Autophagy caused by oxidative stress promotes TGF-ß1-induced epithelial-to-mesenchymal transition in human peritoneal mesothelial cells.

Epithelial-to-mesenchymal transition (EMT) is one of the main causes of peritoneal fibrosis. However, the pathophysiological mechanisms of EMT, specifically its relationship with autophagy, are still unknown. This study aimed to evaluate the role of autophagy in transforming growth factor-beta 1 (TGF-ß1)-induced EMT in human peritoneal mesothelial cells (HPMCs). Primary cultured HPMCs were treated with TGF-ß1 (2 and 5 ng/mL) and changes in autophagy markers and the relationship between autophagy and EMT were evaluated. We also identified changes in EMT- and autophagy-related signaling pathways after autophagy and NADPH oxidase 4 (NOX4) inhibition. TGF-ß1 increased the generation of NOX4 and reactive oxygen species (ROS) in HPMCs, resulting in mitochondrial damage. Treatment with GKT137831 (20 µM), a NOX1/4 inhibitor, reduced ROS in the mitochondria of HPMC cells and reduced TGF-ß1-induced mitochondrial damage. Additionally, the indirect inhibition of autophagy by GKT137831 (20 µM) downregulated TGF-ß1-induced EMT, whereas direct inhibition of autophagy using 3-methyladenine (3-MA) (2 mM) or autophagy-related gene 5 (ATG5) gene silencing decreased the TGF-ß1-induced EMT in HPMCs. The suppressor of mothers against decapentaplegic 2/3 (Smad2/3), autophagy-related phosphoinositide 3-kinase (PI3K) class III, and protein kinase B (Akt) pathways, and mitogen-activated protein kinase (MAPK) signaling pathways, such as extracellular signal-regulated kinase (ERK) and P38, were involved in TGF-ß1-induced EMT. Autophagy and NOX4 inhibition suppressed the activation of these signaling pathways. Direct inhibition of autophagy and its indirect inhibition through the reduction of mitochondrial damage by upstream NOX4 inhibition reduced EMT in HPMCs. These results suggest that autophagy could serve as a therapeutic target for the prevention of peritoneal fibrosis in patients undergoing peritoneal dialysis.

Dual Immunoglobulin Domain-Containing Cell Adhesion Molecule Increases Early in Renal Tubular Cell Injury and Plays Anti-Inflammatory Role.

Dual immunoglobulin domain-containing cell adhesion molecule (DICAM) is a type I transmembrane protein that presents in various cells including renal tubular cells. This study evaluated the expression and protective role of DICAM in renal tubular cell injury. HK-2 cells were incubated and treated with lipopolysaccharide (LPS, 30 µg/mL) or hydrogen peroxide (H2O2, 100 µM) for 24 h. To investigate the effect of the gene silencing of DICAM, small interfering RNA of DICAM was used. Additionally, to explain its role in cellular response to injury, DICAM was overexpressed using an adenoviral vector. DICAM protein expression levels significantly increased following treatment with LPS or H2O2 in HK-2 cells. In response to oxidative stress, DICAM showed an earlier increase (2-4 h following treatment) than neutrophil gelatinase-associated lipocalin (NGAL) (24 h following treatment). DICAM gene silencing increased the protein expression of inflammation-related markers, including IL-1ß, TNF-α, NOX4, integrin ß1, and integrin ß3, in H2O2-induced HK-2 cell injury. Likewise, in the LPS-induced HK-2 cell injury, DICAM knockdown led to a decrease in occludin levels and an increase in integrin ß3, IL-1ß, and IL-6 levels. Furthermore, DICAM overexpression followed by LPS-induced HK-2 cell injury resulted in an increase in occludin levels and a decrease in integrin ß1, integrin ß3, TNF-α, IL-1ß, and IL-6 levels, suggesting an alleviating effect on inflammatory responses. DICAM was elevated in the early stage of regular tubular cell injury and may protect against renal tubular injury through its anti-inflammatory properties. DICAM has a potential as an early diagnostic marker and therapeutic target for renal cell injury.

Lymphoscintigraphy in patients with breast cancer-related lymphedema after sentinel lymph node dissection and axillary radiation therapy.

The purpose of this study was to investigate lymphoscintigraphy pattern according to the presence or absence of axillary site radiation therapy (aRTx) in breast cancer-related lymphedema (BCRL) patients who underwent sentinel lymph node dissection (SLND). The participants were patients who visited our facility from July 2014 to June 2021 due to upper extremity edema. Among them, patients who underwent SLND after the diagnosis of breast cancer were included. The participants were divided into a group without aRTx (group A) and a group with aRTx (group B). In each patient's lymphoscintigraphy, axillary lymph node uptake (ALNU), lymphatic flow delay, dermal back flow, and the presence of any collateral pathway were checked. Thirty-three patients were enrolled. In all, 27 patients were classified in Group A, and 6 patients were classified in Group B. Between the 2 groups, we found a significant difference (P value < .05) between groups at ALNU and lymphatic flow delay. However, there was no significant difference between groups at the dermal backflow and the presence of a collateral pathway (P value > .05). And 24.2% of patients who developed lymphedema after SLND showed normal lymphoscintigraphy. In this study we suggest that SLND and aRTx affects the activity of the axillary lymph node and ultimately adversely affects lymphatic flow, becoming a risk factor for lymphedema. In addition, regardless of SLND or aRTx, lymphedema may eventually occur in the patient with normal lymphoscintigraphy.

Single-Dose Toxicity Study on ML171, a Selective NOX1 Inhibitor, in Mice.

BACKGROUND: ML171 is a potent nicotinamide adenine dinucleotide phosphate oxidase (NOX) inhibitor with isoform selectivity only for NOX1. This study is aimed at investigating the safety of ML171 after a single intraperitoneal (IP) injection in mice. METHODS: The toxicity of a single dose of ML171 was evaluated in 6-week-old Institute of Cancer Research (ICR) mice in a good laboratory practice (GLP) laboratory. Twenty-five mice of each sex were assigned to five groups: negative control, vehicle control, and 125, 250, and 500 mg/kg of ML171. All mice were acclimatized for one week before beginning the study. Mice received an IP injection of ML171 or vehicle. The general condition and mortality of the animals were observed. The mice were sacrificed to evaluate histopathology 14 days after the administration of ML171 or vehicle. RESULTS: Bodyweights were not significantly different in any group. Three males and one female died due to ML171 administration in the 500 mg/kg dose group. Autopsies of the surviving mice did not reveal any significant abnormalities after the injection of 125 mg/kg of ML171. However, the anterior lobe edge of the liver was thickened and adhesions between the liver and adjacent organs were observed in mice treated with 250 or 500 mg/kg of ML171. In addition, hypertrophy of centrilobular hepatocytes and inflammatory cell infiltration were observed after injection of 250 and 500 mg/kg of ML171. CONCLUSION: Our results indicate that the lethal IP injection dose of ML171 is 500 mg/kg for both males and females. Mortality were not observed for lower doses of ML171. The safe dose of single IP ML171 in ICR mice was 250 mg/kg or less. Further studies are needed to confirm the safety of ML171 in the human body.

Comparison of the short-term effects of hyperbaric oxygen therapy and complex decongestive therapy on breast cancer-related lymphedema: A pilot study.

Although there have been some reports that hyperbaric oxygen therapy (HBOT) is effective in treating breast cancer-related lymphedema (BCRL), controversy regarding its therapeutic effects remains.We sought to evaluate the efficacy of HBOT in addition to conventional complex decongestive therapy (CDT) for BCRL.A prospective observational study was conducted on 10 patients with BCRL. After screening, the subjects were stratified into a CDT-only group and a CDT and HBOT combination (CDT-HBOT) group. All patients received a total of 10 treatments over 2 weeks. Changes in the circumference of the upper limbs, quality-of-life questionnaire results, and bioelectrical impedance values were compared between the 2 groups.Between both groups, there were no significant differences in demographic or clinical characteristics and in the quality-of-life outcomes for lymphedema of the limbs. The parameters measured by bioimpedance spectroscopy showed more significant improvements in the CDT-HBOT group than in the CDT-only group.In patients with BCRL, HBOT may be recommended as an adjunct treatment to the existing therapies.

Alpha-1 antitrypsin inhibits formaldehyde-induced apoptosis of human peritoneal mesothelial cells.

BACKGROUND: The alpha-1 antitrypsin (AAT) protein has an important role in the anti-inflammatory and apoptotic response. AAT inhibits not only serine proteases but also cysteine and aspartic proteases. Apoptosis results from the sequential activation of cysteine proteases of the caspase family. This study aimed to evaluate the effect of AAT on formaldehyde-induced apoptosis of human peritoneal mesothelial cells (HPMCs). METHODS: HPMCs were cultured and treated with formaldehyde (250 µM) to induce apoptosis. In the AAT group, the cultured HPMCs were pretreated with AAT (2 mg/mL) for 1 h before formaldehyde treatment. We used 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assays to determine cell viability, and flow cytometry and terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) assays to detect apoptosis. The MTT assays were used to find optimal concentrations of formaldehyde and AAT. We measured caspase-3 activity and used Western blotting to estimate Bcl-2 and Bad expression. RESULTS: Flow cytometry and TUNEL assays revealed that formaldehyde exposure significantly increased apoptosis compared with the control treatment, but pretreatment with AAT significantly inhibited this effect. Compared with the control, caspase-3 activity was significantly increased and the ratio of Bcl-2 to Bad expression significantly decreased following treatment with formaldehyde. However, caspase-3 activity was significantly lower and the Bcl-2 to Bad expression ratio higher in the AAT group than in the formaldehyde-only group. CONCLUSION: AAT inhibits formaldehyde-induced apoptosis of HPMCs via a caspase-mediated pathway. These data support a potential use for AAT as a therapeutic agent for the inhibition of peritoneal cell apoptosis during peritoneal dialysis.

The emerging role of xanthine oxidase inhibition for suppression of breast cancer cell migration and metastasis associated with hypercholesterolemia.

Hypercholesterolemia is reported to increase reactive oxygen species (ROS) and to promote breast cancer progression. ROS play an important role in tumor biology, and xanthine oxidase (XO) is an enzyme that generates ROS. The effects of febuxostat (FBX), an XO inhibitor, on breast cancer cell migration under LDL stimulation in vitro and metastasis of breast cancer associated with hypercholesterolemia in vivo were studied. In vitro, FBX significantly inhibited LDL-induced ROS production and cell migration. Treatment of small interfering RNA against XO was consistent with the findings of FBX treatment. In vivo, a significant increase of tumor growth and pulmonary metastasis was observed in a xenograft mouse model with 4T1 cells on a high cholesterol diet (HCD), both of which were markedly inhibited by FBX or allopurinol treatment. Moreover, ERK represented the main target-signaling pathway that was affected by FBX treatment in a xenograft mouse model on an HCD evaluated by NanoString nCounter analysis. Consistently, MEK/ERK inhibitors directly decreased the LDL-induced cell migration in vitro. In conclusion, FBX mitigates breast cancer cell migration and pulmonary metastasis in the hyperlipidemic condition, associated with decreased ROS generation and MAPK phosphorylation. The inhibition of ERK pathways is likely to underlie the XO inhibitor-mediated suppression of breast cancer cell migration.-Oh, S.-H., Choi, S.-Y., Choi, H.-J., Ryu, H.-M., Kim, Y.-J., Jung, H.-Y., Cho, J.-H., Kim, C.-D., Park, S.-H., Kwon, T.-H., Kim, Y.-L. The emerging role of xanthine oxidase inhibition for suppression of breast cancer cell migration and metastasis associated with hypercholesterolemia.

Effects of Different Bandaging Methods for Treating Patients With Breast Cancer-Related Lymphedema.

OBJECTIVE: To compare the treatment effects, satisfaction with the treatment, and performance improvement following bandage treatment using the spiral method and spica method for breast cancer-related lymphedema (BCRL). METHODS: A prospective study with 46 patients with BCRL was conducted. All patients were divided into either the spiral or spica group for non-elastic bandage therapy and received the same treatment for 2 weeks, apart from the group-specific bandaging method used. For both groups, the Quality of Life Instrument score before treatment, changes in the volume of lymphedema limb and the Disability of the Arm, Shoulder, and Hand (DASH) score before and after treatment, and treatment satisfaction after treatment were compared. The Student t-test was used to compare the parameters between the two different bandage methods. RESULTS: With respect to the treatment outcomes, total volume reduction and proximal part volume reduction after treatment were 98.0±158.3 mL and 56.0±65.4 mL in the spiral method group and 199.0±125.1 mL and 106.1±82.2 mL in the spica method group, respectively. Therefore, the spica method group showed a significantly better improvement (p<0.05). The DASH score changes after treatment showed that the spiral group score increased by 3.8±5.4 and the spica group score increased by 7.7±6.1; thus, a significantly better improvement was noted in the spica group (p<0.05). CONCLUSION: The spica method indicated better volume reduction and DASH score improvement than the spiral method. Therefore, the spica method may be more effective for treating patients with BCRL.

Implementation of Targeted Temperature Management in a Patient with Cerebral Arterial Gas Embolism.

Cerebral arterial gas embolism (CAGE) shows various manifestations according to the quantity of gas and the brain areas affected. The symptoms range from minor motor weakness, headache, and confusion to disorientation, convulsions, hemiparesis, unconsciousness, and coma. A 46-year-old man was transferred to our emergency department due to altered sensorium. Immediately after a controlled ascent from 33 m of seawater, he complained of shortness of breath and rigid extremities, lapsing into unconsciousness. He was intubated at another medical center, where a brain computerized axial tomography scan showed no definitive abnormal findings. Pneumothorax and obstructing lesions were apparent in the left thorax of the computed tomography scan. Following closed thoracostomy, we provided hyperbaric oxygen therapy (HBOT) using U.S. Navy Treatment Table (USN TT) 6A. A brain magnetic resonance imaging diffusion image taken after HBOT showed acute infarction in both middle and posterior cerebral arteries. We implemented targeted temperature management (TTM) to prevent worsening of cerebral function in the intensive care unit. After completing TTM, we repeated HBOT using USN TT5 and started rehabilitation therapy. He fully recovered from the neurological deficits. This is the first case of CAGE treated with TTM and consecutive HBOTs suggesting that TTM might facilitate salvage of the penumbra in severe CAGE.

Hypoxanthine induces cholesterol accumulation and incites atherosclerosis in apolipoprotein E-deficient mice and cells.

Reactive oxygen species (ROS) generation during purine metabolism is associated with xanthine oxidase and uric acid. However, the direct effect of hypoxanthine on ROS generation and atherosclerosis has not been evaluated. Smoking and heavy drinking are associated with elevated levels of hypoxanthine. In this study, we investigated the role of hypoxanthine on cholesterol synthesis and atherosclerosis development, particularly in apolipoprotein E (APOE)-deficient mice. The effect of hypoxanthine on the regulation of cholesterol synthesis and atherosclerosis were evaluated in Apoe knockout (KO) mice and cultured HepG2 cells. Hypoxanthine markedly increased serum cholesterol levels and the atherosclerotic plaque area in Apoe KO mice. In HepG2 cells, hypoxanthine increased intracellular ROS production. Hypoxanthine increased cholesterol accumulation and decreased APOE and ATP-binding cassette transporter A1 (ABCA1) mRNA and protein expression in HepG2 cells. Furthermore, H2 O2 also increased cholesterol accumulation and decreased APOE and ABCA1 expression. This effect was partially reversible by treatment with the antioxidant N-acetyl cysteine and allopurinol. Hypoxanthine and APOE knockdown using APOE-siRNA synergistically induced cholesterol accumulation and reduced APOE and ABCA1 expression. Hypoxanthine induces cholesterol accumulation in hepatic cells through alterations in enzymes that control lipid transport and induces atherosclerosis in APOE-deficient cells and mice. These effects are partially mediated through ROS produced in response to hypoxanthine.