Effects Of Toll-like Receptors Activation On The Biological Function Of Human Endothelial (Progenitor)Cells And Its Mechanism

Charpter I The Possible Mechanisms Responsible for the Effects of PolyⅠ:C on the Proliferation of Human Umbilical Cord Blood-Derived Endothelial Progenitor CellsPart Ⅰ The Expression of Toll-like receptors on the Human Umbilical Cord Blood-Derived Endothelial Progenitor CellsObjective:To observe the expression of Toll-like receptors on the human umbilical cord blood-derived endothelial progenitor cells (EPCs).Methods:Mononuclear cells were isolated from fresh cord blood by density gradient centrifugation and cultured in EBM-2medium. The biological features of the isolated cells were observed at different time point and identified by morphology, immunofluorescence staining and RT-PCR. Furthermore, RT-PCR was performed firstly to detect the mRNA expression of Toll-like receptor subtypes in EPCs at rest, secondly the expression of Toll-like receptor3(TLR3)and inflammatory cytokines induced by PolyⅠ:C at different concentrations.Results:1. Early EPCs changed from small size round cells to spindle shape cells,Late EPCs formed a typical cobblestone-like cells.Adherent EPCs were characterized by DiLDL uptake and concomitant lectin binding, and by expressing of CD34,CD133and KDR, which are markers of stem cells.2. RT-PCR results shown that Under basal condition, except TLR9, TLR1～TLR10were expressed in EPCs with high expression levels of TLR1,TLR3,TLR4. TLR6and low expression levels of TLR2,TLR5, TLR7,TLR8,TLR10. Expression of TLR3was significantly up-regulated by treatment with Poly I:C at both the gene and protein levels and there was dose-effect relationship in experiment range.3. PolyI:C upregulated the mRNA expression of inflammatory cytokines such as IL-1β,IL-6,IL-8,TNF-α,IFN-β by activating TLR3.Conclusions:EPCs expressed functional TLR3; PolyI:C can up-regulate the expression of TLR3at both the gene and protein levels and up-regulate the gene expression of inflammatory cytokines in a dose-dependent manner by activating TLR3. Part Ⅱ The Effect of PolyⅠ:C on the Proliferation and Apoptosis of Human Umbilical Cord Blood-Derived Endothelial Progenitor CellsObjective:To analyze the effect of TLR3agonist PolyⅠ:C on the proliferation and apoptosis of human umbilical cord blood-derived endothelial progenitor cells.Methods:EPCs were treated with different concentrations of PolyⅠ: C sequentially for3days, and then cell numbers were measured by CCK-8assay.EPCs were treated with Polyl:C at different concentrations, the phase of cell cycle was tested by PI staining and cell apoptosis was detected by the PI/Annexin V staining.Results:1.Compared with the control group, PolyⅠ:C at high concentrations of1and10μg/ml significantly inhibited the proliferation of EPCs after3days treatment, and the inhibition of cell proliferation by Poly I:C was time-dependent.2. Poly Ⅰ:C at high concentrations of1and10μg/ml significantly decreased the proportion of cells in S phase and G2/M phase. Moreover, Poly Ⅰ:C down-regulated the gene expression of cyclins A, B1, D1, and E, inducing cell cycle arrest in G0/G1phase, thus inhibiting the proliferation of EPCs.3. PolyI:C induced the apoptosis of EPCs in a dose-dependent manner.Conclusions:PolyⅠ:C at high concentrations inhibited EPCs proliferation by inducing cell cycle arrest in G0/G1phase and inducing cell anoptosis of EPCs. Part Ⅲ The Possible Mechanisms Responsible for the Effects of PolyⅠ:C on the Apoptosis of Human Umbilical Cord Blood-Derived Endothelial Progenitor CellsObjective:To elucidate the mechanism of PolyⅠ:C induced apoptosis in human umbilical cord blood-derived endothelial progenitor cells.Methods:We used specific inhibitors of Caspase8and Caspase9to inhibit the corresponding signal molecules, then tested the effect of caspase inhibition on Poly Ⅰ:C-induced apoptosis. EPCs were treated with different concentrations of TNF-α and IL-1β for24h respectively,then CCK-8assay was used to detect the apoptosis of EPCs. EPCs were pre-treated with IL-1receptor1neutralizing antibody(Anti-IL-1R1),then re-stimulated with PolyⅠ:C, cell apoptosis was measured by CCK-8assay.Results:1.Caspase8and Caspase9inhibitors did not reduce PolyⅠ:C-induced apoptosis of EPCs.2. When EPCs were treated with increasing concentration of TNF-α, cell apoptosis was not increase in TNF-α-treated groups.3. IL-1β induced cell apoptosis in a dose-dependent manner. Moreover, when EPCs pre-treated with Anti-IL-1R1, were re-stimulated with PolyⅠ:C, the cell apoptosis induced by Poly Ⅰ:C was decreased.Conclusions:PolyⅠ:C induced the apoptosis of EPCs through up-regulating the expression of IL-1β via activating TLR3. Endogenous and exogenous apoptosis pathway and TNF-α did not contribute to PolyⅠ:C-induced cell apoptosis. Charpter II Pam3CSK4Induces a Non-tolerant Up-regulation of Cytokine in Human Umbilical Vein Endothelial CellsPart I The Possible Mechanism Responsible for the Effect of Pam3CSK4on the Expression of Cytokines in Human Umbilical Vein Endothelial CellsObjective:To observe the effects of TLR1/TRL2agonist Pam3CSK4on the expression of cytokines in HUVECs and to elucidate the possible mechanism.Methods:HUVECs were treated with different concentrations of Pam3CSK4, RT-PCR was used to detect the mRNA expression of TLR1and TLR2. Then, RT-PCR,Real-time RT-PCR and ELISA were performed to detect the effect of Pam3CSK4on the expression of TNF-a and IL-6at both mRNA and protein level. We used specific inhibitors of TLR1,TLR2and TLR1/TLR2to inhibit the corresponding receptors, then tested the effect of TLR1,TLR2inhibition on the expression of TNF-a and IL-6induced by Pam3CSK4. In order to elucidate the signaling pathways involving in this process, Western-blot was performed to detect the phosphorylation of MAPK and NF-κB.Results:After treated with different concentrations of Pam3CSK4for24h, the RT-PCR results show that Pam3CSK4could significantly up-regulate the mRNA expression of TLR2,the effect of Pam3CSK4on TLR1mRNA expression was not obvious. Pam3CSK4up-regulated the expression of TNF-α and IL-6at both mRNA and protein level in a dose-dependent manner.TLR1,TLR2and TLR1/TLR2inhibitors could significantly inhibit the up-regulation of TNF-α and IL-6induced by Pam3CSK4,and the inhibition induced by TLR1/TLR2inhibitor was most obvious. The Western-blot results showed that Pam3CSK4could induce the phosphorylation of MAPK and NF-κB signal transduction pathways via TLR2activation.Conclusions:HUVECs expressed functional TLR2.Pam3CSK4induced the phosphorylation of MAPK and NF-κB signal transduction pathways via TLR2activation,thus significantly up-regulate the expression of TNF-a and IL-6at both mRNA and protein level which was TLR1and TLR2dependent. Part II Pam3csk4Induces a Non-tolerant Up-regulation of Cytokine IL-6in Human Umbilical Vein Endothelial CellsObjective:To observe the effect of Pam3CSK4pre-treatment on cytokine expression in HUVECs induced by Pam3CSK4re-treatment and to elucidate the possible mechanism.Methods:HUVECs, pre-treated with1μg/ml Pam3CSK4for indicated hours, were re-treated with1μg/ml Pam3CSK4for1hour. The gene and protein expression of cytokine IL-6and TNF-a were detected by Real-time RT-PCR and ELISA respectively.Then Real-time RT-PCR and ELISA were performed to detect the effect of various PAMP pre-treatment on cytokine gene and protein expression induced by Pam3CSK4re-treatment,observing the cross-tolerance between different TLRs. Then we used western-blot to detect the phosphorylation of MAPK and NF-κB signal transduction pathways after Pam3CSK4repeating stimulation. The effect of Pam3CSK4on the gene expression of ubiquitin-editing enzyme A20was detected by RT-PCR, and the effect of A20over-expression on cytokine IL-6expression induced by Pam3CSK4and LPS was detected by plasmid transfection. We used inhibitors of Notch signal transduction pathway to inhibit the corresponding signal molecules, then tested the effect of Notch signal transduction pathway inhibition on the expression of IL-6induced by Pam3CSK4. Results:Pam3CSK4pre-treatment could down-regulate the expression of TNF-a induced by Pam3CSK4at both gene and protein level,but had no effect on the expression of IL-6. LPS pre-treatment could down-regulate the expression of TNF-a induced by Pam3CSK4,but had no effect on the expression of IL-6.The Western-blot results showed that Pam3CSK4repeating stimulation inhibited activation of JNK, without influencing the phospholyration of p38,ERK,NF-κB. Pam3CSK4down-regulated the mRNA expression of ubiquitin-editing enzyme A20in a dose-dependent manner,but A20over-expression had no effect on the the expression of IL-6induced by Pam3CSK4and LPS. Inhibitors of Notch signal transduction pathway could partly inhibit the expression of IL-6induced by Pam3CSK4.Conclusions:Pam3CSK4induced a tolerant up-regulation of cytokine TNF-a, but induced a non-tolerant up-regulation of cytokine IL-6.There was cross-tolerance between TLR2and TLR4. Pam3CSK4probably induced the non-tolerant up-regulation of cytokine IL-6via Notch signal transduction pathway.