| Aims Pulmonary vascular structural remodeling (PVSR) induced by hypoxia is the critical pathologic changes of hypoxic pulmonary hypertension(HPH). Adrenomedullin (ADM) is a potent vasodilator peptide consisting of 52 amino acids. It was originally discovered from a human pheochromocytoma, and belongs to the calcitonin gene-related peptide (CGRP) family. Adrenomedullin is produced and secreted by various types of cells, including fibroblasts. The adrenomedullin induced by hypoxia was observed in cultured normal cells and tumor cells. Adrenomedullin induced by hypoxia may ameliorate hypoxic states through its vasodilator action. In addition to vasodilator action, this peptide also has effects on cell growth and apoptosis. It is reported that transforming growth factor B1 (TGF-B1) may act as a growth factor and increase proliferation and collagen production of fibroblasts. Change of [Ca2+]i is an important step in signal transduction of cells. Our previous studies have shown that ADM level in the plasma of rat and human with HPH is highter than that of control group. This means that ADM takes an important role in HPH. But we don't know why and how ADM works on the PVSR under hypoxia. To understand this, the following experiments have been designed: (1) Investigate the effects of ADM on proliferation and collagen synthesis of human fetal lung fibroblasts under hypoxia (2%O2) . (2) Check the influence of ADM on TGF-B1 production and [Ca2+]i of fibroblasts, which conduce to find out the regulation mechanisms of ADM on the PVSR in hypoxia.Methods Human fetal lung fibroblasts were cultured by outgrowth in vitro. The steptavidin-peroxidase method was used as detection system. A stable model of fibroblasts under hypoxia (2%O2) was established. The expression of ADM protein in fibroblasts was tested by immunohistochemistry analysis. Cells were exposed to hypoxia (2%O2) or normoxia after adding ADM 10-9, 10-8, 10-7mol/L. The effects of ADM and TGF-B1 on proliferation of fibroblasts were determined by manual cell counting under the microscope and MTT assay. Total collagen synthesis and secretion were detected by 3H-prolineincorporation. TGF-B1 production in culture supernatant were measured by ELISA assay. With the help of laser scanning confocal microscope (LSCM) system, concentrations of [Ca2+]i in fibroblasts were detected.Results a -actin staining of fibroblasts were negative. Exposed to both normoxia and hypoxia for 24h and 48h, there were positive staining in fibroblasts incubated with anti-adrenomedullin. Compared to normoxia, Cell number and 490nm OD expressing low oxygen values were significantly increased by the 24h, 48h, 72h exposure to 2% hypoxia respectively. An obvious increase of collagen synthesis and secretion expression was detected after 24h, 48h, 72h exposure to 2% hypoxia. ADM could inhibit proliferation and collagen synthesis of fibroblasts induced by TGF-B1(5ng/ml). TGF-B1 production in culture supernatant also increased after hypoxia. The increase of cell proliferation and collagen synthesis and TGF-pi production induced by hypoxia could be inhibited by ADM (10-7mol/L), respectively. Hypoxia could increase the concentrations of [Ca2+]i in fibroblasts. It could be suppressed by adding ADM (10-7mol/L).Conclusions ADM is produced by human fetal lung fibroblast. Hypoxia is a stimulus for the ADM protein expression in fibroblasts. ADM may attenuate hypoxic pulmonary vascular structural remodeling through inhibiting fibroblasts proliferation and collagen synthesis. The direct actions of ADM on reducing TGF-B1 production and concentration of [Ca2+]i in fibroblasts induced by hypoxia also help to its inhibitory effects on fibroblasts growth and collagen synthesis. This study implies that ADM may work as a protective factor in hypoxic pulmonary hypertension.
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