| Background and objectiveAngiogenesis is the growth of new capillary from pre-exsiting microvasculature, which sustains tissue growth and body metabolism. Angiogenesis is the pro-angiogenic factor and inhibitory factor (anti-angiogenesis) process which are in coordination in the equilibrium state. The emergence of many diseases can be attributed to the break of the balance of angiogenesis, such as excessive angiogenesis: cancer, arthritis, atherosclerosis, diabetic retinopathy; insufficient angiogenesis: heart-brain ischemia, hypertension, diabetes, nephropathy, etc.VEGF (Vascular endothelial growth factor) and its receptor VEGFR1(vascular endothelial growth factor receptor1), VEGFR2(vascular endothelial growth factor receptor2) are the most important factors in regulation of angiogenesis in vivo. VEGFR1and VEGFR2receptor are the main effect of VEGF receptor, they are expressed in vascular endothelial cells mainly and some non vascular cells such as stem cells, megakaryocytes, platelet precursors, mononuclear cells and DC cells, nerve cells. VEGFR2is considered to be the most important vascular growth receptor to promote angiogenesis effect when combined with VEGF. The binding ability of VEGFR1to VEGF is stronger than VEGFR2, but the angiogenic ability of VEGFR1is much weaker than that of VEGFR2, so VEGFR1is considered the factor inhibiting angiogenesis.Transmembrane molecule VEGFR1length is180kDa, easy to splice to a truncated110kDa variant sVEGFR1. sVEGFRl is very easy to combine with VEGF, which hampered the combination of VEGFR2to VEGF, thereby blocking a series of ways to promote angiogenesis, inhibiting vascular growth. So sVEGFR1is considered to be the strongest anti-angiogenesis factor. There is a growing interesting in biomedical research to explore sVEGFRl as a disease marker and a therapeutic vector for angiogenesis inhibition.Mitogen activated protein kinase (MAPK) cascade have very important role on cell growth, differentiation, apoptosis, inflammation and cell response to external stimuli. MAPK family have four members:C-Jun base terminal kinase (JNK) stress activated protein kinase (SAPK), extra cellular signal-regulated protein kinase (ERK), ERK5/big MAP MAP kinase pathway (BMK1), and p38mitogen activated protein kinase(p38MAPK). P38MAPK signaling pathway is found in1993, when the bacteria lipopolysaccharide (LPS) transfec with CD14gene in mice before B lymphocyte, RAW264.7and C3HeB Raw/FeJ (LPS+) it was found that macrophages p38MAPK molecular tyrosine phosphorylation, thus the p38MAPK pathway was found. Itplay an important role in LPS mediated-inflammatory response and regulation mechanism. Moreover p38MAPK pathway was very important in cancer research, it may be involved in the occurrence of tumor, proliferation, movement, and in the regulation of extracellular matrix degradation and plays an important role in angiogenesis of tumor cells.Growth arrest and DNAdamage inducible protein45(Gadd45) family is a pressure signal of body to the outside physical and environmental stress responsef, Gadd45gene is small, their coding are18kDa, its family members including gadd45a, gadd45b, gadd45g, members of the amino acid sequence similarity is55%to58%, having a high homology. The cells can be found in cells’cytoplasm and nucleus. Gadd45protein were induced by changes of environmental and physical conditions such as methyl methane sulfonic acid ester, radiation, ultraviolet light, soft erythromycin and inflammation factor stimulated, they worked as sensors of outside pressure to stimulate. Gadd45a produce physical and chemical changes or interact with other proteins, the role of these proteins result in that cells adjusting their the cell cycle-causes the stagnation of the cell cycle, DNA repair, or make the cell apoptosis. Gadd45proteins in the body are important regulators of inhibition of tumor, if lack of gadd45proteins in mice probability increase its mutation greatly, and itssensitive to ionizing radiation and tumor. Human breast cancer, pituitary adenoma and cancer of the liver may be associated with promoter methylation Gadd45proteins which made Gadd45dysfunction. In the inflammatory immune response,when lack of Gadd45proteins is easy to get lupus-like autoimmune disease in mice, also Gadd45a can make Thl cells polarization reaction disappeared.Dendritic cells (DCs) are the most antigen-presenting cells (APC) can activate specific immune response and naive T cells, has a special role in the study of inflammatory and tumor therapy. The natural state of DCs in the human body is immature state, when they are stimulated by antigens, they will differentiate, antigen present, then start immune response. Some reports has speculated that "cross-talk" can regulate angiogenesis between DCs and endothelial cell system.This study is to investigate the changes of angiogenesis ability andits relation to Gadd45a when monocytes develop into immature DCs through examining the changes in VEGFR1, sVEGFRl, Gadd45a and VEGF levels and explore the underlying signaling mechanisms. It will be important in research the relationship between DCs and endothelial cell system and their application in immune inflammation and cancer therapy.Materials and methodsPart one:1To model imDC differentiation, RAW264.7cell was plated4x105in6-well plate, and treated with2ml media containing IL-4(lOng/ml) and GM-CSF (20ng/ml) at37℃in a humidified atmosphere of95%air and5%CO2incubator for3days, the cell morphology was observed under phase contrast microscope. Cells were collected about1-2x104adding CD86, CD11c, CD80and VEGFR1antibody to examine their express on cell membrane by flow cytometry.2RAW264.7cell was plated4x105in6-well plate, and treated with2ml media containing IL-4(10ng/ml)and GM-CSF (20ng/ml) at37℃in a humidified atmosphere of95%air and5%CO2, the cells were harvested on day3for analysis. Cell culture supernatant, cell RNA and cell protein were collected to detect the expression of VEGF mRNA, VEGFR1mRNA and VEGFR1protein, sVEGFR1by PCR, western blot technology, ELISA technology, VEGFR1protein expression on the cell membrane was detected by flow cytometry analysis.3RAW264.7cell was plated4×105in6-well plate, and treated with2ml media containing IL-4(10ng/ml,20ng/ml,40ng/ml)or GM-CSF (lOng/ml,20ng/ml,50ng/ml) and blank group(RAW264.7only) at37℃in a humidified atmosphere of95%air and5%CO2, the cells were harvested on day3for analysis. Cell culture supernatant, cell RNA and cell protein were collected to detect the expression of VEGF mRNA, VEGFR1mRNA and VEGFR1protein, sVEGFRl by PCR, western blot technology, ELISA technology, VEGFR1protein expression on the cell membrane was detected by flow cytometry analysis.Part two:1RAW264.7cells were incubated with IL-420ng/ml or GM-CSF20ng/ml for15min,30min,1h, then collecting the cells, extracting cell protein, incubated with P-P38, P38and beta-actin antibodies to test the activation of P38signal pathway.2RAW264.7cell were pre-incubated with SB203580(1:1000) which were added into the culture fluid to culture for1h, then removed SB203580, according to the experimental requirements, the cells were treated with IL-4and GM-CSF alone or in combination, cell culture supernatant, cell RNA and cell protein were collected to detect the expression of VEGF mRNA, VEGFR1mRNA and VEGFR1protein, sVEGFRl by PCR, western blot technology, ELISA technology, VEGFR1protein expression on the cell membrane was detected by flow cytometry analysis.Part three:1RAW264.7cell was plated4x105in6-well plate, and treated with2ml media containing IL-4(10ng/ml)and GM-CSF (20ng/ml) at37℃in a humidified atmosphere of95%air and5%CO2, the cells were harvested on day3for analysis. Cell RNA and cell protein were collected to detect the expression of Gadd45a mRNA and protein, by PCR, western blot technology.2RAW264.7cell was plated4x105in6-well plate, and treated with2ml media containing IL-4(10ng/ml,20ng/ml,40ng/ml)or GM-CSF (lOng/ml,20ng/ml,50ng/ml) and blank group(RAW264.7only) at37℃in a humidified atmosphere of95%air and5%CO2, the cells were harvested on day3for analysis. Cell RNA were collected to detect the expression of Gadd45a mRNA, it was analysed by RT-PCR. Cell culture supernatant, cell RNA and cell protein were collected to detect the expression of VEGF mRNA, VEGFR1mRNA and VEGFR1protein, sVEGFR1by PCR, western blot technology, ELISA technology, VEGFR1protein expression on the cell membrane was detected by flow cytometry analysis.3Using the small RNA interference technology, Gadd45a gene was made silence by using Gadd45a-SiRNA to transfect Gadd45a genein RAW264.7cells, cells’ RNA were extracted at needed time to detect the transfection efficiency, and the Gadd45a-silencing RAW264.7cells were plated4×105in6-well plate, stimulatingwith IL-4(10ng/ml) and gm-csf (20ng/ml), Cell culture supernatant and cell RNA were collectedto detected VEGFR1mRNA and sVEGFR1level by RT-PCR and ELISA.Result.1RAW264.7cells were stimulated with IL-4(20ng/ml) and GM-CSF (20ng/ml) for3days. Flow cytometry analysis indicated that the expression of co-stimulatory molecules CD11c, CD80and CD86were up-regulated. Morphological changes in the cells were also observed. The cells exhibited irregular long protrusions similar to dendrites and the nucleus size increased. These data indicated that the differentiated cells possessed features similar to imDCs.2RT-PCR analysis showed that when RAW264.7cells were treated with IL-4and GM-CSF for3days, the VEGFR1mRNA expression increased remarkably. Western blot analysis showed that protein expression of VEGFR1and sVEGFRl also increased. However, VEGFRl expression on the cell membrane did not change significantly. We detected the sVEGFR1level in the supernatants by ELISA. The results showed that the level of sVEGFRl increased significantly (P<0.01).3Three different doses of rmGM-CSF (10ng/ml,20ng/ml,50ng/ml) and rmIL-4(10ng/ml,20ng/ml,40ng/ml) were applied RAW264.7cells to identify their effects on VEGFR1and sVEGFR1levels. RT-PCR showed that the mRNA expression of VEGFR1was induced by IL-4, but not by GM-CSF. However, Western blot analysis showed that the VEGFR1level increased when RAW264.7cells were stimulated by IL-4or GM-CSF. ELISA showed that both IL-4and GM-CSF increased the sVEGFRl levels, although IL-4had a greater effect compared to GM-CSF. There was no significant difference among the various doses of IL-4or GM-CSF. IL-4reduced VEGFR1protein expression on cell membrane surface membrane, while GM-CSF increase VEGFR1protein expression on cell membrane surface membrane a little. VEGF mRNA changed little.4We found that P38MAPK signaling was activated when RAW264.7cells were treated with IL-4or GM-CSF. When pretreated cells with SB203580, VEGFR1mRNA levels in RAW264.7cells treated with IL-4and GM-CSF were significantly reduced, while the sVEGFR1level increased. RAW264.7cells which were pretreated with SB203580stimulated IL-4or GM-CSF alone. The sVEGFR1level was reduced in the cells stimulated with IL-4, but was increased slightly in the cells stimulated with GM-CSF. The change of VEGFR1protein on cell membrane were abolished. VEGF mRNA changed little.5When RAW264.7cells developed into immature DC like cells, the high levels of Gadd45mRNA and Gadd45proteinsexpressed in cells.6VEGFR1mRNA and sVEGFR1levels were down-regulated in the immature DC like cells when cells’Gadd45agene were transfected by Si-RNA to make silence.Conclusion1When RAW264.7cells were stimulated with IL-4and GM-CSF to develop into immature DC accompanying with VEGFR1mRNA expression and sVEGFR1level up-regulated, while VEGF mRNA were down-regulated, showing the anti-angiogenesis ability of cells increase in this progress.2The mechanism of increase sVEGFR1level was different:IL-4can improve VEGFR1mRNA expression, protein translation and sVEGFR1level, while reducing the membrane protein VEGFR1. We deduced that IL-4can improve TACE activity and it led to shedding ectodomain process. GM-CSF did not increase VEGFR1mRNA expression, but improve the protein expression of VEGFR1, makes sVEGFR1level increased.3IL-4improved RAW264.7cells VEGFR1mRNA expression and sVEGFR1level and P38signal pathway played a key role in this progress. Many signaling pathways may involve in the progress of GM-CSF improved RAW264.7cells’sVEGFR1level, SB203580may have cross-talk with other signaling pathways in sVEGFRl secretion mechanism.4when RAW264.7cells were stimulated to develop into immature DC like cells, the expression of Gadd45a mRNA and Gadd45a protein increase. VEGFR1mRNA and sVEGFR1levels were down-regulated in the immature DC like cells when cells’ Gadd45agene were transfected by Si-RNA to make silence. So we can deduced that the increase ability of anti-angiogenesis in the process of RAW264.7developed into immature DC was partly determined by Gadd45a. |
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