Effects of adrenomedullin gene overexpression on biological behavior of hepatic stellate cells.
OBJECTIVE
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).


CONCLUSIONS
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.
Wang
Yi
Y
Department of Pathology, Medical Center, Fudan University, Shanghai 200032, China.
Zhang
Jin-Sheng
JS
Huang
Guang-Cun
GC
Cheng
Qi
Q
Zhao
Zhong-Hua
ZH
D053607:Adrenomedullin; D000818:Animals; D002455:Cell Division; D005109:Extracellular Matrix / Q000502:physiology; D005786:Gene Expression Regulation; D006801:Humans; D008099:Liver / Q000166:cytology / Q000502:physiology*; D010455:Peptides / Q000235:genetics*; D012333:RNA, Messenger / Q000235:genetics; D051381:Rats; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014162:Transfection
D010455:Peptides; D012333:RNA, Messenger; D053607:Adrenomedullin