Milk Fat Globule-EGF Factor 8, Secreted by Mesenchymal Stem Cells, Protects Against Liver Fibrosis in Mice.

BACKGROUND & AIMS: Mesenchymal stem cells (MSCs) mediate tissue repair and might be used to prevent or reduce liver fibrosis. However, little is known about the anti-fibrotic factors secreted from MSCs or their mechanisms. METHODS: Umbilical cord-derived MSCs (UCMSCs) were differentiated into hepatocyte-like cells (hpUCMSCs), medium was collected, and secretome proteins were identified and quantified using nanochip-liquid chromatography/quadrupole time-of-flight mass spectrometry. Liver fibrosis was induced in mice by intraperitoneal injection of thioacetamide or CCl4; some mice were then given injections of secretomes or proteins. Liver tissues were collected and analyzed by histology or polymerase chain reaction array to analyze changes in gene expression patterns. We analyzed the effects of MSC secretomes and potential anti-fibrotic proteins on transforming growth factor ß 1 (TGFß1)-mediated activation of human hepatic stellate cell (HSC) lines (hTert-HSC and LX2) and human primary HSCs. Liver tissues were collected from 16 patients with liver cirrhosis and 16 individuals without cirrhosis (controls) in Korea and analyzed by immunohistochemistry and immunoblots. RESULTS: In mice with fibrosis, accumulation of extracellular matrix proteins was significantly reduced 3 days after injecting secretomes from UCMSCs, and to a greater extent from hpUCMSCs; numbers of activated HSCs that expressed the myogenic marker α-smooth muscle actin (α-SMA, encoded by ACTA2 [actin, alpha 2, smooth muscle]) were also reduced. Secretomes from UCMSCs, and to a greater extent from hpUCMSCs, reduced liver expression of multiple fibrotic factors, collagens, metalloproteinases, TGFß, and Smad proteins in the TGFß signaling pathways. In HSC cell lines and primary HSCs, TGFß1-stimulated upregulation of α-SMA was significantly inhibited (and SMAD2 phosphorylation reduced) by secretomes from UCMSCs, and to a greater extent from hpUCMSCs. We identified 32 proteins in secretomes of UCMSCs that were more highly concentrated in secretomes from hpUCMSCs and inhibited TGFß-mediated activation of HSCs. One of these, milk fat globule-EGF factor 8 (MFGE8), was a strong inhibitor of activation of human primary HSCs. We found MFGE8 to down-regulate expression of TGFß type I receptor by binding to αvß3 integrin on HSCs and to be secreted by MSCs from umbilical cord, teeth, and bone marrow. In mice, injection of recombinant human MFGE8 had anti-fibrotic effects comparable to those of the hpUCMSC secretome, reducing extracellular matrix deposition and HSC activation. Co-injection of an antibody against MFGE8 reduced the anti-fibrotic effects of the hpUCMSC secretome in mice. Levels of MFGE8 were reduced in cirrhotic liver tissue from patients compared with controls. CONCLUSIONS: MFGE8 is an anti-fibrotic protein in MSC secretomes that strongly inhibits TGFß signaling and reduces extracellular matrix deposition and liver fibrosis in mice.

Identification and Validation of SAA4 as a Rheumatoid Arthritis Prescreening Marker by Liquid Chromatography Tandem-mass Spectrometry.

Rheumatoid arthritis (RA) is a chronic autoimmune disease that progresses into systemic inflammation and joint deformity. RA diagnosis is a complicated procedure, and early diagnostic methods are insufficient. Therefore, in this study, we attempted to identify new markers to improve the accuracy of RA prescreening. e identified differentially expressed proteins (DEPs) by using liquid chromatography tandem-mass spectrometry in health-prescreening sera with high rheumatoid factor (RF) values, and compared the findings with those from sera with normal RF values. We identified 93 DEPs; of these, 36 were upregulated, and 57 were downregulated in high-RF sera. Pathway analysis revealed that these DEPs were related to immune responses. Additionally, four DEPs were statistically analyzed by proteomic analysis; of these, SAA4 was significantly validated in individual enzyme-linked immunosorbent assays. Moreover, SAA4 was significantly upregulated in RA patients (n = 40, 66.43 ± 12.97 ng/mL) compared with normal controls (n = 40, 4.79 ± 0.95 ng/mL) and had a higher area under the curve than C-reactive protein. Thus, we identified SAA4 as a protein that was positively correlated with RF and RA. SAA4 may represent a novel prescreening marker for the diagnosis of RA.

Mitochondrial induced and self-monitored intrinsic apoptosis by antitumor theranostic prodrug: in vivo imaging and precise cancer treatment.

Activation of apoptosis, the cell death machinery, in tumor cells by organelle-specific delivery of antitumor theranostic agent is the utmost challenge in cancer therapy. Herein, we developed a highly efficient mitochondria-targeting antitumor theranostic prodrug 7 that contained two molecules of drug 5'-deoxy-5-fluorouridine and an apoptotic marker ethidium for self-monitoring intrinsic (mitochondrial) apoptosis after its activation in tumor cells. Theranostic 7 was activated by endogenously produced mitochondrial-overexpressed H2O2 and released drug 5'-deoxy-5-fluorouridine and apoptotic marker ethidium to the tumor cells. The in vitro and in vivo drug release was monitored by observing the fluorescence changes of ethidium. Theranostic 7 exhibited an enhanced cytotoxicity over commercial 5-fluorouracil (an active drug of 5'-deoxy-5-fluorouridine) leading to intrinsic apoptosis monitored by in situ generated ethidium. Enhanced expression of mitochondria-mediated apoptotic genes (NOXA, PUMA, BID, BAX, and BAK), Cyt C, Caspase-3 and -9, and cell surface death receptors was observed after theranostic 7 activation in tumor cells. In vivo and ex vivo xenografts revealed that theranostic 7 significantly inhibited tumor progression and cured the tumor-bearing mice. Such organelle-specific theranostic strategies have great potential for the early diagnosis and precise treatment of cancer.

Direct and indirect contribution of human embryonic stem cell-derived hepatocyte-like cells to liver repair in mice.

BACKGROUND & AIMS: Many studies of embryonic stem cells have investigated direct cell replacement of damaged tissues, but little is known about how donor cell-derived signals affect host tissue regeneration. We investigated the direct and indirect roles of human embryonic stem cell-derived cells in liver repair in mice. METHODS: To promote the initial differentiation of human embryonic stem cells into mesendoderm, we activated the ß-catenin signaling pathway with lithium; cells were then further differentiated into hepatocyte-like cells. The differentiated cells were purified by indocyanine green staining and laser microdissection and characterized by immunostaining, polymerase chain reaction, biochemical function, electron microscopy, and transplantation analyses. To investigate indirect effects of these cells, secreted proteins (secretomes) were analyzed by a label-free quantitative mass spectrometry. Carbon tetrachloride was used to induce acute liver injury in mice; cells or secreted proteins were administered by intrasplenic or intraperitoneal injection, respectively. RESULTS: The differentiated hepatocyte-like cells had multiple features of normal hepatocytes, engrafted efficiently into mice, and continued to have hepatic features; they promoted proliferation of host hepatocytes and revascularization of injured host liver tissues. Proteomic analysis identified proteins secreted from these cells that might promote host tissue repair. Injection of the secreted proteins into injured livers of mice promoted significant amounts of tissue regeneration without cell grafts. CONCLUSIONS: Hepatocyte-like cells derived from human embryonic stem cells contribute to recovery of injured liver tissues in mice, not only by cell replacement but also by delivering trophic factors that support endogenous liver regeneration.

Spirulina Extract Enhanced a Protective Effect in Type 1 Diabetes by Anti-Apoptosis and Anti-ROS Production.

Interest in the nutritional value and pharmacological activities of blue-green algae has gradually increased. Spirulina extracts show protective effects against apoptosis and inflammatory damage in various cell types. Here, we investigated the protective effects of extracts from Spirulina maxima in a cytokine-mediated type 1 diabetes model in vitro and in streptozotocin-induced diabetic Wistar rats in vivo. Interleukin-1ß and interferon-gamma induced substantial cytotoxicity to RINm5F rat insulinoma cells, increasing nitric oxide (NO) production, nuclear factor-kappa B (NF-κB) activity, the expression of endoplasmic reticulum (ER) stress genes, and activation of mitogen-activated protein kinases and key genes related apoptosis. However, the cytotoxicity of cytokines was significantly attenuated by Spirulina extract, which effectively prevented NO production by inhibiting the synthesis of cytokine-activated NO synthase (iNOS), and apoptosis was suppressed. These results suggest that Spirulina extract might be effective to preserve the viability and function of pancreatic ß-cells against cytotoxic conditions. Moreover, diabetic mice orally administered Spirulina extract showed decreased glucose levels, increased insulin, and improvement in liver enzyme markers. The antioxidant effect of Spirulina extract may be helpful in treating type 1 diabetes by enhancing the survival, and reducing or delaying cytokine-mediated ß-cells destruction.

N-glycoproteomic analysis of human follicular fluid during natural and stimulated cycles in patients undergoing in vitro fertilization.

OBJECTIVE: Hyperstimulation methods are broadly used for in vitro fertilization (IVF) in patients with infertility; however, the side effects associated with these therapies, such as ovarian hyperstimulation syndrome (OHSS), have not been well studied. N-glycoproteomes are subproteomes used for the remote sensing of ovarian stimulation in follicular growth. Glycoproteomic variation in human follicular fluid (hFF) has not been evaluated. In this study, we aimed to identify and quantify the glycoproteomes and N-glycoproteins (N-GPs) in natural and stimulated hFF using label-free nano-liquid chromatography/electrospray ionization-quad time-of-flight mass spectrometry. METHODS: For profiling of the total proteome and glycoproteome, pooled protein samples from natural and stimulated hFF samples were selectively isolated using hydrazide chemistry to obtain the total proteomes and glycoproteomes. N-GPs were validated by the consensus sequence N-X-S/T (92.2% specificity for the N-glycomotif at p<0.05). All data were compared between natural versus hyperstimulated hFF samples. RESULTS: We detected 41 and 44 N-GPs in the natural and stimulated hFF samples, respectively. Importantly, we identified 11 N-GPs with greater than two-fold upregulation in stimulated hFF samples compared to natural hFF samples. We also validated the novel N-GPs thyroxine-binding globulin, vitamin D-binding protein, and complement proteins C3 and C9. CONCLUSION: We identified and classified N-GPs in hFF to improve our understanding of follicular physiology in patients requiring assisted reproduction. Our results provided important insights into the prevention of hyperstimulation side effects, such as OHSS.

Proteomic analysis of serum from patients with major depressive disorder to compare their depressive and remission statuses.

OBJECTIVE: Currently, there are a few biological markers to aid in the diagnosis and treatment of depression. However, it is not sufficient for diagnosis. We attempted to identify differentially expressed proteins during depressive moods as putative diagnostic biomarkers by using quantitative proteomic analysis of serum. METHODS: Blood samples were collected twice from five patients with major depressive disorder (MDD) at depressive status before treatment and at remission status during treatment. Samples were individually analyzed by liquid chromatography-tandem mass spectrometry for protein profiling. Differentially expressed proteins were analyzed by label-free quantification. Enzyme-linked immunosorbent assay (ELISA) results and receiver-operating characteristic (ROC) curves were used to validate the differentially expressed proteins. For validation, 8 patients with MDD including 3 additional patients and 8 matched normal controls were analyzed. RESULTS: The quantitative proteomic studies identified 10 proteins that were consistently upregulated or downregulated in 5 MDD patients. ELISA yielded results consistent with the proteomic analysis for 3 proteins. Expression levels were significantly different between normal controls and MDD patients. The 3 proteins were ceruloplasmin, inter-alpha-trypsin inhibitor heavy chain H4 and complement component 1qC, which were upregulated during the depressive status. The depressive status could be distinguished from the euthymic status from the ROC curves for these proteins, and this discrimination was enhanced when all 3 proteins were analyzed together. CONCLUSION: This is the first proteomic study in MDD patients to compare intra-individual differences dependent on mood. This technique could be a useful approach to identify MDD biomarkers, but requires additional proteomic studies for validation.

Interleukin-8 is related to poor chemotherapeutic response and tumourigenicity in hepatocellular carcinoma.

AIM: Interleukin-8 (IL-8) has been suggested as a prognostic biomarker for human hepatocellular carcinoma (HCC), but its roles in HCC progression and drug resistance have not been studied. This study investigates the role and underlying mechanism of IL-8 in the chemoresistance and progressive growth of HCC. METHODS: The change of chemosensitivity and proportion of side population in hepatoma cells was examined by cell growth and flow cytometric analyses after anti-cancer treatments or knockdown of IL-8. Expression of IL-8 and ATP-binding cassette (ABC) transporters in hepatoma cells, xenograft and clinical HCC tissues was determined by Western blot and immunohistochemical analyses. Tumourigenicity of hepatoma cells was evaluated in vivo after silencing IL-8 gene. RESULTS: Treatment of hepatoma cells with anti-cancer drugs increased the production of IL-8 and its receptor, as well as the proportion of side population (SP). Exogenous IL-8 increased the SP fraction and expression of multidrug resistance-1, decreasing the drug sensitivity. Silencing of IL-8 gene decreased the ratio of SP cells and drug resistance properties. Both IL-8 and ABC transporters were highly expressed in xenograft and clinical HCC tissues, and knockdown of IL-8 significantly reduced tumour size in vivo. CONCLUSION: Anti-cancer drug-induced IL-8 secretion increased the expression of ABC transporters and SP cells, promoting the growth of HCC in vitro. Thus IL-8 may be a potential therapeutic target in the treatment of HCC.

Valproic acid promotes differentiation of hepatocyte-like cells from whole human umbilical cord-derived mesenchymal stem cells.

Mesenchymal stem cells (MSCs) are mesoderm-derived cells that are considered a good source of somatic cells for treatment of many degenerative diseases. Previous studies have reported the differentiation of mesodermal MSCs into endodermal and ectodermal cell types beyond their embryonic lineages, including hepatocytes and neurons. However, the molecular pathways responsible for the direct or indirect cell type conversion and the functional ability of the differentiated cells remain unclear and need further research. In the present study, we demonstrated that valproic acid (VPA), which is a histone deacetylase inhibitor, induced an increase in the expression of endodermal genes including CXCR4, SOX17, FOXA1, FOXA2, GSC, c-MET, EOMES, and HNF-1ß in human umbilical cord derived MSCs (hUCMSCs). In addition, we found that VPA is able to increase these endodermal genes in hUCMSCs by activating signal transduction of AKT and ERK. VPA pretreatment increased hepatic differentiation at the expense of adipogenic differentiation. The effects of VPA on modulating hUCMSCs fate were diminished by blocking AKT and ERK activation using specific signaling inhibitors. Together, our results suggest that VPA contributes to the lineage conversion of hUCMSCs to hepatic cell fate by upregulating the expression of endodermal genes through AKT and ERK activation.

Biochemical and morphological effects of hypoxic environment on human embryonic stem cells in long-term culture and differentiating embryoid bodies.

The mammalian reproductive tract is known to contain 1.5-5.3% oxygen (O(2)), but human embryonic stem cells (hESCs) derived from preimplantation embryos are typically cultured under 21% O(2) tension. The aim of this study was to investigate the effects of O(2) tension on the long-term culture of hESCs and on cell-fate determination during early differentiation. hESCs and embryoid bodies (EBs) were grown under different O(2) tensions (3, 12, and 21% O(2)). The expression of markers associated with pluripotency, embryonic germ layers, and hypoxia was analyzed using RTPCR, immunostaining, and Western blotting. Proliferation, apoptosis, and chromosomal aberrations were examined using BrdU incorporation, caspase-3 immunostaining, and karyotype analysis, respectively. Structural and morphological changes of EBs under different O(2) tensions were comparatively examined using azan- and hematoxylineosin staining, and scanning and transmission electron microscopy. Mild hypoxia (12% O(2)) increased the number of cells expressing Oct4/Nanog and reduced BrdU incorporation and aneuploidy. The percentage of cells positive for active caspase-3, which was high during normoxia (21% O(2)), gradually decreased when hESCs were continuously cultured under mild hypoxia. EBs subjected to hypoxia (3% O(2)) exhibited well-differentiated microvilli on their surface, secreted high levels of collagen, and showed enhanced differentiation into primitive endoderm. These changes were associated with increased expression of Foxa2, Sox17, AFP, and GATA4 on the EB periphery. Our data suggest that mild hypoxia facilitates the slow mitotic division of hESCs in long-term culture and reduces the frequency of chromosomal abnormalities and apoptosis. In addition, hypoxia promotes the differentiation of EBs into extraembryonic endoderm.

Reprogramming fibroblasts into induced pluripotent stem cells with Bmi1.

Somatic cells can be reprogrammed into induced pluripotent stem (iPS) cells by the transcription factors Oct4, Sox2, and Klf4 in combination with c-Myc. Recently, Sox2 plus Oct4 was shown to reprogram fibroblasts and Oct4 alone was able to reprogram mouse and human neural stem cells (NSCs) into iPS cells. Here, we report that Bmi1 leads to the transdifferentiation of mouse fibroblasts into NSC-like cells, and, in combination with Oct4, can replace Sox2, Klf4 and c-Myc during the reprogramming of fibroblasts into iPS cells. Furthermore, activation of sonic hedgehog signaling (by Shh, purmorphamine, or oxysterol) compensates for the effects of Bmi1, and, in combination with Oct4, reprograms mouse embryonic and adult fibroblasts into iPS cells. One- and two-factor iPS cells are similar to mouse embryonic stem cells in their global gene expression profile, epigenetic status, and in vitro and in vivo differentiation into all three germ layers, as well as teratoma formation and germline transmission in vivo. These data support that converting fibroblasts with Bmi1 or activation of the sonic hedgehog pathway to an intermediate cell type that expresses Sox2, Klf4, and N-Myc allows iPS generation via the addition of Oct4.