[Effects of human tissue kallikrein gene delivery on the proliferation of vascular smooth muscle cells].
OBJECTIVE
Tissue kallikrein cleaves kininogen substrate to produce vasoactive kinin peptides that have been implicated in the proliferation of vascular smooth muscle cells. We investigated the effects of adenovirus-mediated human tissue kallikrein (Ad-hKLK1) gene delivery on the proliferation of vascular smooth muscle cells of SHR (VSMCs(SHR)) induced by platelet derived growth factor-BB (PDGF-BB).


METHODS
Primary VSMCs(SHR) were isolated and cultured from thoracic aorta of male SHR. The VSMCs(SHR) proliferation induced by PDGF-BB was accessed by cell counting and methyl thiazolyl tetrazolium (MTT). Western blot was used to determine the protein expression of hKLK1, the cycle-independent kinase inhibitors p27(Kip1) and p21(Cip1). The mRNA expressions of bradykinin B1 receptor and B2 receptor were detected by RT-PCR in VSMCs(SHR).


RESULTS
Proliferation of VSMCs(SHR) induced by PDGF-BB was significantly inhibited post transfection of Ad-hKLK1 (20-100 MOI) in a MOI-dependent manner. The peak inhibition titer of Ad-hKLK1 was 100 MOI with peak inhibition rate of 39.3% (cell counting, n = 3, P < 0.01), 30.2% (MTT, n = 3, P < 0.01) and 36.4% (peak stunning rate of cell-cycle in phase G(0)/G(1)). The inhibitory effects of proliferation and cell-cycle caused by hKLK1 gene delivery could be abolished by Hoe140, a bradykinin B2 receptor antagonist. The protein expression of p27(Kip1) and p21(Cip1) increased significantly after the hKLK1 gene delivery, whereas Hoe140 nearly completely blocked these effects (n = 3, P < 0.001, respectively). PDGF-BB also significantly upregulated the mRNA expression of B2 receptor but not B1 receptor in VSMCs(SHR).


CONCLUSIONS
The hKLK1 gene delivery could inhibit PDGF-BB induced proliferation in VSMCs(SHR) through Bradykinin B2 receptor and up-regulate expression of p27(Kip1) and p2l(Cip1).
Fujian Provincial Hospital, Provincial Clinical College, Fujian Medical University, Fujian Institute of Clinical Geriatrics, Fuzhou 350001, China. zpl7755@126.com
Peng-Li
PL
Zhu
Hui-Zhen
HZ
Yu
Liang-di
LD
Xie
Jing-Ming
JM
Ruan
Chang-Sheng
CS
Xu
Ti-Yuan
TY
Li
D000818:Animals; D002455:Cell Division / Q000187:drug effects; D049109:Cell Proliferation / Q000187:drug effects*; D050759:Cyclin-Dependent Kinase Inhibitor p21 / Q000378:metabolism; D050760:Cyclin-Dependent Kinase Inhibitor p27 / Q000378:metabolism; D006801:Humans; D007610:Kallikreins / Q000235:genetics* / Q000494:pharmacology*; D008297:Male; D009131:Muscle, Smooth, Vascular / Q000166:cytology* / Q000187:drug effects; D051381:Rats; D011918:Rats, Inbred SHR; D011995:Recombination, Genetic
C493958:Cdkn1a protein, rat; D050759:Cyclin-Dependent Kinase Inhibitor p21; D050760:Cyclin-Dependent Kinase Inhibitor p27; D007610:Kallikreins