[Effects of injuries pulmonary arterial endothelial cell induced by endotoxin on proliferation of pulmonary artery smooth muscle cells and interference effect of bone morphogenetic protein-2].

OBJECTIVE: To investigate the effects of injuries pulmonary arterial endothelial cell induced by endotoxin on proliferation of pulmonary artery smooth muscle cells and interference effect of bone morphogenetic protein-2, and at the same time explore its possible mechanism.

METHODS: Upper liquid of normal PAEC incubated by DMEM for 24 h without serum (EC-CMI) or that of PAEC incubated by the same DMEM and same period after incubating with endotoxin at concentration of 1 microg/ml for 1 h (EM-CMII) were collected and preserved for PASMC incubating. Confluent monolayer PASMC of rats were incubated with EC-CMI (group I), EC-CMII (group II), EC-DMII + BMP-2 1 ng/ml (group III), EC-DMII + BMP-2 10 ng/ml (group IV) and EC-DMII + BMP-2 100 ng/ml (group V) for 24 h. The cultured cells were identified by phase-contrast microscope and immunofluorescent stain of SMC specific antigen. The proliferation of PASMC were evaluated by the BrdU assay. The cell cycle analysis including proliferation index (PI) and S-phase cell fraction (SPF) were performed by the flow cytometry. the DNA synthesis was detected by [methyl-(3)H]-thymidine incorporation; the expression of cyclinD1 mRNA was elucidated by RT-PCR technology; the analysis of phosphorylated Smad1 (pSmad1) and expression of cyclinD1 protein were detected by Western-blot experiment.

RESULTS: Cells were confirmed as smooth muscle by their typical "hill-valley" morphological features displayed under phase-contrast microscope and by immunofluorescent staining of smooth muscle, Compared with group I, there were a significant increase in proliferation rate, S-phase cell fraction (SPF), DNA synthesis in group II. Compared with group II, there were no significant changes in group III about that data. But the groups with 10 or 100 ng/ml (group IV and group V) of BMP-2 significantly attenuated proliferation rate, S-phase cell fraction (SPF), DNA synthesis and expression of cyclinD1 mRNA and protein compared with EC-CM2 alone (group II, P < 0.01). Phosphorylated Smad1 increased in group IV and group V compared with group II, (P < 0.01).

CONCLUSION: The BMP-2 could downregulate expression of cyclinD1 gene by smad signaling transconduction system, then depressed proliferation of PASMCs by EC-CM2 in vitro culture. And this effect is dependent in the concentration range of 1 - 100 ng/ml.