Clinical transplantation has indicated that cord blood (CB) can be used in the hematopoietic reconstitution in the children, but not well used in the adult patients because of the low cell amount. The present study aimed to explore the capability of proliferation and differentiation of the hematopoietic stem/progenitor cells derived from human mature placenta tissue (PT) in vitro, and to find a new source of hematopoietic/progenitor cells for clinical transplantation. CD34(+) cells in human mature placenta tissue were isolated and characterized by using enzyme-digestion method and flow cytometry. A long culture system without cytokines was established with human mature placenta tissue-derived mononucleated cells and cord blood mononuclear cells. The number of nucleated cells was weekly counted in culture for 14 weeks. The number of CFC was counted in culture for 2 weeks. The results showed that the CFC yields (CFU-GM, 186.90 +/- 24.52; BFU-E, 101.40 +/- 13.35) and the percentage of CD34(+) cells (2.74 +/- 0.61%) and CD34(+)/CD38(-) cells (2.46 +/- 0.42%) in placenta tissue (PT) were higher than CFC (CFU-GM, 136.90 +/- 25.15; BFU-E, 49.20 +/- 8.13), CD34(+) cells (1.73 +/- 0.32%) and CD34(+)/CD38(-) cells (0.80 +/- 0.25%) in cord blood (CB). The MNCs from PT have shown more survival ability than the cells from CB in the long-term cell culture condition; and the cells from PT increased by 2 times. It is concluded that the placenta may be another hematopoietic organ in ontogeny. The cells from placenta were more juvenile, and may be favorable source for clinical stem cell transplantation.
Antigens, CD34 ; analysis ; CD36 Antigens ; analysis ; Cell Differentiation ; physiology ; Cells, Cultured ; Hematopoietic Stem Cell Transplantation ; Hematopoietic Stem Cells ; cytology ; Humans ; Leukocytes, Mononuclear ; cytology ; Placenta ; cytology

BACKGROUND: Scavenger receptors are thought to be involved in the recognition of oxidized low-density lipoprotein (oxLDL) and oxidized erythrocyte (oxRBC). However, there are controversies about the kind of receptors and ligands related to the binding. Macrophages lacking class A scavenger receptor show identical binding of oxRBC with wild-type ones. METHODS: RBCs were oxidized with ascorbic acid and CuSO4. Lipid oxidation was measured indirectly by measuring TBARS semiquantitatively. The binding and phagocytosis were measured by counting the number of oxRBC bound or taken up after incubation at 4 degrees C or 37 degrees C for 60 minutes to 100 macrophages differentiated from human monocytic leukemia cell line. RESULTS: The degree of oxidation and the binding of oxRBCs were dependent on the concentration of CuSO4. The binding and phagocytosis of oxRBC were inhibited by 99% with oxLDL. Fucoidan, competing class A scavenger receptor, inhibited the binding by more than 90%. The binding of oxRBC was higher at 37 degrees C than at 4 degrees C by 3 times. The binding of oxRBCs was maximal at pH 6.5 and higher than at physiologic pH by 2.8 times. At pH 8.5 and 9.5, binding decreased by 67 and 88%, respectively. CONCLUSION: OxRBCs might bind and be taken up to macrophages not mainly through class A nor B scavenger receptors, but through other scavenger receptors and/or pathways. These processes are dynamic and ionic strength might be involved.
Antigens, CD36 ; *Erythrocyte Aging ; Erythrocytes/*metabolism ; Human ; Lipoproteins, LDL/metabolism ; Macrophages/*metabolism ; Oxidation-Reduction ; Phagocytosis/*physiology ; Receptors, Immunologic/metabolism ; Support, Non-U.S. Gov't ; Tumor Cells, Cultured/metabolism

Oxidized LDL (OxLDL), a causal factor in atherosclerosis, induces the expression of heat shock proteins (Hsp) in a variety of cells. In this study, we investigated the role of CD36, an OxLDL receptor, and peroxisome proliferator-activated receptor gamma (PPAR gamma) in OxLDL-induced Hsp70 expression. Overexpression of dominant-negative forms of CD36 or knockdown of CD36 by siRNA transfection increased OxLDL-induced Hsp70 protein expression in human monocytic U937 cells, suggesting that CD36 signaling inhibits Hsp70 expression. Similar results were obtained by the inhibition of PPAR gamma activity or knockdown of PPAR gamma expression. In contrast, overexpression of CD36, which is induced by treatment of MCF-7 cells with troglitazone, decreased Hsp70 protein expression induced by OxLDL. Interestingly, activation of PPAR gamma through a synthetic ligand, ciglitazone or troglitazone, decreased the expression levels of Hsp70 protein in OxLDL-treated U937 cells. However, major changes in Hsp70 mRNA levels were not observed. Cycloheximide studies demonstrate that troglitazone attenuates Hsp70 translation but not Hsp70 protein stability. PPAR gamma siRNA transfection reversed the inhibitory effects of troglitazone on Hsp70 translation. These results suggest that CD36 signaling may inhibit stress- induced gene expression by suppressing translation via activation of PPAR gamma in monocytes. These findings reveal a new molecular basis for the anti-inflammatory effects of PPAR gamma.
Antigens, CD36/*physiology ; Cell Line, Tumor ; Chromans/pharmacology ; Cycloheximide/pharmacology ; HSP70 Heat-Shock Proteins/*biosynthesis ; Humans ; Lipoproteins, LDL/pharmacology/*physiology ; Monocytes/drug effects/metabolism ; PPAR gamma/agonists/antagonists & inhibitors/*physiology ; Protein Synthesis Inhibitors/pharmacology ; Signal Transduction ; Thiazolidinediones/pharmacology

During chronic inflammatory response, mono- cytes/macrophages produce 92-kDa matrix metalloproteinase-9 (MMP-9), which may contribute to their extravasation, migration and tissue remodeling. Activation of peroxisome proliferator- activated factor receptor-gamma (PPAR-gamma) has been shown to inhibit MMP-9 activity. To evaluate whether ox-LDL, a PPAR-gamma activator, inhibits PMA-induced MMP-9 expression and activity, and if so, whether CD36 and PPAR-gamma are involved in this process, we investigated the effect of ox-LDL on MMP-9 expression and activity in PMA-activated human monocytic cell line U937. PMA-induced MMP-9 expression and activity were suppressed by the treatment with ox-LDL (50 micrigram/ml) or PPAR-gamma activators such as troglitazone (5 micrometer), ciglitazone (5 micrometer), and 15d- PGJ2 (1 micrometer) for 24 h. This ox-LDL or PPAR-gamma activator-mediated inhibition of micrometer P-9 activity was diminished by the pre-treatment of cells with a blocking antibody to CD36, or PGF2a (0.3 micrometer), which is a PPAR-gamma inhibitor, as well as overexpression of a dominant-negative form of CD36. Taken together, these results suggest that ox-LDL suppresses PMA-induced MMP-9 expression and activity through CD36-mediated activation of PPAR-gamma.
Antibodies, Blocking/pharmacology ; Antigens, CD36/immunology/*physiology ; Cells, Cultured ; Chromans/pharmacology ; Gelatinase B/antagonists & inhibitors/genetics/*metabolism ; Humans ; Lipoproteins, LDL/pharmacology/*physiology ; Monocytes/drug effects/*enzymology/metabolism ; NF-kappa B/antagonists & inhibitors ; PPAR gamma/*metabolism ; Prostaglandin D2/*analogs & derivatives/pharmacology ; RNA, Messenger/analysis/metabolism ; Research Support, Non-U.S. Gov't ; Tetradecanoylphorbol Acetate/antagonists & inhibitors/pharmacology ; Thiazolidinediones/pharmacology ; Transcription, Genetic/drug effects

Both hydropathy plot and in vitro translation results predict the topology of SR-BI; the receptor is an integral membrane protein of 509 amino acids, consisting of a short cytoplasmic N-terminus of 9 amino acids followed by a first transmembrane domain of 22 amino acids, the extracellular domain of 408 amino acids, the second transmembrane domain of 22 amino acids, and the cytoplasmic C-terminus of 47 amino acids. The immunoblot of rBBMV in the presence or absence of pAb589 peptide antigen (the C-terminal 22 amino acid residues of SR-BI) confirmed that the bands at apparent molecular weight of 140 and 210 kDa are SR-BI related protein which might be multimeric forms of SR-BI. 125I apo A-I overlay analysis showed that SR-BI can bind to its ligand, apo A-I, only when it is thoroughly matured - glycosylated and dimerized. The antibody which was generated against extracellular domain of SR-BI (pAb230) not only prevented 125I-labeled apo A-I from binding to 140 kDa band but also inhibited the esterified cholesterol uptake of rabbit BBMV with its IC50 value of 40 microgram/ml of IgG. In contrast, the antibody generated against the C-terminal domain of SR-BI (pAb589) did not show any effect either on cholesterol uptake of rabbit BBMV or 125I-labeled apo A-I binding to 140 kDa band. Overall results show that the ligand binding site of SR-BI in rabbit BBMV is located in extracellular domain, and SR-BI is only functional when it is part of dimeric forms which rationalize the previously found cooperative nature of the binding interaction and maybe a fundamental finding towards the so far poorly understood mechanism of SR-BI function.
Amino Acid Sequence ; Animals ; Antigens, CD36/*metabolism ; Apolipoprotein A-I/metabolism ; Binding Sites/physiology ; Blotting, Western ; Caco-2 Cells ; Cholesterol Esters/metabolism ; Humans ; Intestinal Mucosa/metabolism ; Intestine, Small/*metabolism/ultrastructure ; Iodine Radioisotopes ; Membrane Proteins/*metabolism ; Microvilli/metabolism ; Molecular Sequence Data ; Rabbits ; *Receptors, Immunologic ; Receptors, Lipoprotein/*metabolism ; Receptors, Scavenger ; Scavenger Receptors, Class B ; Surface Properties

The purpose of the present study is to explore the effect of oxidized low density lipoprotein (ox-LDL) on the induction of endoplasmic reticulum stress (ERS) and the underlying mechanisms in ox-LDL-induced macrophage foam-forming process. RAW264.7 macrophages were cultured in DMEM medium containing 10% fetal bovine serum, and then treated with ox-LDL (25, 50 and 100 mg/L), anti-CD36 monoclonal antibody+ox-LDL and tunicamycin (TM), respectively. After incubation for 24 h, the cells were collected. The cellular lipid accumulation was showed by oil red O staining and the content of cellular total cholesterol was quantified by enzymatic colorimetry. The expression of glucose-regulated protein 94 (GRP94), a molecular marker of ERS, was determined by immunocytochemistry assay. The levels of GRP94 protein, phosphorylated inositol-requiring enzyme 1 (p-IRE1) and X box binding protein 1 (XBP1) in RAW264.7 cells were detected by Western blotting. The results indicated that after incubation with ox-LDL (25, 50 and 100 mg/L) for 24 h, a large amount of lipid droplets were found in the cytoplasm, and the contents of cellular total cholesterol were increased by 2.1, 2.8 and 3.1 folds compared with the control, respectively. Anti-CD36 antibody decreased markedly the cellular lipid accumulation induced by ox-LDL at 100 mg/L. Both ox-LDL and TM, a specific ERS inducer, could up-regulate the protein expression of GRP94 in a dose-dependent manner. Furthermore, p-IRE1 and XBP1, two key components of the unfolded protein response, were also significantly induced by the treatment with ox-LDL. The up-regulations of the three proteins induced by ox-LDL were inhibited significantly when the macrophages were pre-incubated with anti-CD36 antibody. These results suggest that ox-LDL may induce ERS in a dose-dependent way and subsequently activate the unfolded protein response signaling pathway in RAW264.7 macrophages, which is potentially mediated by scavenger receptor CD36.
Animals ; CD36 Antigens ; physiology ; Cell Line ; Cells, Cultured ; DNA-Binding Proteins ; metabolism ; Endoplasmic Reticulum ; drug effects ; Foam Cells ; cytology ; Lipoproteins, LDL ; pharmacology ; Macrophages ; cytology ; Membrane Glycoproteins ; metabolism ; Membrane Proteins ; metabolism ; Mice ; Protein-Serine-Threonine Kinases ; metabolism ; Regulatory Factor X Transcription Factors ; Stress, Physiological ; drug effects ; Transcription Factors ; metabolism ; X-Box Binding Protein 1