| Part1 Study on the correlation between endothelial microparticles and endothelial damage in aGVHDObjective:To investigate the correlation between endothelial microparticles and endothelial damage in the aGVHD murine model in vivo.Methods:The recipient BALB/c mice were intravenously injected with bone marrow cells and spleen cells from the syngenic mice (BALB/c) or allogenic mice (C57BL/6) to construct the non-aGVHD murine model or aGVHD murine model, respectively. The BALB/c mice that did not receive a transplant were regarded as the normal control. The histopathological signs of aGVHD were analyzed in the HE stained sections of liver, spleen, skin and small intestine under a light microscope. The BALB/c mice were humanely sacrificed before and after transplantation. And then the level of plasma EMPs was determined quantitatively by flow cytomety; the apoptosis of aortic endothelial cell was estimated by TUNEL assay; the reactive oxygen species (ROS) production in aorta was detected by DHE staining; the plasma nitric oxide (NO) production was determined with Griess Reagents.Results:(1) We have construct successfully a aGVHD murine model by injecting intravenously bone marrow cells and spleen cells from C57BL/6 mice to BALB/c mice. (2) The EMP levels in aGVHD mice were increased after transplantation and continued to the time of aGVHD attack. However, the EMP levels in non-aGVHD mice showed a transient elevation at the early post-transplantation period. After transplantation, the EMP levels in aGVHD mice were significantly higher than those in non-aGVHD mice. (3) In the aGVHD mice, the gradually aggravated endothelial damage including EC apoptosis, increased ROS production in the aorta and decreased NO concentrations in the plasma, persisted until the time of aGVHD attack after transplantation. On the contrary, these manifestations of endothelial damage in the non-aGVHD mice were temporary and were only observed at +4 days after transplantation.Conclusion:The persistently increased EMP level and endothelial damage after transplantation were closely related to the occurrence of aGVHD.Part 2 The effect of TNF-a-induced endothelial microparticles on endothelial damageObjective:To investigate the effect of endothelial microparticles derived from TNF-a-stimulated endothelial cells on endothelial damage.Methods:Primary human umbilical vein endothelial cells (HUVECs) and human umbilical vein endothelial cell line (EA.hy926) were stimulated with TNF-a to release EMPs, the EMPs number were assessed by flow cytometry. The EA.hy926 cells were treated with EMPs obtained from injured EA.hy926 cells in a range of concentration (0、5、10、20μg/ml). Then the cell apoptosis was estimated by Annexin V-FITC/PI flow cytometry analysis; the intracellular NO production and NO content in the culture medium were assessed by DAF-FM DA staining and Griess Reagents, respectively; the ROS production was evaluated by DCFH-DA staining; the expression of ICAM-1 and VCAM-1 on cell surface were analyzed by flow cytometry; the effect of EMPs on endothelial cell angiogenesis was measured by Matrigel tubulogenesis assay.Results:(1) TNF-a was found to stimulate both HUVECs and EA.hy926 cells to release a large number of EMPs. (2) The EMPs obtained from injured endothelial cells were observed to induce endothelial damage in a concentration dependent manner. EMPs were found to induce endothelial cell apoptosis at the concentration of 20μg/ml, and trigger reduced NO production, increased ROS production, enhanced expression of ICAM-1 and VCAM-1, and impaired angiogenesis at the concentration of 10μg/ml.Conclusion:EMPs obtained from injured endothelial cells were able to induce endothelial damage, thus expanding the signal of endothelial damage.Part 3 The expression and transfer of Shh inhibitory signal carried by endothelial microparticlesObjective:To investigate the expression and transfer of Shh inhibitory signal carried by endothelial microparticles.Methods:The expression of Hip in the plasma MPs in patients and healthy volunteers were examined by western blotting. The expression of Hip in aortic endothelial cells was detected by immunohistochemistry analysis. A siRNA special for Hip (siRNA-Hip) or a control siRNA (siRNA-NC) was transfected into EA.hy926 cells. The silencing efficiency was assessed by qRT-PCR at 24 h and western blotting at 48 h after transfection. EMPs are collected from cell culture medium, the EMPs shedding from the siRNA-NC transfected cells were referred to as EMPs, and the EMPs shedding from the siRNA-Hip transfected cells are referred to as EMPsHip-.The EA.hy926 cells were treated with the two types of EMPs, pre-incubated with or without the agonist of the SHH pathway (SAG), and then qRT-PCR and western blotting were used to evaluate the expression of Hip on target cells.Results:(1) the Hip expression in plasma MPs from the aGVHD patients was significantly higher compared the non-aGVHD patients and the control. (2) The expression of Hip in aortic endothelial cells was also significantly increased in the aGVHD mice compared with the non-aGVHD mice and normal mice. (3) The level of Hip on EMPs obtained from TNF-a-stimulated HUVEC and EA.hy 926 cells were increased compared with EMPs from resting cells. (4)The expression of Hip protein on EMPs was remarked decreased by siRNA-Hip transfection, and the EMPs from siRNA-Hip transfected cells were referred to EMPsHip-. (5) EMPs was fovmd to increased the expression of Hip on protein level without affecting its mRNA level; while EMPsHip- had no effects on the expression of Hip at both mRNA and protein levels.Conclusion:The Shh inhibitory signal is closely related with the occurrence of aGVHD, and the signal was carried and transferred to target cells by EMPs which is obtained from injured endothelial cells.Part 4 Study on the effects and related mechanisms of the Shh inhibitory signal carried by endothelial microparticles on endothelial damageObjective:To investigate the effects and mechanisms of the Shh inhibitory signal carried by endothelial micropaiticles on endothelial damage in vitro.Methods:The siRNA special for Hip (siRNA-Hip) or the control siRNA (siRNA-NC) was transfected into EA.hy926 cells as described at part 3, the EMPs shedding from the siRNA-NC transfected cells are referred to as EMPs, and the EMPs shedding from the siRNA-Hip transfected cells are referred to as EMPsHip-. The EA.hy926 cells were treated with the two types of EMPs, pre-incubated with or without the agonist of the SHH pathway (SAG). qRT-PCR and western blotting were used to evaluate the expression levels of Fas, Bcl-2, Bax, eNOS, AKT, Ang-1 and Ang-2 at mRNA level and protein level, respectively; Annexin V-FITC/PI flow cytometry analysis was performed to estimate the cell apoptosis; DAF-FM DA staining and Griess Reagents were applied to assess the intracellular NO production and NO content in the culture medium, respectively; DCFH-DA staining was used to detect intracellular ROS production; flow cytometry was used to analyze the expression of ICAM-1 and VCAM-1 on cell surface; Matrigel tubulogenesis assay was performed to measure the angiogenic activity of endothelial cell.Results:(1) EMPs were found to induced increased endothelial cell apoptosis, accompanied by enhanced Bax expression and reduced Bcl-2 expression both at the mRNA and protein levels. However, EMPsHip- did not induce cell apoptosis and affect the expression of related protein, and SAG reversed the effect of EMPs on cell apoptosis and related protein expression. (2) EMPs enhanced the expression of Fas, but EMPsHip- and EMPs+SAG had no effect on its expression. (3) EMPs decreased Intracellular and extracellular NO production by decreasing eNOS expression and AKT1 phosphorylation at its activator site (Ser-473). However, EMPsHip- and EMPs+SAG had no effect on NO production, eNOS expression and AKT1 phosphorylation. (4) EMPs impaired angiogenic activity of endothelial cell, accompanied by decreased Ang-1 expression and increased Ang-2 expression both at the mRNA and protein levels. Nevertheless, EMPsHip- and EMPs+SAG did not affect endothelial cell angiogenesis and the expression of Ang-1 and Ang-2. (5) EMPs increased ROS production and enhanced the expression of ICAM-1 and VCAM-1, without relying on the Hip-mediated inhibition of the Shh pathway.Conclusion:EMPs obtained from injured endothelial cells induced endothelial cells apoptosis and endothelial dysfunction through Hip-mediated inhibition of the SHH pathway. |
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