Shen Shuai II recipe improves renal hypoxia to attenuate renal injury in 5/6 renal ablation/infarction rats and effect evaluation using blood oxygenation level-dependent functional magnetic resonance imaging.

Background: Renal hypoxia plays a key role in the progression of chronic kidney disease (CKD). Shen Shuai II Recipe (SSR) has shown good results in the treatment of CKD as a common herbal formula. This study aimed to explore the effect of SSR on renal hypoxia and injury in CKD rats. Methods: Twenty-five Wistar rats underwent 5/6 renal ablation/infarction (A/I) surgery were randomly divided into three groups: 5/6 (A/I), 5/6 (A/I) + losartan (LOS), and 5/6 (A/I) + SSR groups. Another eight normal rats were used as the Sham group. After 8-week corresponding interventions, blood oxygenation level-dependent functional magnetic resonance imaging (BOLD-fMRI) was performed to evaluate renal oxygenation in all rats, and biochemical indicators were used to measure kidney and liver function, hemoglobin, and proteinuria. The expression of fibrosis and hypoxia-related proteins was analyzed using immunoblotting examination. Results: Renal oxygenation, evaluated by BOLD-fMRI as cortical and medullary T2* values (COT2* and MET2*), was decreased in 5/6 (A/I) rats, but increased after SSR treatment. SSR also downregulated the expression of hypoxia-inducible factor-1α (HIF-1α) in 5/6 (A/I) kidneys. With the improvement of renal hypoxia, renal function and fibrosis were improved in 5/6 (A/I) rats, accompanied by reduced proteinuria. Furthermore, the COT2* and MET2* were significantly positively correlated with the levels of creatinine clearance rate (Ccr) and hemoglobin, but negatively associated with the levels of serum creatinine (SCr), blood urea nitrogen (BUN), serum cystatin C (CysC), serum uric acid (UA), 24-h urinary protein (24-h Upr), and urinary albumin:creatinine ratio (UACR). Conclusion: The degree of renal oxygenation reduction is correlated with the severity of renal injury in CKD. SSR can improve renal hypoxia to attenuate renal injury in 5/6 (A/I) rats of CKD.

IL-6/gp130 signaling in CD4+ T cells drives the pathogenesis of pulmonary hypertension.

Pulmonary arterial hypertension (PAH) is characterized by stenosis and occlusions of small pulmonary arteries, leading to elevated pulmonary arterial pressure and right heart failure. Although accumulating evidence shows the importance of interleukin (IL)-6 in the pathogenesis of PAH, the target cells of IL-6 are poorly understood. Using mice harboring the floxed allele of gp130, a subunit of the IL-6 receptor, we found substantial Cre recombination in all hematopoietic cell lineages from the primitive hematopoietic stem cell level in SM22α-Cre mice. We also revealed that a CD4+ cell-specific gp130 deletion ameliorated the phenotype of hypoxia-induced pulmonary hypertension in mice. Disruption of IL-6 signaling via deletion of gp130 in CD4+ T cells inhibited phosphorylation of signal transducer and activator of transcription 3 (STAT3) and suppressed the hypoxia-induced increase in T helper 17 cells. To further examine the role of IL-6/gp130 signaling in more severe PH models, we developed Il6 knockout (KO) rats using the CRISPR/Cas9 system and showed that IL-6 deficiency could improve the pathophysiology in hypoxia-, monocrotaline-, and Sugen5416/hypoxia (SuHx)-induced rat PH models. Phosphorylation of STAT3 in CD4+ cells was also observed around the vascular lesions in the lungs of the SuHx rat model, but not in Il6 KO rats. Blockade of IL-6 signaling had an additive effect on conventional PAH therapeutics, such as endothelin receptor antagonist (macitentan) and soluble guanylyl cyclase stimulator (BAY41-2272). These findings suggest that IL-6/gp130 signaling in CD4+ cells plays a critical role in the pathogenesis of PAH.

Morusin suppresses the stemness characteristics of gastric cancer cells induced by hypoxic microenvironment through inhibition of HIF-1α accumulation.

Gastric cancer (GC) is a common, life-threatening malignancy that contributes to the global burden of cancer-related mortality, as conventional therapeutic modalities show limited effects on GC. Hence, it is critical to develop novel agents for GC therapy. Morusin, a typical prenylated flavonoid, possesses antitumor effects against various cancers. The present study aimed to demonstrate the inhibitory effect and mechanism of morusin on the stemness characteristics of human GC in vitro under hypoxia and to explore the potential molecular mechanisms. The effects of morusin on cell proliferation and cancer stem cell-like properties of the human GC cell lines SNU-1 and AGS were assessed by MTT assay, colony formation test, qRT-PCR, flow cytometry analysis, and sphere formation test under hypoxia or normoxia condition through in vitro assays. The potential molecular mechanisms underlying the effects of morusin on the stem-cell-like properties of human GC cells in vitro were investigated by qRT-PCR, western blotting assay, and immunofluorescence assay by evaluating the nuclear translocation and expression level of hypoxia-inducible factor-1α (HIF-1α). The results showed that morusin exerted growth inhibitory effects on SNU-1 and AGS cells under hypoxia in vitro. Moreover, the proportions of CD44+/CD24- cells and the sphere formation ability of SNU-1 and AGS reduced in a dose-dependent manner following morusin treatment. The expression levels of stem cell-related genes, namely Nanog, OCT4, SOX2, and HIF-1α, gradually decreased, and the nuclear translocation of the HIF-1α protein was apparently attenuated. HIF-1α overexpression partially reversed the abovementioned effects of morusin. Taken together, morusin could restrain stemness characteristics of GC cells by inhibiting HIF-1α accumulation and nuclear translocation and could serve as a promising compound for GC treatment.

Equivalent uniform aerobic dose in radiotherapy for hypoxic tumors.

Objective.Equivalent uniform aerobic dose (EUAD) is proposed for comparison of integrated cell survival in tumors with different distributions of hypoxia and radiation dose.Approach.The EUAD assumes that for any non-uniform distributions of radiation dose and oxygen enhancement ratio (OER) within a tumor, there is a uniform distribution of radiation dose under hypothetical aerobic conditions with OER = 1 that produces equal integrated survival of clonogenic cells. This definition of EUAD has several advantages. First, the EUAD allows one to compare survival of clonogenic cells in tumors with intra-tumor and inter-tumor variation of radio sensitivity due to hypoxia because the cell survival is recomputed under the same benchmark oxygen level (OER = 1). Second, the EUAD for homogeneously oxygenated tumors is equal to the concept of equivalent uniform dose.Main results. We computed the EUAD using radiotherapy dose and the OER derived from the18F-Fluoromisonidazole PET (18F-FMISO PET) images of hypoxia in patients with glioblastoma, the most common and aggressive type of primary malignant brain tumor. The18F-FMISO PET images include a distribution of SUV (Standardized Uptake Value); therefore, the SUV is converted to partial oxygen pressure (pO2) and then to the OER. The prognostic value of EUAD in radiotherapy for hypoxic tumors is demonstrated using correlation between EUAD and overall survival (OS) in radiotherapy for glioblastoma. The correction to the EUAD for the absolute hypoxic volume that traceable to the tumor control probability improves the correlation with OS.Significance. While the analysis proposed in this research is based on the18F-FMISO PET images for glioblastoma, the EUAD is a universal radiobiological concept and is not associated with any specific cancer or any specific PET or MRI biomarker of hypoxia. Therefore, this research can be generalized to other cancers, for example stage III lung cancer, and to other hypoxia biomarkers.

RAS protein activator-like 2 (RASAL2) initiates peritubular capillary rarefaction in hypoxic renal interstitial fibrosis.

The progression of chronic kidney disease (CKD) often involves renal interstitial fibrosis (RIF) and subsequent loss of peritubular capillaries (PTCs), which enhances disease severity. Despite advancements in our understanding of fibrosis, effective interventions for reversing capillary loss remain elusive. Notably, RIF exhibits reduced capillary density, whereas renal cell carcinoma (RCC) shows robust angiogenesis under hypoxic conditions. Using RNA sequencing and bioinformatics, we identified differentially expressed genes (DEGs) in hypoxic human renal tubular epithelial cells (HK-2) and renal cancer cells (786-0). Analysis of altered Ras and PI3K/Akt pathways coupled with hub gene investigation revealed RAS protein activator-like 2 (RASAL2) as a key candidate. Subsequent in vitro and in vivo studies confirmed RASAL2's early-stage response in RIF, which reduced with fibrosis progression. RASAL2 suppression in HK-2 cells enhanced angiogenesis, as evidenced by increased proliferation, migration, and branching of human umbilical vein endothelial cells (HUVECs) co-cultured with HK-2 cells. In mice, RASAL2 knockdown improved Vascular endothelial growth factor A (VEGFA) and Proliferating cell nuclear antigen (PCNA) levels in unilateral ureteral occlusion (UUO)-induced fibrosis (compared to wild type). Hypoxia-inducible factor 1 alpha (HIF-1α) emerged as a pivotal mediator, substantiated by chromatin immunoprecipitation (ChIP) sequencing, with its induction linked to activation. Hypoxia increased the production of RASAL2-enriched extracellular vesicles (EVs) derived from tubular cells, which were internalized by endothelial cells, contributing to the exacerbation of PTC loss. These findings underscore RASAL2's role in mediating reduced angiogenesis in RIF and reveal a novel EV-mediated communication between hypoxic tubular- and endothelial cells, demonstrating a complex interplay between angiogenesis and fibrosis in CKD pathogenesis.

Infertility in Fabry's Disease: role of hypoxia and inflammation in determining testicular damage.

Introduction: Fabry's disease (FD) is a genetic X-linked systemic and progressive rare disease characterized by the accumulation of globotriaosylceramide (GB3) into the lysosomes of many tissues. FD is due to loss-of-function mutations of α-galactosidase, a key-enzyme for lysosomal catabolism of glycosphingolipids, which accumulate as glycolipid bodies (GB). In homozygous males the progressive deposition of GB3 into the cells leads to clinical symptoms in CNS, skin, kidney, etc. In testis GB accumulation causes infertility and alterations of spermatogenesis. However, the precise damaging mechanism is still unknown. Our hypothesis is that GB accumulation reduces blood vessel lumen and increases the distance of vessels from both stromal cells and seminiferous parenchyma; this, in turn, impairs oxygen and nutrients diffusion leading to subcellular degradation of seminiferous epithelium and sterility. Methods: To test this hypothesis, we have studied a 42-year-old patient presenting a severe FD and infertility, with reduced number of spermatozoa, but preserved sexual activity. Testicular biopsies were analyzed by optical (OM) and transmission electron microscopy (TEM). Activation and cellular localization of HIF-1α and NFκB was analyzed by immunofluorescence (IF) and RT-PCR on homogeneous tissue fractions after laser capture microdissection (LCMD). Results: OM and TEM showed that GB were abundant in vessel wall cells and in interstitial cells. By contrast, GB were absent in seminiferous epithelium, Sertoli's and Leydig's cells. However, seminiferous tubular epithelium and Sertoli's cells showed reduced diameter, thickening of basement membrane and tunica propria, and swollen or degenerated spermatogonia. IF showed an accumulation of HIF-1α in stromal cells but not in seminiferous tubules. On the contrary, NFκB fluorescence was evident in tubules, but very low in interstitial cells. Finally, RT-PCR analysis on LCMD fractions showed the expression of pro-inflammatory genes connected to the HIF-1α/NFκB inflammatory-like pathway. Conclusion: Our study demonstrates that infertility in FD may be caused by reduced oxygen and nutrients due to GB accumulation in blood vessels cells. Reduced oxygen and nutrients alter HIF-1α/NFκB expression and localization while activating HIF-1α/NFκB driven-inflammation-like response damaging seminiferous tubular epithelium and Sertoli's cells.

WSB1, a Hypoxia-Inducible E3 Ligase, Promotes Myofibroblast Accumulation and Attenuates Alveolar Epithelial Regeneration in Mouse Lung Fibrosis.

Idiopathic pulmonary fibrosis is a progressive interstitial lung disease for which there is no curative therapy available. Repetitive alveolar epithelial injury repair, myofibroblast accumulation, and excessive collagen deposition are key pathologic features of idiopathic pulmonary fibrosis, eventually leading to cellular hypoxia and respiratory failure. The precise mechanism driving this complex maladaptive process remains inadequately understood. WD repeat and suppressor of cytokine signaling box containing 1 (WSB1) is an E3 ubiquitin ligase, the expression of which is associated strongly with hypoxia, and forms a positive feedback loop with hypoxia-inducible factor 1α (HIF-1α) under anoxic condition. This study explored the expression, cellular distribution, and function of WSB1 in bleomycin (BLM)-induced mouse lung injury and fibrosis. WSB1 expression was highly induced by BLM injury and correlated with the progression of lung fibrosis. Significantly, conditional deletion of Wsb1 in adult mice ameliorated BLM-induced pulmonary fibrosis. Phenotypically, Wsb1-deficient mice showed reduced lipofibroblast to myofibroblast transition, but enhanced alveolar type 2 proliferation and differentiation into alveolar type 1 after BLM injury. Proteomic analysis of mouse lung tissues identified caveolin 2 as a potential downstream target of WSB1, contributing to BLM-induced epithelial injury repair and fibrosis. These findings unravel a vital role for WSB1 induction in lung injury repair, thus highlighting it as a potential therapeutic target for pulmonary fibrosis.

Hypoxia-adenosine axis as therapeutic targets for acute respiratory distress syndrome.

The human respiratory and circulatory systems collaborate intricately to ensure oxygen delivery to all cells, which is vital for ATP production and maintaining physiological functions and structures. During limited oxygen availability, hypoxia-inducible factors (HIFs) are stabilized and play a fundamental role in maintaining cellular processes for hypoxia adaptation. First discovered during investigations of erythropoietin production regulation, HIFs influence physiological and pathological processes, including development, inflammation, wound healing, and cancer. HIFs promote extracellular adenosine signaling by enhancing adenosine generation and receptor signaling, representing an endogenous feedback mechanism that curbs excessive inflammation, supports injury resolution, and enhances hypoxia tolerance. This is especially important for conditions that involve tissue hypoxia, such as acute respiratory distress syndrome (ARDS), which globally poses significant health challenges without specific treatment options. Consequently, pharmacological strategies to amplify HIF-mediated adenosine production and receptor signaling are of great importance.

Secretome of Hypoxia-Preconditioned Mesenchymal Stem Cells Promotes Liver Regeneration and Anti-Fibrotic Effect in Liver Fibrosis Animal Model.

<b>Background and Objective:</b> Liver fibrosis (LF) is a most common pathological process characterized by the activation of hepatocytes leading to the accumulation of extracellular matrix (ECM). Hypoxia precondition treated in MSCs (H-MSCs) could enhance their immunomodulatory and regeneration capability, through expressing robust anti-inflammatory cytokines and growth factors, known as H-MSCs secretome (SH-MSCs) that are critical for the improvement of liver fibrosis. However, the study regarding the efficacy and mechanism of action of SH-MSCs in ameliorating liver fibrosis is still inconclusive. In this study, the therapeutic potential and underlying mechanism for SH-MSCs in the treatment of liver fibrosis were investigated. <b>Materials and Methods:</b> A rat model with liver fibrosis induced by CCl₄ was created and maintained for 8 weeks. The rats received intravenous doses of SH-MSCs and secretome derived from normoxia MSCs (SN-MSCs), filtered using a tangential flow filtration (TFF) system with different molecular weight cut-off categories, both at a dosage of 0.5 mL. The ELISA assay was employed to examine the cytokines and growth factors present in both SH-MSCs and SN-MSCs. On the ninth day, the rats were euthanized and liver tissues were collected for subsequent histological examination and analysis of mRNA expression. <b>Results:</b> The ELISA test revealed that SH-MSCs exhibited higher levels of VEGF, PDGF, bFGF, IL-10, TGF-ß and IL-6 compared to SN-MSCs. <i>In vivo</i>, administration of SH-MSCs notably decreased mortality rates. It also demonstrated a reduction in liver fibrosis, collagen fiber areas, α-SMA positive staining and relative mRNA expression of TGF-ß. Conversely, SN-MSCs also contributed to liver fibrosis improvement, although SH-MSCs demonstrated more favorable outcomes. <b>Conclusion:</b> Current findings suggested that SH-MSCs could improve CCl₄-induced liver fibrosis and decrease α-SMA and TGF-ß expression.

Strategies to Reverse Hypoxic Tumor Microenvironment for Enhanced Sonodynamic Therapy.

Sonodynamic therapy (SDT) has emerged as a highly effective modality for the treatment of malignant tumors owing to its powerful penetration ability, noninvasiveness, site-confined irradiation, and excellent therapeutic efficacy. However, the traditional SDT, which relies on oxygen availability, often fails to generate a satisfactory level of reactive oxygen species because of the widespread issue of hypoxia in the tumor microenvironment of solid tumors. To address this challenge, various approaches are developed to alleviate hypoxia and improve the efficiency of SDT. These strategies aim to either increase oxygen supply or prevent hypoxia exacerbation, thereby enhancing the effectiveness of SDT. In view of this, the current review provides an overview of these strategies and their underlying principles, focusing on the circulation of oxygen from consumption to external supply. The detailed research examples conducted using these strategies in combination with SDT are also discussed. Additionally, this review highlights the future prospects and challenges of the hypoxia-alleviated SDT, along with the key considerations for future clinical applications. These considerations include the development of efficient oxygen delivery systems, the accurate methods for hypoxia detection, and the exploration of combination therapies to optimize SDT outcomes.

Hypoxia-Induced Signaling in Gut and Liver Pathobiology.

Oxygen (O2) is essential for cellular metabolism and biochemical reactions. When the demand for O2 exceeds the supply, hypoxia occurs. Hypoxia-inducible factors (HIFs) are essential to activate adaptive and survival responses following hypoxic stress. In the gut (intestines) and liver, the presence of oxygen gradients or physiologic hypoxia is necessary to maintain normal homeostasis. While physiologic hypoxia is beneficial and aids in normal functions, pathological hypoxia is harmful as it exacerbates inflammatory responses and tissue dysfunction and is a hallmark of many cancers. In this review, we discuss the role of gut and liver hypoxia-induced signaling, primarily focusing on HIFs, in the physiology and pathobiology of gut and liver diseases. Additionally, we examine the function of HIFs in various cell types during gut and liver diseases, beyond intestinal epithelial and hepatocyte HIFs. This review highlights the importance of understanding hypoxia-induced signaling in the pathogenesis of gut and liver diseases and emphasizes the potential of HIFs as therapeutic targets.

TAM-Hijacked Immunoreaction Rescued by Hypoxia-Pathway-Intervened Strategy for Enhanced Metastatic Cancer Immunotherapy.

Immunotherapy is regarded as a prospective strategy against metastatic cancer. However, tumor-associated macrophages (TAMs), which accumulate in hypoxic tumor microenvironment, reduce the effectiveness of immunotherapy by blocking or "hijacking" the initiation of the immune response. Here, a novel tumor-targeted nanoplatform loaded with hypoxia-pathway-intervened docosahexaenoic acid (DHA) and chemotherapeutic drug carfilzomib (CFZ) is developed, which realizes the rescue of TAM-hijacked immune response and effective metastatic cancer immunotherapy. DHA is conjugated to fucoidan (Fuc) via a reduction cleavable selenylsulfide bond (SSe) for micelle preparation, and CFZ is encapsulated in the hydrophobic cores of micelles. The functionalized nanoplatforms (Fuc─SSe─DHA (FSSeD)-CFZs) induce immunogenic cell death, inhibit hypoxia-inducible factor-1α expression, and improve immunosuppression by TAM suppression. FSSeD-CFZs enhance immune response against primary tumor development and metastasis formation. In brief, the novel rescue strategy for TAM-hijacked immunoreaction by inhibiting hypoxia pathway has the potential and clinically translational significance for enhanced metastatic cancer immunotherapy.

Cobalt Chloride as a Hypoxia Mimicking Agent Induced HIF-1α and mTOR Expressions of Human Umbilical Cord Mesenchymal Stem Cells

ABSTRACT Objective: To assess the effects of cobalt chloride (CoCl2) as a hypoxia mimicking agent on human umbilical cord mesenchymal stem cells (hUCMSCs) expression of HIF-1α and mTOR for use in regenerative dentistry. Material and Methods: Human umbilical cord mesenchymal stem cells were isolated and then cultured. The characteristics of stemness were screened and confirmed by flow cytometry. The experiment was conducted on hypoxia (H) and normoxia (N) groups. Each group was divided and incubated into 24-, 48-, and 72-hours observations. Hypoxic treatment was performed using 100 µM CoCl2 on 5th passage cells in a conventional incubator (37°C; 5CO2). Then, immunofluorescence of HIF-1α and mTOR was done. Data was analyzed statistically using One-way ANOVA and Tukey's HSD. Results: Significant differences were found between normoxic and hypoxic groups on HIF-1α (p=0.015) and mTOR (p=0.000) expressions. The highest HIF-1α expression was found at 48 hours in the hypoxia group, while for mTOR at 24 hours in the hypoxia group. Conclusion: Hypoxia using cobalt chloride was able to increase human umbilical cord mesenchymal stem cells expression of HIF-1α and mTOR.

ADAR1 regulates macrophage polarization and is protective against liver ischemia and reperfusion injury.

Liver ischemia and reperfusion injury (LIRI) is a major risk for the poor prognosis of patients receiving liver transplantation. The molecular mechanism involved in LIRI is complex and related to various cellular components. We previously reported that adenosine deaminase acting on RNA 1 (ADAR1) alleviated the allogeneic skin graft rejection by regulating macrophage polarization. However, the regulatory effects of ADAR1 on liver macrophages after LIRI remain largely unknown. In this study, we mainly adopted a mouse model of LIRI and cellular experiments with hypoxia and reoxygenation (HR) treatment to explore the regulatory roles of ADAR1 on liver macrophages under LIRI conditions. We found that IRI caused decreased ADAR1 in liver tissues and remarkable changes of liver macrophage polarization and profiles. ADAR1 supplementation alleviated the pathological injury caused by IRI and accelerated the activation of M2 macrophages in the liver of IRI mice. Increased hypoxia duration reduced ADAR1 expression levels in murine RAW264.7 macrophages at the transcriptional level. Further overexpression of ADAR1 significantly increased the expressions of anti-inflammatory cytokines and promoted M2 polarization of macrophages under HR exposure. ADAR1 knockdown exhibited opposite effects on macrophage polarization. Hence, ADAR1 promotes the M2 polarization of liver macrophages that may further alleviate LIRI. The protective effects of ADAR1 against LIRI provide a novel insight into the prevention and treatment of LIRI.

Phenotypic Alterations in Erythroid Nucleated Cells of Spleen and Bone Marrow in Acute Hypoxia.

Hypoxia leads to metabolic changes at the cellular, tissue, and organismal levels. The molecular mechanisms for controlling physiological changes during hypoxia have not yet been fully studied. Erythroid cells are essential for adjusting the rate of erythropoiesis and can influence the development and differentiation of immune cells under normal and pathological conditions. We simulated high-altitude hypoxia conditions for mice and assessed the content of erythroid nucleated cells in the spleen and bone marrow under the existing microenvironment. For a pure population of CD71+ erythroid cells, we assessed the production of cytokines and the expression of genes that regulate the immune response. Our findings show changes in the cellular composition of the bone marrow and spleen during hypoxia, as well as changes in the composition of the erythroid cell subpopulations during acute hypoxic exposure in the form of a decrease in orthochromatophilic erythroid cells that are ready for rapid enucleation and the accumulation of their precursors. Cytokine production normally differs only between organs; this effect persists during hypoxia. In the bone marrow, during hypoxia, genes of the C-lectin pathway are activated. Thus, hypoxia triggers the activation of various adaptive and compensatory mechanisms in order to limit inflammatory processes and modify metabolism.

Hypoxia-inducible factor 1α modulates interstitial pneumonia-mediated lung cancer progression.

BACKGROUND: The prognosis of patients with lung cancer accompanied by interstitial pneumonia is poorer than that of patients with lung cancer but without interstitial pneumonia. Moreover, the available therapeutic interventions for lung cancer patients with interstitial pneumonia are limited. Therefore, a new treatment strategy for these patients is required. The aim of the present study was to investigate the pathophysiological relationship between interstitial pneumonia and lung cancer and explore potential therapeutic agents. METHODS: A novel hybrid murine model of lung cancer with interstitial pneumonia was established via bleomycin-induced pulmonary fibrosis followed by orthotopic lung cancer cell transplantation into the lungs. Changes in tumor progression, lung fibrosis, RNA expression, cytokine levels, and tumor microenvironment in the lung cancer with interstitial pneumonia model were investigated, and therapeutic agents were examined. Additionally, clinical data and samples from patients with lung cancer accompanied by interstitial pneumonia were analyzed to explore the potential clinical significance of the findings. RESULTS: In the lung cancer with interstitial pneumonia model, accelerated tumor growth was observed based on an altered tumor microenvironment. RNA sequencing analysis revealed upregulation of the hypoxia-inducible factor 1 signaling pathway. These findings were consistent with those obtained for human samples. Moreover, we explored whether ascorbic acid could be an alternative treatment for lung cancer with interstitial pneumonia to avoid the disadvantages of hypoxia-inducible factor 1 inhibitors. Ascorbic acid successfully downregulated the hypoxia-inducible factor 1 signaling pathway and inhibited tumor progression and lung fibrosis. CONCLUSIONS: The hypoxia-inducible factor 1 pathway is critical in lung cancer with interstitial pneumonia and could be a therapeutic target for mitigating interstitial pneumonia-mediated lung cancer progression.

Hypoxic tumour-derived exosomal miR-1225-5p regulates M2 macrophage polarisation via toll-like receptor 2 to promote ovarian cancer progress.

Tumor-secreted exosomes are critical for the functional regulation of tumor-associated macrophages (TAMs). This study aimed to explore how exosomes secreted by ovarian carcinoma cells regulate the phenotype and function of macrophages. Hypoxic treatment of A2780 cells was postulated to mimic the tumor microenvironment, and exosomes were co-cultured with TAMs. miR-1225-5p was enriched in hypoxic exosomes and contributed to M2 macrophage polarizationby modulating Toll-like receptor 2 expression (TLR2). Furthermore, hypoxia-treated macrophages promote ovarian cancer cell viability, migration, and invasion via the wnt/ß-catenin pathway. This study clarified that exosomal miR-1225-5p promotes macrophage M2-like polarization by targeting TLR2 to promote ovarian cancer, which may via the wnt/ß-catenin pathway.

Hypoxia Modulates Cellular Endocytic Pathways and Organelles with Enhanced Cell Migration and 3D Cell Invasion.

Hypoxia, a decrease in cellular or tissue level oxygen content, is characteristic of most tumors and has been shown to drive cancer progression by altering multiple subcellular processes. We hypothesized that the cancer cells in a hypoxic environment might have slower proliferation rates and increased invasion and migration rates with altered endocytosis compared to the cancer cells in the periphery of the tumor mass that experience normoxic conditions. We induced cellular hypoxia by exposing cells to cobalt chloride, a chemical hypoxic mimicking agent. This study measured the effect of hypoxia on cell proliferation, migration, and invasion. Uptake of fluorescently labeled transferrin, galectin3, and dextran that undergo endocytosis through major endocytic pathways (Clathrin-mediated pathway (CME), Clathrin-independent pathway (CIE), Fluid phase endocytosis (FPE)) were analyzed during hypoxia. Also, the organelle changes associated with hypoxia were studied with organelle trackers. We found that the proliferation rate decreased, and the migration and invasion rate increased in cancer cells in hypoxic conditions compared to normoxic cancer cells. A short hypoxic exposure increased galectin3 uptake in hypoxic cancer cells, but a prolonged hypoxic exposure decreased clathrin-independent endocytic uptake of galectin 3. Subcellular organelles, such as mitochondria, increased to withstand the hypoxic stress, while other organelles, such as Endoplasmic reticulum (ER), were significantly decreased. These data suggest that hypoxia modulates cellular endocytic pathways with reduced proliferation and enhanced cell migration and invasion.

Dose-Specific Intratumoral GM-CSF Modulates Breast Tumor Oxygenation and Antitumor Immunity.

GM-CSF has been employed as an adjuvant to cancer immunotherapy with mixed results based on dosage. We previously showed that GM-CSF regulated tumor angiogenesis by stimulating soluble vascular endothelial growth factor (VEGF) receptor-1 from monocytes/macrophages in a dose-dependent manner that neutralized free VEGF, and intratumoral injections of high-dose GM-CSF ablated blood vessels and worsened hypoxia in orthotopic polyoma middle T Ag (PyMT) triple-negative breast cancer (TNBC). In this study, we assessed both immunoregulatory and oxygen-regulatory components of low-dose versus high-dose GM-CSF to compare effects on tumor oxygen, vasculature, and antitumor immunity. We performed intratumoral injections of low-dose GM-CSF or saline controls for 3 wk in FVB/N PyMT TNBC. Low-dose GM-CSF uniquely reduced tumor hypoxia and normalized tumor vasculature by increasing NG2+ pericyte coverage on CD31+ endothelial cells. Priming of "cold," anti-PD1-resistant PyMT tumors with low-dose GM-CSF (hypoxia reduced) sensitized tumors to anti-PD1, whereas high-dose GM-CSF (hypoxia exacerbated) did not. Low-dose GM-CSF reduced hypoxic and inflammatory tumor-associated macrophage (TAM) transcriptional profiles; however, no phenotypic modulation of TAMs or tumor-infiltrating lymphocytes were observed by flow cytometry. In contrast, high-dose GM-CSF priming increased infiltration of TAMs lacking the MHC class IIhi phenotype or immunostimulatory marker expression, indicating an immunosuppressive phenotype under hypoxia. However, in anti-PD1 (programmed cell death 1)-susceptible BALB/c 4T1 tumors (considered hot versus PyMT), high-dose GM-CSF increased MHC class IIhi TAMs and immunostimulatory molecules, suggesting disparate effects of high-dose GM-CSF across PyMT versus 4T1 TNBC models. Our data demonstrate a (to our knowledge) novel role for low-dose GM-CSF in reducing tumor hypoxia for synergy with anti-PD1 and highlight why dosage and setting of GM-CSF in cancer immunotherapy regimens require careful consideration.

Morphological and molecular-biological features of glioblastoma progression in tolerant and susceptible to hypoxia Wistar rats.

Hypoxia is a major pathogenetic factor in many cancers. Individual resistance to suboptimal oxygen availability is subject to broad variation and its possible role in tumorigenesis remains underexplored. This study aimed at specific characterization of glioblastoma progression in male tolerant and susceptible to hypoxia Wistar rats. Hypoxia resistance was assessed by gasping time measurement in an 11,500 m altitude-equivalent hypobaric decompression chamber. Based on the outcome, the animals were assigned to three groups termed 'tolerant to hypoxia' (n = 13), 'normal', and 'susceptible to hypoxia' (n = 24). The 'normal' group was excluded from subsequent experiments. One month later, the animals underwent inoculation with rat glioblastoma 101.8 followed by monitoring of survival, body weight dynamics and neurological symptoms. The animals were sacrificed on post-inoculation days 11 (subgroup 1) and 15 (subgroup 2). Relative vessels number, necrosis areas and Ki-67 index were assessed microscopically; tumor volumes were determined by 3D reconstruction from histological images; serum levels of HIF-1α, IL-1ß, and TNFα were determined by ELISA. None of the tolerant to hypoxia animals died of the disease during observation period, cf. 85% survival on day 11 and 55% survival on day 15 in the susceptible group. On day 11, proliferative activity of the tumors in the tolerant animals was higher compared with the susceptible group. On day 15, proliferative activity, necrosis area and volume of the tumors in the tolerant to hypoxia animals were higher compared with the susceptible group. ELISA revealed no dynamics in TNFα levels, elevated levels of IL-1ß in the susceptible animals on day 15 in comparison with day 11 and tolerant ones. Moreover, there were elevated levels of HIF-1α in the tolerant animals on day 15 in comparison with day 11. Thus, the proliferative activity of glioblastoma cells and the content of HIF-1α were higher in tolerant to hypoxia rats, but the mortality associated with the tumor process and IL-1ß level in them were lower than in susceptible animals. Specific features of glioblastoma 101.8 progression in tolerant and susceptible to hypoxia rats, including survival, tumor growth rates and IL-1ß level, can become the basis of new personalized approaches for cancer diseases treatment in accordance to individual hypoxia resistance.