxia. As significant morphological and biochemical changes were proven, it can be used to investigate therapeutic and preventive drugs for global cerebral hypoxia.

Chronic hypoxia impairs skeletal muscle repair via HIF-2α stabilization.

Chronic hypoxia and skeletal muscle atrophy commonly coexist in patients with COPD and CHF, yet the underlying physio-pathological mechanisms remain elusive. Muscle regeneration, driven by muscle stem cells (MuSCs), holds therapeutic potential for mitigating muscle atrophy. This study endeavours to investigate the influence of chronic hypoxia on muscle regeneration, unravel key molecular mechanisms, and explore potential therapeutic interventions. Experimental mice were exposed to prolonged normobaric hypoxic air (15% pO2, 1atm, 2weeks) to establish a chronic hypoxia model. The impact of chronic hypoxia on body composition, muscle mass, muscle strength, and the expression levels of hypoxia-inducible factors HIF-1α and HIF-2α in MuSC was examined. The influence of chronic hypoxia on muscle regeneration, MuSC proliferation, and the recovery of muscle mass and strength following cardiotoxin-induced injury were assessed. The muscle regeneration capacities under chronic hypoxia were compared between wildtype mice, MuSC-specific HIF-2α knockout mice, and mice treated with HIF-2α inhibitor PT2385, and angiotensin converting enzyme (ACE) inhibitor lisinopril. Transcriptomic analysis was performed to identify hypoxia- and HIF-2α-dependent molecular mechanisms. Statistical significance was determined using analysis of variance (ANOVA) and Mann-Whitney U tests. Chronic hypoxia led to limb muscle atrophy (EDL: 17.7%, P<0.001; Soleus: 11.5% reduction in weight, P<0.001) and weakness (10.0% reduction in peak-isometric torque, P<0.001), along with impaired muscle regeneration characterized by diminished myofibre cross-sectional areas, increased fibrosis (P<0.001), and incomplete strength recovery (92.3% of pre-injury levels, P<0.05). HIF-2α stabilization in MuSC under chronic hypoxia hindered MuSC proliferation (26.1% reduction of MuSC at 10 dpi, P<0.01). HIF-2α ablation in MuSC mitigated the adverse effects of chronic hypoxia on muscle regeneration and MuSC proliferation (30.9% increase in MuSC numbers at 10 dpi, P<0.01), while HIF-1α ablation did not have the same effect. HIF-2α stabilization under chronic hypoxia led to elevated local ACE, a novel direct target of HIF-2α. Notably, pharmacological interventions with PT2385 or lisinopril enhanced muscle regeneration under chronic hypoxia (PT2385: 81.3% increase, P<0.001; lisinopril: 34.6% increase in MuSC numbers at 10 dpi, P<0.05), suggesting their therapeutic potential for alleviating chronic hypoxia-associated muscle atrophy. Chronic hypoxia detrimentally affects skeletal muscle regeneration by stabilizing HIF-2α in MuSC and thereby diminishing MuSC proliferation. HIF-2α increases local ACE levels in skeletal muscle, contributing to hypoxia-induced regenerative deficits. Administration of HIF-2α or ACE inhibitors may prove beneficial to ameliorate chronic hypoxia-associated muscle atrophy and weakness by improving muscle regeneration under chronic hypoxia.

Comparison and discussion of behavior and pathology of four kinds of cerebral palsy disease models.

Explore the differences in behavioral and pathological manifestations of rat models of cerebral palsy made by different methods and discuss what types of studies these models are suitable for. Behavioral evaluation and pathological section observation were used to observe and evaluate the model. Conclusion: except for the absence of data of bilateral common carotid artery ligation rats, the other three methods could all achieve a successful cerebral palsy disease model for both behavioral and pathological. For researchers, the selection of intraperitoneal infection model in pregnant rats or unilateral ischemia and hypoxia model in infant rats is sufficient to meet the experimental needs, whereas the selection of the combined method for modeling does not show enough advantages, which not only causes the waste of financial and human resources but also increases the possibility of experimental error made by intervention factors.

Low Fetal Resistance to Hypoxia as a Cause of Stillbirth and Neonatal Encephalopathy

Objective: Low fetal resistance to hypoxia is a factor in stillbirth and neonatal encephalopathy. This review examines fetal movement patterns in response to hypoxia as a predictor of the likelihood of stillbirth. Monitoring the dynamics of fetal movements during maternal apnea could allow the assessment of fetal resistance to hypoxia. The goal of this study is to describe the practical application of this method by doctors and pregnant women. Mechanism: We searched relevant keywords in the international scientific literature databases Scopus and Web of Science, as well as databases for patents granted in China, India, USA, Japan, Germany, Russia and other countries. Devices, drugs and medical technologies that provide diagnosis, modeling, prevention and treatment of intrauterine fetal hypoxia, stillbirth and neonatal encephalopathy were considered. Findings in Brief: During apnea by a pregnant woman in the second half of normal pregnancy, if the maximum duration of fetal immobility exceeds 30 seconds from the onset of breath-holding, then the fetus is considered to show good resistance to hypoxia, thus preserving its health and life during vaginal delivery. On the other hand, excessive fetal movements &lt;10 seconds after the onset of apnea in a pregnant woman indicates low fetal resistance to hypoxia. When fetal resistance to hypoxia is low, there is no alternative to immediate cesarean section for the preservation of fetal life and health. Conclusions: The monitoring of fetal movements during apnea in pregnant women allows real-time assessment of fetal resistance to intrauterine hypoxia. Obtaining timely information on fetal resistance to hypoxia is critical for determining the optimal timing and type of delivery in order to prevent encephalopathy and stillbirth.