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Carbon monoxide inhibits proliferation of pulmonary smooth muscle cells under hypoxia.

OBJECTIVE
To investigate the expression of inducible heme oxygenase (HO-1) gene in pulmonary artery smooth muscle cells (PASMCs) exposed to hypoxia, and the influence of carbon monoxide (CO) on the proliferation of PASMCs under hypoxic conditions.


METHODS
Primary culture of rat PASMCs were passed every 3 days, and the 3 - 5 passages were used. After exposure to hypoxic conditions (95% N2, 5% CO(2)) 0, 12, 24 and 48 hours, the level of HO-1 mRNA was examined by reverse transcriptase polymerase chain reaction (RT-PCR). The volume of COHb in the medium was measured spectrophotometrically. The cyclic guanosine mono-phosphate (cGMP) concentration of cell extracts was determined by radioimmunoassay. PASMCs were divided into 5 groups, cultured under normoxia and hypoxia and treated with hemin, hemoglobin (Hb) and exogenous CO respectively. Then 3-(4, 5-cimethylthiazol-2-yl)-2, 5-diphenyl tetrazolium bromide (MTT) colorimetric assay and immunocytochemical staining were used to study the energy metabolism and the expression of proliferating cell nuclear antigen (PCNA) in PASMCs. Flow cytometry was used to analyze the cell cycle of PASMCs.


RESULTS
After exposure to hypoxic conditions for 12, 24, and 48 hours, the HO-1 mRNA increased by 2.7%, 5.7% and 27.1% respectively (P < 0.01). The carboxy-hemoglobin (COHb) in the medium increased by 13.8%, 31.0% and 93.1% (P < 0.01); the cGMP concentrations were 2.7, 4.0 and 6.8-fold compared with the control group (P < 0.01 and P < 0.05). In comparison with the control group, the value of MTT colorimetric assay, the immunocytochemical staining of PCNA and the percentages of PASMCs in S and G2M phases in the hypoxic group were significantly higher (P < 0.01). After treatment with Hemin and CO, the results of the above analysis decreased significantly (P < 0.01 and P < 0.05), but increased significantly after treatment with Hb (P < 0.01 and P < 0.05).


CONCLUSIONS
The expression of HO-1 gene in PASMCs is upregulated by hypoxia and the production of endogenous CO is elevated as well. The endogenous CO suppresses the proliferation of PASMC in an autocrine way. Both the induction of endogenous CO by Hemin and the treatment with exogenous CO can suppress the proliferation of rat PASMCs of under hypoxic conditions.