[The effects of Ad-Gax transfection on apoptosis and related gene expression in pulmonary arterial smooth muscle cells in hypoxic rat model].
Maneno muhimu
Kikemikali
OBJECTIVE
To explore the effects of growth arrest-specific homeobox (Gax) transfection on apoptosis and expressions of Bcl-2 and Bax proteins of rat pulmonary arterial smooth muscle cells (PASMCs) exposed to hypoxia.
METHODS
PASMCs were transfected with Gax gene by a replication-deficient adenovirus expressing the hemagglutinin-tagged Gax cDNA (Ad-Gax). After exposure to normal oxygenation (21% O(2)) or hypoxia (2.5% O(2)) for 2 h, 6 h, 12 h, 24 h and 48 h, apoptosis was observed by transmission electronic microscope and TUNEL-positive staining. The expressions of Bcl-2 and Bax proteins in PASMCs were detected by immunocytochemistry.
RESULTS
Before Ad-Gax transfection, none or few of TUNEL-positive PASMCs were detected. After Ad-Gax transfection, the PASMCs unexposed to hypoxia displayed none or few TUNEL-positive stain, while the PASMCs exposed to hypoxia displayed a marked increase in TUNEL-positive stain, especially in cells exposed to hypoxia for 24 h to 48 h. The ratio of apoptosis of PASMCs at normoxic, hypoxia 2 h, 6 h, 12 h, 24 h and 48 h were (9.11 +/- 1.21)%, (34.13 +/- 1.02)%, (39.12 +/- 0.43)%, (50.09 +/- 0.13)%, (60.04 +/- 1.12)% and (55.47 +/- 0.03)% (P < 0.01), respectively. Before Ad-Gax transfection, the level of Bax protein showed no significant increase in PASMCs exposed to hypoxia, while that of Bcl-2 protein increased significantly, as compared to that in PASMCs under normoxic condition. The average optical density (AOD) of Bcl-2 was 2.31 +/- 0.12, 2.35 +/- 0.23, 2.49 +/- 0.27, 2.51 +/- 0.19, 2.54 +/- 0.25 and 2.53 +/- 0.20 at normoxic, hypoxia for 2 h, 6 h, 12 h, 24 h and 48 h before Ad-Gax transfection, respectively (P < 0.05, P < 0.01). After Ad-Gax transfection to PASMCs exposed to hypoxia, Bax protein increased; the AOD of Bax was 3.82 +/- 0.38, 3.12 +/- 0.42, 3.53 +/- 0.61, 4.52 +/- 0.23, 4.25 +/- 0.76 and 4.03 +/- 0.38 at normoxic, hypoxia for 2 h, 6 h, 12 h, 24 h and 48 h (P < 0.01), respectively. But Bcl-2 protein decreased significantly; the AOD of Bcl-2 was 9.11 +/- 1.21, 34.13 +/- 1.02, 39.12 +/- 0.43, 50.09 +/- 0.13, 60.04 +/- 1.12 and 55.47 +/- 0.03, respectively (P < 0.01). After Ad-Gax transfection, the Bcl-2/Bax ratio was negatively correlated with the rate of apoptotic PASMCs (r = -0.53, P < 0.01).
CONCLUSIONS
Ad-Gax transfection induced PASMC apoptosis after exposure to hypoxia. A possible mechanism is that Gax can downregulate Bcl-2 expression and upregulate Bax expression, especially by decrease of the Bcl-2/Bax ratio.
