AIM: To investigate the effects of adrenomedullin (AM) gene overexpression on the biological characteristics of human hepatic stellate cells (hHSCs) by stable transfection. METHODS: hHSCs which express low basal levels of AM were stably transfected with an expression construct containing rat AM gene or with an empty expression vector. Expression of AM in hHSCs was determined by reverse transcription (RT)-polymerase chain reaction (PCR) and radioimmunoassay (RIA). Cell proliferation was evaluated by 5-bromo-2'-deoxyuridine (BrdU) incorporation and immunocytochemistry. RT-PCR and Western blot were used to test the expression of procollagen types I and III. Protein expressions of interstitial collagenase (MMP-1), gelatinase (MMP-2) and tissue inhibitors of matrix metalloproteinases-2 (TIMP-2) were assessed by Western blot. RESULTS: Two cell clones (A-2, A-8) transfected with the AM gene expressed higher levels of AM mRNA (non-transfected group: 0.86+/-0.11, empty vector group: 1.01+/-0.11, A-2 clone group: 1.44+/-0.08 and A-8 clone group: 1.36+/-0.05) and protein (12.31+/-0.17, 12.35+/-0.12, 12.56+/-0.06 and 12.62+/-0.07) (P<0.05). AM gene overexpression had inhibitory effects on cell proliferation of hHSCs (29.6%, 30.9%, 18.9% and 21.8%, respectively. P<0.05) and expression of procollagen type I (0.58+/-0.1, 0.48+/-0.11, 0.3+/-0.06 and 0.31+/-0.07 at mRNA level) (0.27+/-0.07, 0.3+/-0.06, 0.14+/-0.05 and 0.13+/-0.05 at protein level) (P<0.05) and procollagen type III (0.17+/-0.04, 0.15+/-0.03, 0.1+/-0.02 and 0.09+/-0.02 at mRNA level) (0.22+/-0.04, 0.2+/-0.03, 0.11+/-0.04 and 0.13+/-0.03 at protein level) (P<0.05). Compared with cells non-transfected (TIMP2: 2.77+/-0.03, MMP-2: 0.5+/-0.04, MMP-1: 0.49+/-0.07) and transfected with empty vector (TIMP2: 2.79+/-0.04, MMP-2: 0.48+/-0.03, MMP-1: 0.45+/-0.09), these two clones had lower expression levels of TIMP2(A-2 clone group: 2.7+/-0.02 and A-8 clone group: 2.71+/-0.02) (P<0.05) and MMP-2(A-2 clone group: 0.15+/-0.05 and A-8 clone group: 0.13+/-0.04) (P<0.05) but displayed a higher expression level of MMP-1(A-2 clone group: 0.68+/-0.06 and A-8 clone group: 0.81+/-0.09) (P<0.05). CONCLUSION: AM gene exerts negative influence to some extent on hHSCs by inhibiting proliferation and production of extracellular matrix (ECM) in addition to inducing MMP-1 expression.
AIM: To investigate the effects of adrenomedullin (AM) gene overexpression on the biological characteristics of human hepatic stellate cells (hHSCs) by stable transfection. METHODS: hHSCs which express low basal levels of AM were stably transfected with an expression construct containing rat AM gene or with an empty expression vector. Expression of AM in hHSCs was determined by reverse transcription (RT)-polymerase chain reaction (PCR) and radioimmunoassay (RIA). Cell proliferation was evaluated by 5-bromo-2'-deoxyuridine (BrdU) incorporation and immunocytochemistry. RT-PCR and Western blot were used to test the expression of procollagen types I and III. Protein expressions of interstitial collagenase (MMP-1), gelatinase (MMP-2) and tissue inhibitors of matrix metalloproteinases-2 (TIMP-2) were assessed by Western blot. RESULTS: Two cell clones (A-2, A-8) transfected with the AM gene expressed higher levels of AM mRNA (non-transfected group: 0.86+/-0.11, empty vector group: 1.01+/-0.11, A-2 clone group: 1.44+/-0.08 and A-8 clone group: 1.36+/-0.05) and protein (12.31+/-0.17, 12.35+/-0.12, 12.56+/-0.06 and 12.62+/-0.07) (P<0.05). AM gene overexpression had inhibitory effects on cell proliferation of hHSCs (29.6%, 30.9%, 18.9% and 21.8%, respectively. P<0.05) and expression of procollagen type I (0.58+/-0.1, 0.48+/-0.11, 0.3+/-0.06 and 0.31+/-0.07 at mRNA level) (0.27+/-0.07, 0.3+/-0.06, 0.14+/-0.05 and 0.13+/-0.05 at protein level) (P<0.05) and procollagen type III (0.17+/-0.04, 0.15+/-0.03, 0.1+/-0.02 and 0.09+/-0.02 at mRNA level) (0.22+/-0.04, 0.2+/-0.03, 0.11+/-0.04 and 0.13+/-0.03 at protein level) (P<0.05). Compared with cells non-transfected (TIMP2: 2.77+/-0.03, MMP-2: 0.5+/-0.04, MMP-1: 0.49+/-0.07) and transfected with empty vector (TIMP2: 2.79+/-0.04, MMP-2: 0.48+/-0.03, MMP-1: 0.45+/-0.09), these two clones had lower expression levels of TIMP2(A-2 clone group: 2.7+/-0.02 and A-8 clone group: 2.71+/-0.02) (P<0.05) and MMP-2(A-2 clone group: 0.15+/-0.05 and A-8 clone group: 0.13+/-0.04) (P<0.05) but displayed a higher expression level of MMP-1(A-2 clone group: 0.68+/-0.06 and A-8 clone group: 0.81+/-0.09) (P<0.05). CONCLUSION: AM gene exerts negative influence to some extent on hHSCs by inhibiting proliferation and production of extracellular matrix (ECM) in addition to inducing MMP-1 expression.
Authors: N Théret; O Musso; B Turlin; D Lotrian; P Bioulac-Sage; J P Campion; K Boudjéma; B Clément Journal: Hepatology Date: 2001-07 Impact factor: 17.425
Authors: Suzy M Salama; Mahmood A Abdulla; Ahmed S Alrashdi; A Hamid A Hadi Journal: Evid Based Complement Alternat Med Date: 2013-08-07 Impact factor: 2.629