Effect Of Adrenomedullin On Lipopolysaccharide-inhibited Tissue Factor Pathway Inhibitor Expression In Cultured Human Umbilical Vein Endothelial Cells And Its Molecular Mechanism

【Backgrounds】Adrenomedullin (ADM) is a vasodilator peptide isolated from human pheochromocytoma tissue, which has a variety of effects, including vasorelaxation, diuretic action, inhibition of aldosteron production, etc. Recent studies showed that it also has antithrombotic activities and resistant roles to lipopolysaccharide (LPS)-induced septic shock. However, the mechanism underlying ADM-induced resistance to sepsis is poorly understood. One of the frequent complications of septic shock is disseminated intravascular coagulation (DIC). LPS is a major trigger of sepsis-induced DIC via the tissue factor (TF)/factorâ…¦a-dependent pathway of coagulation. It is unknown whether the antithrombotic activities of ADM contribute to its resistance to sepsis. In the present study, we investigated the effects of ADM on tissue factor pathway inhibitor (TFPI) (primary anticoagulant factor) expression in human umbilical vein endothelial cells (HUVECs) exposed to LPS, and the possible underlying mechanism for these effects.【Methods】HUVECs were used as target cells and the research of four aspects were carried out by the following methods:1. Effect of ADM and/or LPS on TFPI expression in HUVECs(1) The human umbilical vein endothelial cells were isolated, cultured and identified;(2) With chromogenic assays, the TPFI protein activity in culture medium was determined. The best time and concentration of ADM or LPS used to treat HUVECs were selected. And the effect of ADM on TFPI activity in HUVECs exposed to LPS was investigated.(3) Using RT-PCR technique, the effect of ADM on TFPI mRNA expression in HUVECs exposed to LPS was investigated.2. The signal transduction pathways involved in counteraction role of ADM on LPS-inhibited TFPI expression(1) The role of MAPK (mitogen-activated protein kinase) pathway in TFPI expression changes induced by ADM and/or LPS was observed with MAPKK inhibitor PD98059 intervention experiment.(2) The role of PKC (protein kinase C) pathway in TFPI expression changes induced by ADM and/or LPS was observed with PKC inhibitor H7 or Staurosporine intervention experiment.(3) The role of cAMP pathway in TFPI expression changes induced by ADM and/or LPS was observed with cAMP antagonist Rp-isomer-8-bromo-cAMP (Rp-cAMP) or cAMP analogue 8-bromo-cAMP intervention experiment.(4) The changes of calcium concentration in a single HUVEC induced by ADM and/or LPS were observed by using Laser-Scanning Confocal Microscope. The role of calcium ion in TFPI inducible expression was examined with calcium antagonist Amlodipine intervention experiment.3. Roles of nuclear transcriptional factor GATA-2, SP1 and c-Myc in TFPI inducible expression in HUVECs(1) The activities of nuclear transcriptional factor GATA-2, SP1 and c-Myc binding with corresponding regulatory sequences in TFPI gene promoter were analyzed by EMSA (Electrophoresis mobility shift assay) and Super Shift assays.(2) The effect of ADM and/or LPS on nuclear transcriptional factor GATA-2, SP1 and c-Myc related with TFPI expression in HUVECs with Western Blot technique.4. Roles of GATA-2, SP1 and c-Myc motifs of TFPI5' upstream promoter region in TFPI gene transcription in HUVECs(1) Using PCR and gene recombination techniques, luciferase reporter gene plasmids containing different length of human TFPI gene promoter were constructed.(2) With eukaryon gene transfection techniques, the effect of different length of human TFPI gene promoter on TFPI transcription induced by ADM and/or LPS.(3) The three GATAo2 and one SP1 motifs in TFPI gene promoter -1247 to -381bp were site-directed mutated, respectively, and then luciferase reporter gene plasmids containing site-directed mutanted GATA-2 or SP1 sequence were constructed by gene recombination techniques.(4) The role of GATA-2 and SP1 motif located in TFPI gene promoter on TFPI transcription affected by ADM and/or LPS.【Results】1. ADM increased TFPI protein activities and mRNA expression levels in HUVECs in a dose-dependent and time-dependent manner. Contrarily, LPS inhibited TFPI protein activities dose-dependently, and ADM antagonized the role of LPS on TFPI expression.2. MAPKK inhibitor PD98059 and cAMP antagonist Rp-cAMP could markedly inhibit the counteraction role of ADM on LPS-decreased TFPI expression. And c-AMP analogue could directly inhibit the effect of LPS on TFPI expression.3. PKC inhibitor H7 and Staurosporine had no significant effect on the the counteraction role of ADM on LPS-decreased TFPI expression.4. Laser-Scanning Confocal Microscope investigation showed a slow and steady decrease of【Ca²⁺】i in HUVEC was found after ADM treatment, while a quick and short increase was found after LPS exposure. After HUVEC was pretreated with ADM, the speed and extent of【Ca²⁺】i increase induced by LPS decrease significantly. The calcium antagonist Amlodipine could increase LPS-inhibited TFPI expression in HUVECs.5. EMSA and Super Shift assays indicated that nuclear transcriptional factor GATA-2, SP1 and c-Myc could bind to the corresponding regulatory sequences specifically. ADM increased the activities of GATA-2 and SP1 factor binding to the corresponding GATA-2 and SP1 motif, contrary to the role of LPS. And ADM could counteract the role of LPS.6. Western Blot results showed that ADM increased the protein content of nuclear transcriptional factor GATA-2 and SP1. On the contrary, LPS decreased the GATA-2 and SP1 protein content, which could be antaganized by ADM.7. The transient transfection analysis showed that the sequence from -1247bp to -381 bp played important role in tuciferase expression induced by AND and/or LPS. This sequence contains three GATA-2 and one SP1 regulatory sequences.8. The site-directed results showed that the above GATA-2 and SP1 elements all play important role in TFPI expression induced by ADM and/or LPS. In addition, the mutation of all the three GATA-2 elements decreased the basic luciferase expression activities in HUVECs, while the mutation of SP1 motif had no effect on the basic luciferase activities.【Conclusions】In conclusion, our data demonstrated that ADM could antagonize the effect of LPS on TFPI expression through cAMP-MAPK and calcium signaling pathway. The nuclear transcriptional factor GATA-2 and SP1, and GATA-2 and SP1 motif in TFPI gene promoter -1247bp to -381 bp played an important role in the regulation of TFPI expression in HUVECs exposed to LPS with or without ADM. This counteraction effect of ADM on LPS-inhibited TFPI expression and the clarification of TFPI expression regulation mechanism might contribute to improve coagulant status of septic stock by applying ADM or interfering with the expression pathway of TFPI.