OBJECTIVE: To determine the effect of pregnancy-associated plasma protein A (PAPP-A) on the function of vascular endothelial cells (VEC). METHODS: Human umbilical vein endothelial cell (HUVEC) line, derived from human umbilical vein, was cultured in vitro with PAPP-A at 0, 50, 100, and 200 ng/mL for 0, 12, 24, and 48 hours, respectively. Nitric oxide (NO) levels and endothlin-1 (ET-1) levels were determined by spectrophotometer and immunehistory. RESULTS: The NO levels of HUVECs in the PAPP-A groups were significantly lower than those in the control group (P<0.05). The ET-1 levels of HUVECs in the PAPP-A groups were significantly higher than those in the control group (P<0.05). The changes were all dose-dependent. CONCLUSION PAPP-A may affect the function of vascular endothelial cells by reducing the secretion of NO and increasing the level of ET-1.
Cell Line ; Endothelial Cells ; cytology ; metabolism ; physiology ; Endothelin-1 ; biosynthesis ; Female ; Humans ; Nitric Oxide ; biosynthesis ; Pregnancy-Associated Plasma Protein-A ; pharmacology ; Umbilical Veins ; cytology ; metabolism

Previously, we demonstrated that the p190 Rho guanine nucleotide exchange factor (p190RhoGEF) was induced following CD40 stimulation of B cells. In this study, we examined whether p190RhoGEF and a downstream effector molecule RhoA are required for B cell differentiation. Expression of p190RhoGEF positively correlated with the expression of surface markers and transcriptional regulators that are characteristic of mature B cells with plasma cell (PC) phenotypes. Moreover, either the overexpression of p190RhoGEF or the expression of a constitutively active RhoA drove cellular differentiation toward PC phenotypes. B cell maturation was abrogated in cells that overexpressed p190RhoGEF and a dominant-negative form of RhoA simultaneously. CD40-mediated maturation events were also abrogated in cells that overexpressed either dominant-negative p190RhoGEF or RhoA. Together, these data provide evidence that p190RhoGEF signaling through RhoA in CD40-activated B cells drives the induction of the PC differentiation.
Animals ; B-Lymphocytes/*cytology/*metabolism ; Cell Differentiation/genetics/*physiology ; Cell Line ; Cells, Cultured ; Female ; Guanine Nucleotide Exchange Factors/genetics/*metabolism ; Humans ; Lymphocyte Activation/genetics/*physiology ; Mice ; Mice, Inbred BALB C ; Plasma Cells/*cytology/*metabolism ; rhoA GTP-Binding Protein/genetics/metabolism

OBJECTIVETo investigate the location and distribution of plasma membrane Ca²⁺ -ATPase isoform 2(PMCA2) in the cochleas of C57BL/6J mice at various ages (4w, 14w, 22w, 45w), and to reveal the relationship of PMCA2 and age-related hearing loss (AHL).

METHODSThe distribution of PMCA2 in the cochleas of C57BL/6J mice was detected by immunohistochemistry at various ages (4w, 14w, 22w, 45w). Real-time polymerase chain reaction (Rt-PCR) was used to detect the level of PMCA2 mRNA in the cochleas of C57BL/6J mice at the ages of 4, 14, 22 and 45 weeks old respectively. Using SPSS17.0 software for statistical analysis.

RESULTSPMCA2 was mainly located in the hear cells, stria vascularis, and spiral ganglion cells. Faint labeling of PMCA2 was also observed in spiral ligament. Hair cells missed and the number of spiral ganglion cells reduced with age. Expression of PMCA2 in the cochleas of C57BL/6J mice also showed age-related decreasing. The results of Rt-PCR demonstrated the expression of mRNA of gene (Atp2b2) at 14 weeks age was significantly less than 4 week-old mice cochlears (P<0.05). The expression of mRNA of gene (Atp2b2) at 22 weeks age was significantly less than 14 week-old mice cochlears (P<0.05). The expression of mRNA of gene (Atp2b2) at 45 weeks age was significantly less than 14 week-old mice cochlears (P<0.01).

CONCLUSIONSPMCA2 is mainly located in the hear cells, stria vascularis, and spiral ganglion cells. Faint labeling of PMCA2 is also observed in spiral ligament. The expression of PMCA2 demonstrates an age-related decrease with age. The mRNA expression level of PMCA2 gene(Atp2b2) in the cochleas of C57BL/6J mice displayed an age-related decrease. PMCA2 transporters may play a critical role in maintaining the normal morphology of the inner ear and it may be related to AHL.

Aging ; Animals ; Cochlea ; enzymology ; Hair Cells, Auditory ; metabolism ; Isoenzymes ; metabolism ; Mice ; Mice, Inbred C57BL ; Plasma Membrane Calcium-Transporting ATPases ; metabolism ; RNA, Messenger ; metabolism ; Real-Time Polymerase Chain Reaction ; Spiral Ganglion ; cytology ; metabolism ; Stria Vascularis ; cytology ; metabolism

OBJECTIVETo study the effect of angiotensin II (Ang II) on pregnancy-associated plasma protein A (PAPP-A) and insulin-like growth factor 1 (IGF-1) mRNA expressions in human umbilical artery smooth muscle cells (HUVSMCs).

METHODSIn the presence or absence of Ox-LDL, HUVSMCs were cultured with Ang II of 10(-5) mol/L for 0, 6, 12, 24, 36, and 48 h, or with Ang II at the concentrations of 10(-7), 10(-6), 10(-5), and 10(-4) mol/L for 24 h, after which the cells were then collected to detect PAPP-A and IGF-1 mRNA expressions in the cells using RT-PCR.

RESULTSAt the concentration of 10(-5) mol/L, Ang II showed a time-dependent effect in inducing PAPP-A and IGF-1 mRNA expressions, which began to increase at 12 h of culture and reaching the highest level at 24 h. Ang II also dose-dependently induced PAPP-A and IGF-1 mRNA expressions, and 10(-5) mol/L Ang II induced the highest expression levels of the two genes. Ox-LDL exposure significantly further increased the expression levels of PAPP-A and IGF-1 mRNA in the cells regardless of the Ang II concentration or duration for cell treatment (P<0.05).

CONCLUSIONAng II can time- and dose-dependently induces PAPP-A and IGF-1 mRNA expression in HUVSMCs and is responsible for inducing platelet activity and inflammatory reaction in acute coronary syndromes, and the effects of Ang II can be enhanced by Ox-LDL.

Angiotensin II ; pharmacology ; Cells, Cultured ; Drug Synergism ; Humans ; Insulin-Like Growth Factor I ; genetics ; metabolism ; Lipoproteins, LDL ; pharmacology ; Myocytes, Smooth Muscle ; cytology ; metabolism ; Pregnancy-Associated Plasma Protein-A ; genetics ; metabolism ; RNA, Messenger ; genetics ; metabolism ; Umbilical Arteries ; cytology ; metabolism

Multiple myeloma (MM) is a malignant tumor, characterized by dysplasia of clonal plasma cells in the bone marrow secreting large amounts of monoclonal immunoglobulin or fragments (M protein), resulting in damage in relevant organs or tissues. The biological complexity of MM is based on disrupted cancer pathways. Except the central role of cytogenetic abnormalities, epigenetic aberrations have also been shown to be involved in the occurrence and development of MM. Epigenetics of MM is mainly concentrated in the ways of DNA methylation, histone modifications and noncoding RNA, which have generated abnormal signaling pathways to regulate cell cycle and apoptosis of MM. In this article, advances of research on epigenetics of development, clinical diagnosis and treatments of MM are reviewed.
Apoptosis ; Bone Marrow ; metabolism ; Cell Cycle ; Chromosome Aberrations ; DNA Methylation ; Epigenesis, Genetic ; Humans ; Multiple Myeloma ; genetics ; Myeloma Proteins ; metabolism ; Plasma Cells ; cytology ; Signal Transduction

BACKGROUND: The objective of this study was to explore whether individual variations in the concentration of growth factors (GFs) influence the biologic effects of platelet-rich plasma (PRP) on human mesenchymal stem cells (HMSCs). METHODS: The concentrations of 7 representative GFs in activated PRP (aPRP) were measured using ELISA. The effects of PRP on the proliferation and alkaline phosphatase (ALP) activity of HMSCs were examined using several concentrations of aPRP from 3 donors; the relationships between the GF levels and these biologic effects were then evaluated using 10% aPRP from 5 subgroups derived from 39 total donors. HMSCs were cultured in DMEM with the addition of aPRP for 4 or 12 days; then, DNA content and ALP activity were measured. RESULTS: The quantity of DNA increased significantly at a 10% concentration of aPRP, but the ALP activity was suppressed at this concentration of aPRP. The GF concentrations varied among donors, and 5 subgroups of characteristic GF release patterns were identified via cluster analysis. DNA levels differed significantly between groups and tended to be higher in groups with higher concentrations of transforming growth factor-beta1 (TGF-beta1) and platelet-derived growth factors (PDGFs). DNA quantity was positively correlated with TGF-beta1 concentration, and was negatively correlated with donor age. ALP activity was negatively correlated with PDGF-BB concentration. CONCLUSIONS: The varying GF concentrations may result in different biologic effects; thus, individual differences in GF levels should be considered for reliable interpretation of the biologic functions and standardized application of PRP.
Alkaline Phosphatase/metabolism ; Blood Donors ; Cell Differentiation ; Cells, Cultured ; Culture Media/chemistry ; DNA/analysis ; Humans ; Intercellular Signaling Peptides and Proteins/*pharmacology ; Mesenchymal Stem Cells/*cytology/drug effects ; Platelet-Derived Growth Factor/pharmacology ; Platelet-Rich Plasma/*metabolism ; Transforming Growth Factor beta1/pharmacology

Auto-transplantation of adipose tissue is commonly used for the treatment of tissue defects in plastic surgery. The survival of the transplanted adipose tissue is not always constant, and one of reasons is the accelerated apoptosis of the implanted preadipocytes. We have recently established highly homogeneous preadipocytes, named ccdPAs. The aim of the current study was to evaluate the regulation of the potency of platelet-rich plasma (PRP) on the apoptosis of ccdPAs in vitro. PRP stimulated the proliferation of the preadipocytes in a dose-dependent manner, and the stimulatory activity of 2% PRP was significantly higher than that of 2% FBS or 2% platelet-poor plasma (PPP). The presence of 2% PRP significantly inhibited serum starvation- or TNF-alpha/cycloheximide-induced apoptosis in comparison to 2% FBS or 2% PPP. DAPK1 and Bcl-2-interacting mediator of cell death (BIM) mRNAs were reduced in the preadipocytes cultured with 2% PRP in comparison to those cultured in 2% FBS. The gene expression levels were significantly higher in cells cultured without serum in comparison to cells cultured with 2% FBS, and the levels in the cells with 2% PRP were reduced to 5-10% of those in the cells without serum. These results indicated that ccdPAs exhibit anti-apoptotic activities, in addition to increased proliferation, when cultured in 2% PRP in comparison to the same concentration of FBS, and that this was accompanied with reduced levels of DAPK1 and BIM mRNA expression in in vitro culture. PRP may improve the outcome of transplantation of adipose tissue by enhancing the anti-apoptotic activities of the implanted preadipocytes.
Adipocytes/*cytology ; Adipose Tissue/cytology/metabolism ; Apoptosis/*physiology ; Apoptosis Regulatory Proteins/antagonists & inhibitors/metabolism ; Calcium-Calmodulin-Dependent Protein Kinases/antagonists & inhibitors/metabolism ; Cell Culture Techniques/*methods ; *Cell Differentiation ; Cell Proliferation ; Cells, Cultured ; Gene Expression Regulation ; Humans ; Membrane Proteins/antagonists & inhibitors/metabolism ; *Platelet-Rich Plasma/metabolism/physiology ; Proto-Oncogene Proteins/antagonists & inhibitors/metabolism ; Tissue Transplantation

The activation and deactivation of Ca(2+)- and calmodulindependent neuronal nitric oxide synthase (nNOS) in the central nervous system must be tightly controlled to prevent excessive nitric oxide (NO) generation. Considering plasma membrane calcium ATPase (PMCA) is a key deactivator of nNOS, the present investigation aims to determine the key events involved in nNOS deactivation of by PMCA in living cells to maintain its cellular context. Using time-resolved Förster resonance energy transfer (FRET), we determined the occurrence of Ca(2+)-induced protein-protein interactions between plasma membrane calcium ATPase 4b (PMCA4b) and nNOS in living cells. PMCA activation significantly decreased the intracellular Ca(2+) concentrations ([Ca(2+)]i), which deactivates nNOS and slowdowns NO synthesis. Under the basal [Ca(2+)]i caused by PMCA activation, no protein-protein interactions were observed between PMCA4b and nNOS. Furthermore, both the PDZ domain of nNOS and the PDZ-binding motif of PMCA4b were essential for the protein-protein interaction. The involvement of lipid raft microdomains on the activity of PMCA4b and nNOS was also investigated. Unlike other PMCA isoforms, PMCA4 was relatively more concentrated in the raft fractions. Disruption of lipid rafts altered the intracellular localization of PMCA4b and affected the interaction between PMCA4b and nNOS, which suggest that the unique lipid raft distribution of PMCA4 may be responsible for its regulation of nNOS activity. In summary, lipid rafts may act as platforms for the PMCA4b regulation of nNOS activity and the transient tethering of nNOS to PMCA4b is responsible for rapid nNOS deactivation.
Animals ; Brain ; metabolism ; Calcium ; metabolism ; Cells, Cultured ; Cerebellum ; cytology ; Fluorescence Resonance Energy Transfer ; HEK293 Cells ; Humans ; Nitric Oxide ; metabolism ; Nitric Oxide Synthase Type I ; metabolism ; PDZ Domains ; Plasma Membrane Calcium-Transporting ATPases ; metabolism ; Protein Interaction Maps ; Protein Isoforms ; metabolism ; Rats ; Rats, Sprague-Dawley

PURPOSE: To distinguish lupus flare-up from infection in systemic lupus erythematosus (SLE), we analyze the expression of circulating CD27(high) plasma cells in SLE patients with and without infection, in comparison to non-SLE patients with infection. MATERIALS AND METHODS: The percentage of circulating CD27(high) plasma cells was measured by flow cytometry in the following four groups: 36 SLE patients without infection, 23 SLE patients with infection, eight non-SLE patients with infection, and 26 healthy controls. RESULTS: The frequency of CD27(high) plasma cells had a correlation with the SLE disease activity index (SLEDAI) (r = 0.866, p < 0.05), level of anti-dsDNA (r = 0.886, p < 0.05), C3 (r = - 0.392, p < 0.05), and C4 (r = - 0.337, p < 0.05) in SLE patients without infection, but there was no correlation with disease activity in SLE patients with infection. Among three groups in particular-SLE without infection, SLE with infection, and non-SLE with infection-the percentages of CD27(high) plasma cells were elevated. The percentage of CD27(high) plasma cells was higher in SLE patients with infection, when compared to SLE patients without infection. CONCLUSION: The percentage of CD27(high) plasma cells is a biomarker for disease activity of SLE without infection, under correlation with SLEDAI, anti-dsDNA, and C3 and C4 level. However, when the SLE patients have an infection, the percentage of CD27(high) plasma cells is not an adequate biomarker for the survey of disease activity. The percentage of CD27(high) plasma cells may serve as a potential parameter to distinguish a lupus flare-up from infection.
Adult ; Antigens, CD27/*biosynthesis ; Bacterial Infections/complications ; Biological Markers/metabolism ; Case-Control Studies ; Female ; Flow Cytometry/methods ; Humans ; Lupus Erythematosus, Systemic/*blood/immunology ; Male ; Plasma Cells/cytology/*immunology ; Virus Diseases/complications

OBJECTIVEWe explored the expressions of the Notch and Wnt signaling pathways and their significance in the repair process of alveolar bone defects by establishing animal models with a composite of autologous bone marrow mesenchymal stem cells (BMSCs) and platelet-rich fibrin (PRF) to repair bone defects in the extraction sockets of rabbits.

METHODSA total of 36 two-month-old male New Zealand white rabbits were randomly divided into four groups, and the left mandibular incisors of all the rabbits were subjected to minimally invasive removalunder general anesthesia. BMSC-PRF compounds, single PRF, and single BMSC were implanted in Groups A, B, and C. No material was implanted in Group D (blank control). The animals were sacrificed at 4, 8 and 12 weeks after surgery, the bone defect was immediately drawn, and the bone specimens underwent surgery after four, eight, and twelve weeks, with three rabbits per time point. The expressions of Notch1 and Wnt3a in the repair process of the bone defect were measured via immunohistochemical and immunofluorescence detection.

RESULTSImmunohistochemistry showed that the expressions of Notch1 and Wnt3a in Groups A, B, and C were higher than that in Group D at the fourth and eighth week after operation (P<0.05). By contrast, the expressions of Notch1 and Wnt3a in Group D were higher than those in Groups A, B, and C at the twelfth week (P<0.05). Immunofluorescence showed that the expressions of both Notch1 and Wnt3a reached their peaks in the new bone cells of the bone defect after four weeks following surgery and gradually disappeared when the bone was repaired completely.

CONCLUSIONNotch1 and Wnt3a signaling molecules are expressed in the process of repairing bone defects using BMSC-PRF composites and can accelerate the healing by regulating the proliferation and differentiation of BMSCs. Moreover, the expressions of Notch and Wnt are similar, and a crosstalk between them may exist it.

Alveolar Bone Grafting ; methods ; Animals ; Blood Platelets ; Bone Marrow Cells ; cytology ; Bone Transplantation ; methods ; Bone and Bones ; abnormalities ; Cell Differentiation ; Fibrin ; administration & dosage ; Male ; Mesenchymal Stem Cell Transplantation ; methods ; Mesenchymal Stromal Cells ; Platelet-Rich Plasma ; Rabbits ; Random Allocation ; Receptor, Notch1 ; metabolism ; Tissue Engineering ; Wnt Signaling Pathway ; Wnt3A Protein ; metabolism ; Wound Healing