Mexenone protects mice from LPS-induced sepsis by EC barrier stabilization.

Blood vessels permit the selective passage of molecules and immune cells between tissues and circulation. Uncontrolled inflammatory responses from an infection can increase vascular permeability and edema, which can occasionally lead to fatal organ failure. We identified mexenone as a vascular permeability blocker by testing 2,910 compounds in the Clinically Applied Compound Library using the lipopolysaccharide (LPS)-induced vascular permeability assay. Mexenone suppressed the LPS-induced downregulation of junctional proteins and phosphorylation of VE-cadherin in Bovine Aortic Endothelial Cells (BAECs). The injection of mexenone 1 hr before LPS administration completely blocked LPS-induced lung vascular permeability and acute lung injury in mice after 18hr. Our results suggest that mexenone-induced endothelial cell (EC) barrier stabilization could be effective in treating sepsis patients.

Bioprinting of pre-vascularized constructs for enhancedin vivoneo-vascularization.

Pre-vascularization has been receiving significant attention for developing implantable engineered 3D tissues. While various pre-vascularization techniques have been developed to improve graft vascularization, the effect of pre-vascularized patterns onin vivoneo-vessel formation has not been studied. In this study, we developed a functional pre-vascularized construct that significantly promotes graft vascularization and conductedin vivoevaluations of the micro-vascular patterns (µVPs) in various printed designs.µVP formation, composed of high-density capillaries, was induced by the co-printing of endothelial cells and adipose-derived stem cells (ADSC). We implanted the printed constructs with variousµVP designs into a murine femoral arteriovenous bundle model and evaluated graft vascularization via 3D visualization and immune-histological analysis of the neo-vessels. TheµVP-distal group (µVP located away from the host vessel) showed approximately two-fold improved neo-vascularization compared to theµVP-proximal group (µVP located near the host vessel). Additionally, we confirmed that theµVP-distal group can generate the angiogenic factor gradient spatial environment for graft vascularization via computational simulations. Based on these results, the ADSC mono pattern (AMP), which secretes four times higher angiogenic factors thanµVP, was added to theµVP + AMP group design. TheµVP + AMP group showed approximately 1.5- and 1.9-fold higher total sprouted neo-vessel volume than theµVP only and AMP only groups, respectively. In immunohistochemical staining analysis, theµVP + AMP group showed two-fold improved density and diameter of the matured neo-vessels. To summarize, these findings demonstrate graft vascularization accelerated due to design optimization of our pre-vascularized constructs. We believe that the developed pre-vascularization printing technique will facilitate new possibilities for the upscaling of implantable engineered tissues/organs.

Quantification and visualization of metastatic lung tumors in mice.

Histopathological examination is important for the diagnosis of various diseases. Conventional histopathology provides a two-dimensional view of the tissues, and requires the tissue to be extracted, fixed, and processed using histotechnology techniques. However, there is an increasing need for three-dimensional (3D) images of structures in biomedical research. The objective of this study was to develop reliable, objective tools for visualizing and quantifying metastatic tumors in mouse lung using micro-computed tomography (micro-CT), optical coherence tomography (OCT), and field emission-scanning electron microscopy (FE-SEM). Melanoma cells were intravenously injected into the tail vein of 8-week-old C57BL/6 mice. The mice were euthanized at 2 or 4 weeks after injection. Lungs were fixed and examined by micro-CT, OCT, FE-SEM, and histopathological observation. Micro-CT clearly distinguished between tumor and normal cells in surface and deep lesions, thereby allowing 3D quantification of the tumor volume. OCT showed a clear difference between the tumor and surrounding normal tissues. FE-SEM clearly showed round tumor cells, mainly located in the alveolar wall and growing inside the alveoli. Therefore, whole-tumor 3D imaging successfully visualized the metastatic tumor and quantified its volume. This promising approach will allow for fast and label-free 3D phenotyping of diverse tissue structures.

Tonicity-responsive enhancer-binding protein promotes stemness of liver cancer and cisplatin resistance.

BACKGROUND: High recurrence and chemoresistance drive the high mortality in hepatocellular carcinoma (HCC). Although cancer stem cells are considered to be the source of recurrent and chemoresistant tumors, they remain poorly defined in HCC. Tonicity-responsive enhancer binding protein (TonEBP) is elevated in almost all HCC tumors and associated with recurrence and death. We aimed to identify function of TonEBP in stemness and chemoresistance of liver cancer. METHODS: Tumors obtained from 280 HCC patients were analyzed by immunohistochemical analyses. Stemness and chemoresistance of liver CSCs (LCSCs) were investigated using cell culture. Tumor-initiating activity was measured by implanting LCSCs into BALB/c nude mice. FINDINGS: Expression of TonEBP is higher in LCSCs in HCC cell lines and correlated with markers of LCSCs whose expression is significantly associated with poor prognosis of HCC patients. TonEBP mediates ATM-mediated activation of NF-κB, which stimulates the promoter of a key stem cell transcription factor SOX2. As expected, TonEBP is required for the tumorigenesis and self-renewal of LSCSs. Cisplatin induces the recruitment of the ERCC1/XPF dimer to the chromatin in a TonEBP-dependent manner leading to DNA repair and cisplatin resistance. The cisplatin-induced inflammation in LSCSs is also dependent on the TonEBP-ERCC1/XPF complex, and leads to enhanced stemness via the ATM-NF-κB-SOX2 pathway. In HCC patients, tumor expression of ERCC1/XPF predicts recurrence and death in a TonEBP-dependent manner. INTERPRETATION: TonEBP promotes stemness and cisplatin resistance of HCC via ATM-NF-κB. TonEBP is a key regulator of LCSCs and a promising therapeutic target for HCC and its recurrence.

Label-free optical projection tomography for quantitative three-dimensional anatomy of mouse embryo.

Recent progress in three-dimensional optical imaging techniques allows visualization of many comprehensive biological specimens. Optical clearing methods provide volumetric and quantitative information by overcoming the limited depth of light due to scattering. However, current imaging technologies mostly rely on the synthetic or genetic fluorescent labels, thus limits its application to whole-body visualization of generic mouse models. Here, we report a label-free optical projection tomography (LF-OPT) technique for quantitative whole mouse embryo imaging. LF-OPT is based on the attenuation contrast of light rather than fluorescence, and it utilizes projection imaging technique similar to computed tomography for visualizing the volumetric structure. We demonstrate this with a collection of mouse embryo morphologies in different stages using LF-OPT. Additionally, we extract quantitative organ information applicable toward high-throughput phenotype screening. Our results indicate that LF-OPT can provide multi-scale morphological information in various tissues including bone, which can be difficult in conventional optical imaging technique.

G-protein-coupled receptor 81 promotes a malignant phenotype in breast cancer through angiogenic factor secretion.

G-protein-coupled receptor 81 (GPR81) functions as a receptor for lactate and plays an important role in the regulation of anti-lipolytic effects in adipocytes. However, to data, a role for GPR81 in the tumor microenvironment has not been clearly defined. Here, GPR81 expression in breast cancer patients and several breast cancer cell lines was significantly increased compared with normal mammary tissues and cells. GPR81 knockdown resulted in impaired breast cancer growth and led to apoptosis both in vitro and in vivo. Furthermore, the inhibition of GPR81 signaling suppressed angiogenesis through a phosphoinositide 3-OH kinase (PI3K)/Akt-cAMP response element binding protein (CREB) pathway, which led to decreased production of the pro-angiogenic mediator amphiregulin (AREG). Overall, these findings identify GPR81 as a tumor-promoting receptor in breast cancer progression and suggest a novel mechanism that regulates GPR81-dependent activation of the PI3K/Akt signaling axis in tumor microenvironment.

Spiraeoside inhibits mast cells activation and IgE-mediated allergic responses by suppressing phospholipase C-γ-mediated signaling.

Mast cells are responsible for IgE-mediated allergic responses through the secretion of various inflammatory cytokines and mediators. Therefore, the pharmacological regulation of mast cell activation is an important goal in the development of novel anti-allergic drugs. In this study, we found that spiraeoside (SP) inhibits mast cell activation and allergic responses in vivo. SP dose-dependently inhibited the degranulation induced by IgE-antigen (Ag) stimulation in RBL-2H3 mast cells without cytotoxic effects. At the molecular level, SP reduced the Ag-induced phosphorylation and subsequent activation of phospholipase C-γ2 (PLC-γ2). Moreover, SP inhibited the phosphorylation of spleen tyrosine kinase (Syk), linker for activation of T cells (LAT), and downstream MAPKs, such as ERK1/2, p38, and JNK, eventually attenuating expression of TNF-α and IL-4. Finally, we found that SP significantly inhibited IgE-mediated passive cutaneous anaphylaxis (PCA) in mice. Taken together, our results strongly suggest that SP suppresses IgE-mediated mast cell activation and allergic responses by inhibiting Lyn-induced PLC-γ2/MAPK signaling in mast cells.

DJ-1 contributes to adipogenesis and obesity-induced inflammation.

Adipose tissue functions as an endocrine organ, and the development of systemic inflammation in adipose tissue is closely associated with metabolic diseases, such as obesity and insulin resistance. Accordingly, the fine regulation of the inflammatory response caused by obesity has therapeutic potential for the treatment of metabolic syndrome. In this study, we analyzed the role of DJ-1 (PARK7) in adipogenesis and inflammation related to obesity in vitro and in vivo. Many intracellular functions of DJ-1, including oxidative stress regulation, are known. However, the possibility of DJ-1 involvement in metabolic disease is largely unknown. Our results suggest that DJ-1 deficiency results in reduced adipogenesis and the down-regulation of pro-inflammatory cytokines in vitro. Furthermore, DJ-1-deficient mice show a low-level inflammatory response in the high-fat diet-induced obesity model. These results indicate previously unknown functions of DJ-1 in metabolism and therefore suggest that precise regulation of DJ-1 in adipose tissue might have a therapeutic advantage for metabolic disease treatment.

Combination treatment with doxorubicin and gamitrinib synergistically augments anticancer activity through enhanced activation of Bim.

BACKGROUND: A common approach to cancer therapy in clinical practice is the combination of several drugs to boost the anticancer activity of available drugs while suppressing their unwanted side effects. In this regard, we examined the efficacy of combination treatment with the widely-used genotoxic drug doxorubicin and the mitochondriotoxic Hsp90 inhibitor gamitrinib to exploit disparate stress signaling pathways for cancer therapy. METHODS: The cytotoxicity of the drugs as single agents or in combination against several cancer cell types was analyzed by MTT assay and the synergism of the drug combination was evaluated by calculating the combination index. To understand the molecular mechanism of the drug synergism, stress signaling pathways were analyzed after drug combination. Two xenograft models with breast and prostate cancer cells were used to evaluate anticancer activity of the drug combination in vivo. Cardiotoxicity was assessed by tissue histology and serum creatine phosphokinase concentration. RESULTS: Gamitrinib sensitized various human cancer cells to doxorubicin treatment, and combination treatment with the two drugs synergistically increased apoptosis. The cytotoxicity of the drug combination involved activation and mitochondrial accumulation of the proapoptotic Bcl-2 family member Bim. Activation of Bim was associated with increased expression of the proapoptotic transcription factor C/EBP-homologous protein and enhanced activation of the stress kinase c-Jun N-terminal kinase. Combined drug treatment with doxorubicin and gamitrinib dramatically reduced in vivo tumor growth in prostate and breast xenograft models without increasing cardiotoxicity. CONCLUSIONS: The drug combination showed synergistic anticancer activities toward various cancer cells without aggravating the cardiotoxic side effects of doxorubicin, suggesting that the full therapeutic potential of doxorubicin can be unleashed through combination with gamitrinib.

Periostin-binding DNA aptamer inhibits breast cancer growth and metastasis.

Periostin is an extracellular matrix (ECM) protein that is overexpressed in a variety of human cancers, and its functions appear to be linked to tumor growth, metastasis, and angiogenesis. Recent clinical evidence suggests that aberrant periostin expression is correlated with poor outcome in patients with breast cancer. To identify novel tools to regulate the functional role of periostin, we generated benzyl-d(U)TP-modified DNA aptamers that were directed against human periostin (PNDAs) and characterized their functional roles in breast cancer progression. PNDA-3 selectively bound to the FAS-1 domain of periostin with nanomolar affinity and disrupted the interaction between periostin and its cell surface receptors, αvß3 and αvß5 integrins. PNDA-3 markedly antagonized the periostin-induced adhesion, migration, and invasion of breast cancer cells and blocked the activation of various components of the αvß3 and αvß5 integrin signal transduction pathways. In a 4T1 orthotopic mouse model, PNDA-3 administration significantly reduced primary tumor growth and distant metastasis. Thus, our results demonstrated that periostin-integrin signaling regulates breast cancer progression at multiple levels in tumor cells and the tumor microenvironment. DNA aptamers targeting periostin may potentially be used to inhibit breast cancer progression.

Kinetic modeling of 3'-deoxy-3'-18F-fluorothymidine for quantitative cell proliferation imaging in subcutaneous tumor models in mice.

UNLABELLED: 3'-Deoxy-3'-(18)F-fluorothymidine ((18)F-FLT) is a thymidine analog that was developed for measuring tumor proliferation with PET. The aim of this study was to establish a kinetic modeling analysis method for quantitative (18)F-FLT PET studies in subcutaneous tumor models in mice. METHODS: To explore the validity of an image-derived left ventricular input function, we measured equilibrium constants for plasma and whole blood and metabolite fractions in blood after (18)F-FLT injection. In parallel, dynamic (18)F-FLT PET scans were acquired in 24 mice with a small-animal dedicated PET scanner to compare arterial blood activities obtained by PET and blood sampling. We then investigated kinetic models for (18)F-FLT in human epithelial carcinoma (A431) and Lewis lung carcinoma tumor models in mice. Three-compartment models with reversible phosphorylation (k(4) not equal 0, 3C5P) and irreversible phosphorylation (k(4) = 0, 3C4P) and a 2-compartment model (2C3P) were examined. The Akaike information criterion and F statistics were used to select the best model for the dataset. Gjedde-Patlak graphic analysis was performed, and standardized uptake values in the last frame were calculated for comparison purposes. In addition, quantitative PET parameters were compared with Ki-67 immunostaining results. RESULTS: (18)F-FLT equilibrated rapidly (within 30 s) between plasma and whole blood, and metabolite fractions were negligible during PET scans. A high correlation between arterial blood sampling and PET data was observed. For 120-min dynamic PET data, the 3C5P model best described tissue time-activity curves for tumor regions. The net influx of (18)F-FLT (K(FLT)) and k(3) obtained with this model showed reasonable intersubject variability and discrimination ability for tumor models with different proliferation properties. The K(FLT) obtained from the 60- or 90-min data correlated well with that obtained from the 120-min data as well as with the Ki-67 results. CONCLUSION: The image-derived arterial input function was found to be feasible for kinetic modeling studies of (18)F-FLT PET in mice, and kinetic modeling analysis with an adequate compartment model provided reliable kinetic parameters for measuring tumor proliferation.