Mesenchymal stem cells (MSCs) can inhibit T cell proliferation; however, the underlying mechanisms are not clear. In this study, we investigated the mechanisms of the immunoregulatory activity of MSCs on T cells. Irradiated MSCs co-cultured with either naive or pre-activated T cells in a mixed lymphocyte reaction (MLR) significantly suppressed T cell proliferation in a dose-dependent manner, irrespective of allogeneic disparity between responders and MSCs. Transwell assays revealed that the suppressive effect was primarily mediated by soluble factors that induced apoptosis. Splenocytes stimulated with alloantigen in the presence of the MSC culture supernatant (CS) produced a significant amount of IL-10, which was attributed to an increase in the number of IL-10 secreting cells, confirmed by an ELISPOT assay. The blockade of IL-10 and IL-10 receptor interaction by anti-IL-10 or anti-IL-10-receptor antibodies abrogated the suppressive capacity of MSC CS, indicating that IL-10 plays a major role in the suppression of T cell proliferation. The addition of 1-methyl-DL-tryptophan (1-MT), an indoleamine 2,3-dioxygenase (IDO) inhibitor, also restored the proliferative capacity of T cells. In conclusion, we demonstrated that soluble mediators from culture supernatant of MSCs could suppress the proliferation of both naive and pre-activated T cells in which IL-10 and IDO play important roles.
Animals ; Cell Proliferation ; Cells, Cultured ; Indoleamine-Pyrrole 2,3,-Dioxygenase/antagonists & inhibitors/metabolism ; Interleukin-10/*biosynthesis ; *Lymphocyte Activation ; Lymphokines/pharmacology ; Mesenchymal Stem Cells/cytology/*metabolism ; Mice ; Mice, Inbred BALB C ; Mice, Inbred C57BL ; Receptors, Interleukin-10/metabolism ; T-Lymphocytes/cytology/*immunology/metabolism ; Tryptophan/analogs & derivatives/pharmacology

Mesenchymal stem cells (MSCs) can inhibit T cell proliferation; however, the underlying mechanisms are not clear. In this study, we investigated the mechanisms of the immunoregulatory activity of MSCs on T cells. Irradiated MSCs co-cultured with either naive or pre-activated T cells in a mixed lymphocyte reaction (MLR) significantly suppressed T cell proliferation in a dose-dependent manner, irrespective of allogeneic disparity between responders and MSCs. Transwell assays revealed that the suppressive effect was primarily mediated by soluble factors that induced apoptosis. Splenocytes stimulated with alloantigen in the presence of the MSC culture supernatant (CS) produced a significant amount of IL-10, which was attributed to an increase in the number of IL-10 secreting cells, confirmed by an ELISPOT assay. The blockade of IL-10 and IL-10 receptor interaction by anti-IL-10 or anti-IL-10-receptor antibodies abrogated the suppressive capacity of MSC CS, indicating that IL-10 plays a major role in the suppression of T cell proliferation. The addition of 1-methyl-DL-tryptophan (1-MT), an indoleamine 2,3-dioxygenase (IDO) inhibitor, also restored the proliferative capacity of T cells. In conclusion, we demonstrated that soluble mediators from culture supernatant of MSCs could suppress the proliferation of both naive and pre-activated T cells in which IL-10 and IDO play important roles.
Animals ; Cell Proliferation ; Cells, Cultured ; Indoleamine-Pyrrole 2,3,-Dioxygenase/antagonists & inhibitors/metabolism ; Interleukin-10/*biosynthesis ; *Lymphocyte Activation ; Lymphokines/pharmacology ; Mesenchymal Stem Cells/cytology/*metabolism ; Mice ; Mice, Inbred BALB C ; Mice, Inbred C57BL ; Receptors, Interleukin-10/metabolism ; T-Lymphocytes/cytology/*immunology/metabolism ; Tryptophan/analogs & derivatives/pharmacology

To investigate the effect of arsenic trioxide (As(2)O(3)) on vascular endothelial growth factor (VEGF) expression in different courses of chronic myeloid leukemia (CML), VEGF level was measured with ELISA in the cultural supernatants of bone marrow mononuclear cells from CML patients. The results showed that supernatants of cultured bone marrow cells from 35 CML patients (20 chronic, 8 accelerated and 7 blast crisis phases) contained significantly higher VEGF levels (649.16 +/- 382.20 pg/ml, 560.27 +/- 409.14 pg/ml and 587.18 +/- 415.28 pg/ml, respectively) than that in 15 normal control samples (152.16 +/- 150.09 pg/ml; P < 0.01), but no significant differences were found in VEGF levels among different phases of CML. The bone marrow cells treated with As(2)O(3) (5 x 10(-6)mol/L) for 72 hours resulted in significant reduction of VEGF levels (down to 396.66 +/- 257.47 pg/ml, 363.42 +/- 239.85 pg/ml and 407.47 +/- 219.38 pg/ml, respectively) (P < 0.05). In conclusion, abnormal high expression of VEGF plays a role in the pathogenetic course of CML and it is probably an additional anticancer mechanism for As(2)O(3) to inhibit VEGF expression of leukemic cells.
Adolescent ; Adult ; Aged ; Arsenicals ; pharmacology ; Bone Marrow Cells ; drug effects ; metabolism ; Cells, Cultured ; Child ; Culture Media, Conditioned ; chemistry ; Down-Regulation ; drug effects ; Endothelial Growth Factors ; metabolism ; Enzyme-Linked Immunosorbent Assay ; Female ; Humans ; Intercellular Signaling Peptides and Proteins ; metabolism ; Leukemia, Myelogenous, Chronic, BCR-ABL Positive ; blood ; pathology ; Lymphokines ; metabolism ; Male ; Middle Aged ; Oxides ; pharmacology ; Vascular Endothelial Growth Factor A ; Vascular Endothelial Growth Factors

Notch signal path plays important roles in the regulation of proliferation and differentiation of hematopoietic stem cells. An extracellular domain of human Delta-like-1 (hDll-1(ext)), one of Notch ligands, was cloned and expressed in CHO cells, and the effect of hDll-1(ext) on expansion of hematopoietic stem/progenitor cells was investigated in this study. Total RNA was isolated from human marrow mononuclear cells. hDll-1(ext) was amplified by RT-PCR and cloned to T vector, then the gene was sequenced and subcloned to pcDNA3.1/Myc-His(+)A expression vector. The constructed plasmid was transfected into CHO cells with lipofectin and the expression of secreted hDll-1(ext) in G418-resistant clones was assayed by Western blot. hDll-1(ext) high-expressed clone was cultured to collect supernatant. Fusion protein hDll-1(ext) was purified from the supernatant by immobilized metal affinity chromatography (IMAC). The results showed that expression of Notch-1 receptor was detected in cord blood-derived CD34(+) cells by RT-PCR. Human umbilical blood CD34(+) cells were cultured in serum-free medium containing SCF, IL-3, VEGF, and with or without purified hDll-1(ext) for 4 or 8 days. Effect of hDll-1(ext) on the expansion of progenitor cells was analyzed then by clonogenic assays. The number of CFU-Mix and HPP-CFC generated from the culture system containing hDll-1(ext) was 1.5 times of that from the control. In conclusion, the recombinant hDll-1(ext) promotes the expansion of primitive hematopoietic progenitors.
Animals ; Antigens, CD34 ; immunology ; Binding Sites ; genetics ; CHO Cells ; Cell Division ; drug effects ; physiology ; Colony-Forming Units Assay ; Cricetinae ; Endothelial Growth Factors ; pharmacology ; Fetal Blood ; cytology ; immunology ; metabolism ; Gene Expression ; Genetic Vectors ; genetics ; Glycoproteins ; genetics ; pharmacology ; physiology ; Hematopoietic Stem Cells ; cytology ; drug effects ; Humans ; Intercellular Signaling Peptides and Proteins ; pharmacology ; Interleukin-3 ; pharmacology ; Lymphokines ; pharmacology ; Membrane Proteins ; genetics ; RNA ; genetics ; metabolism ; Receptor, Notch1 ; Receptors, Cell Surface ; Recombinant Proteins ; isolation & purification ; pharmacology ; Reverse Transcriptase Polymerase Chain Reaction ; Stem Cell Factor ; pharmacology ; Transcription Factors ; Transfection ; Vascular Endothelial Growth Factor A ; Vascular Endothelial Growth Factors

OBJECTIVETo explore the response of nasal mucosa epithelial cells to hypoxia in terms of formation of nasal polyps (NP).

METHODSEpithelial cells of NP and inferior turbinate (IT) were cultured serum-free under normal oxygen and hypoxic circumstances with stimulation of IL-1 beta and TNF alpha. The vascular endothelial growth factor (VEGF) mRNA and VEGF protein levels of the cultured cells were detected using in situ hybridization and ELISA, respectively.

RESULTSThe expression of VEGF mRNA was significantly higher in epithelial cells of NP than in IT exposed to pro-inflammatory cytokines or hypoxia (P < 0.01). VEGF levels were higher in NP epithelial cells than those of IT (P < 0.01) under hypoxia.

CONCLUSIONVEGF-induced by hypoxia is very important for the early stages of forming polyps.

Cell Hypoxia ; physiology ; Cells, Cultured ; Endothelial Growth Factors ; genetics ; Enzyme-Linked Immunosorbent Assay ; Erythropoietin ; genetics ; Humans ; Intercellular Signaling Peptides and Proteins ; genetics ; Interleukin-1 ; pharmacology ; Lymphokines ; genetics ; Nasal Mucosa ; metabolism ; Nasal Polyps ; etiology ; metabolism ; RNA, Messenger ; analysis ; Tumor Necrosis Factor-alpha ; pharmacology ; Vascular Endothelial Growth Factor A ; Vascular Endothelial Growth Factors

OBJECTIVETo investigate the pro-angiogenic effects of several multiple myeloma (MM) cell line culture supernatants on human bone marrow endothelial cell (HBMEC) proliferation, migration, and capillary formation, and the anti-angiogenic effects of thalidomide.

METHODSHBMEC was cultured in the presence of MM cell lines (IM9, XG1, U266 and MOLP-5) supernatants. Proliferation and migration of HBMEC were determined, capillary-like tubule formation of HBMEC was examined in fibrin and Matrigel. The inhibiting effect of thalidomide was investigated by adding it into myeloma cell line culture supernatants. Vascular endothelial growth factor (VEGF) was measured by ELISA.

RESULTS(1) MM cell lines culture supernatants promoted HBMEC proliferation and migration. (2) In fibrin and Matrigel, capillary-like tubule network formation promoted by the supernatants. (3) All of these effects could be inhibited by thalidomide. (4) This effect was not related to VEGF in the supernatants.

CONCLUSIONSMM cell line promote proliferation, migration and tubule formation by secreting VEGF or other several cytokines. Thalidomide can inhibit these effects.

Angiogenesis Inhibitors ; pharmacology ; Bone Marrow ; blood supply ; Cell Division ; drug effects ; Cell Movement ; drug effects ; Cells, Cultured ; Culture Media, Conditioned ; chemistry ; pharmacology ; Endothelial Growth Factors ; metabolism ; Endothelium, Vascular ; cytology ; drug effects ; physiology ; Enzyme-Linked Immunosorbent Assay ; Humans ; Intercellular Signaling Peptides and Proteins ; metabolism ; Lymphokines ; metabolism ; Multiple Myeloma ; pathology ; secretion ; Neovascularization, Physiologic ; drug effects ; Thalidomide ; pharmacology ; Tumor Cells, Cultured ; Vascular Endothelial Growth Factor A ; Vascular Endothelial Growth Factors

OBJECTIVETo investigate the effect of antisense vascular endothelial growth factor (VEGF)(121) cDNA transfection on the growth of K562 cells in nude mice.

METHODSK562 cells transfected with the antisense (AS) or sense (S) VEGF(121) cDNA, and the vector (V, pcDNA3) alone were transplanted subcutaneously into nude mice and the growth of the transfected cells in vivo was investigated. The effects of transfected K562 cells on human bone marrow endothelial cells (BMEC) were analyzed by MTT assay, the microvessel density (MVD) in tumor mass by vWF immunohistochemistry stain.

RESULTSK562/V tumor grew more slowly [(207.5 +/- 192.9) mm(3) vs (445.0 +/- 150.9) mm(3), P < 0.05] and K562/S tumor more rapidly than K562/V tumor did [(1 174.6 +/- 508.7)/mm(3) vs (445.0 +/- 150.9) mm(3), P < 0.01]. K562/S cell culture supernatant was more strongly in promoting the proliferation of BMEC than K562/V supernatant did, but K562/AS supernatant resulted in a marked decrease of the promoting effect as compared with K562/V's. The MVDs in K562/AS, K562/S, and K562/V tumors were [(11.0 +/- 7.6)/0.72 mm(2) vs (50.8 +/- 11.7)/0.72 mm(2) vs (18.9 +/- 7.0)/0.72 mm(2)], respectively.

CONCLUSIONSAntisense VEGF(121) cDNA transfected K562 cells show growth retardation in transplanted nude mice, decrease of tumor MVD, and decrease of promoting BMEC proliferation capacity.

Animals ; Bone Marrow Cells ; cytology ; drug effects ; Cell Division ; genetics ; physiology ; Culture Media, Conditioned ; pharmacology ; DNA, Antisense ; genetics ; DNA, Complementary ; genetics ; Endothelial Growth Factors ; genetics ; physiology ; Endothelium, Vascular ; cytology ; drug effects ; Female ; Humans ; K562 Cells ; Lymphokines ; genetics ; physiology ; Mice ; Mice, Inbred BALB C ; Mice, Nude ; Neoplasm Transplantation ; Neoplasms, Experimental ; blood supply ; genetics ; pathology ; Neovascularization, Pathologic ; genetics ; physiopathology ; Transfection ; Vascular Endothelial Growth Factor A ; Vascular Endothelial Growth Factors

The present study identified the favorable environment conditions for Spermatogomial stem cells in vitro according to their unique biological properties. Three growth factors, stem cell factor (SCF), leukemia inhibitory factor (LIF) and basic fibroblast growth factor (bFGF) were all found to independently contribute to the proliferation of mouse spermatogonial stem cell. The percentage of cell proliferation significantly enhanced by SCF at 30 ng/mL but decreased with heightening its combination after cultured 120 hours. The mice spermatogonial stem cells were significantly proliferated after 120 hours' culture with 10 ng/mL and 20 ng/mL (P < 0.01) of LIF, between 20 ng/mL and 50 ng/mL (P < 0.01) for bFGF. SCF and bFGF were significantly enhanced mice spermatogonial stem cells proliferation after these three factors combination. For LIF, no obvious effect was observed.
Animals ; Cell Division ; drug effects ; Cells, Cultured ; Dose-Response Relationship, Drug ; Fibroblast Growth Factor 2 ; pharmacology ; Growth Inhibitors ; pharmacology ; Interleukin-6 ; Leukemia Inhibitory Factor ; Lymphokines ; pharmacology ; Male ; Mice ; Spermatogonia ; drug effects ; physiology ; Stem Cell Factor ; pharmacology ; Stem Cells ; drug effects ; physiology

Nitric oxide (NO) seems to play a pivotal role in the vascular endothelial growth factor (VEGF)-induced endothelial cell proliferation. This study was designed to investigate the role and intracellular signal pathway of endothelial nitric oxide synthase (eNOS) activation induced by VEGF. ECV 304 cells were treated with betaVEGF(165) and then cell proliferation, eNOS protein and mRNA expression levels were analyzed to elucidate the functional role of eNOS in cell proliferation induced by VEGF. After exposure of cells to betaVEGF(165) , eNOS activity and cell growth were increased by approximately two-fold in the betaVEGF(165) -treated cells compared to the untreated cells. In addition, VEGF stimulated eNOS expression at both the mRNA and protein levels in a dose-dependent manner. Phosphatidylinositol-3 kinase (PI-3K) inhibitors were used to assess PI-3K involvement in eNOS regulation. LY294002 was found to attenuate VEGF-stimulated eNOS expression. Wortmannin was not as effective as LY294002, but the reduction effect was detectable. Cells activated by VEGF showed increased ERK1/2 levels. Moreover, the VEGF-induced eNOS expression was reduced by the PD98059, MAPK pathway inhibitor. This suggests that eNOS expression might be regulated by PI-3K and the ERK1/2 signaling pathway. In conclusion, betaVEGF(165) induces ECV 304 cell proliferation via the NO produced by eNOS. In addition, eNOS may be regulated by the PI-3K or mitogen-activated protein kinase pathway.
1-Phosphatidylinositol 3-Kinase/*antagonists & inhibitors ; Cell Division/drug effects ; Cell Line ; Endothelial Growth Factors/*metabolism/pharmacology ; Endothelium, Vascular/cytology ; *Gene Expression Regulation, Enzymologic ; Lymphokines/*metabolism/pharmacology ; MAP Kinase Signaling System ; Mitogen-Activated Protein Kinase 1/*antagonists & inhibitors ; Mitogen-Activated Protein Kinase 3 ; Mitogen-Activated Protein Kinases/*antagonists & inhibitors ; Nitric Oxide Synthase/*genetics/metabolism ; Nitric Oxide Synthase Type III ; Signal Transduction ; Vascular Endothelial Growth Factor A ; Vascular Endothelial Growth Factors

OBJECTIVESTo investigate mechanism for the increasing level of serum vascular endothelial growth factor(VEGF) in tumour patients during radiotherapy and the inhibitory action of the antisense oligodeoxynucleotide (AS-ODN) to the expression of VEGF protein by radiotherapy in the prostate cancer cell line (PC3M).

METHODSTo observe the changes of serum VEGF in the prostate cancer patients during radiotherapy dynamically and the inhibitory action of the antisense oligodeoxynucleotide to the expression of VEGF by radiotherapy in PC3M.

RESULTSThe changes of serum VEGF in three patients receiving radiotherapy had been observed continuously. The levels of serum VEGF began to increase when the patients received radiotherapy and rised up to peak value after fifteen days, then declined to the range of pre-radiotherapy. Irradiating the PC3M cells with X-rays significantly increased the VEGF expression and secretion. The expression of VEGF protein in the group treated by VEGF AS-ODNs and X-ray irradiation decreased significantly than the group treated only by X-ray irradiation.

CONCLUSIONSThe induction of VEGF protein expression by X-ray irradiation in tumor cells may result in the increasing of the VEGF in the prostate cancer patients during radiotherapy and the induction can be blocked by VEGF AS-ODNs.

DNA, Antisense ; pharmacology ; Endothelial Growth Factors ; antagonists & inhibitors ; blood ; genetics ; Gene Expression ; drug effects ; radiation effects ; Humans ; Intercellular Signaling Peptides and Proteins ; blood ; genetics ; Lymphokines ; antagonists & inhibitors ; blood ; genetics ; Male ; Prostatic Neoplasms ; blood ; pathology ; Radiotherapy ; adverse effects ; Tumor Cells, Cultured ; Vascular Endothelial Growth Factor A ; Vascular Endothelial Growth Factors