Objective:: Firstly, to construct recombinant adenovirus vector of human tissue kallikrein 1(hKLK1) gene and test the expression of hKLK1 gene on vascular smooth muscle cells (VSMCS) derived from spontaneously hypertensive rats(SHR). Secondly, to investigate the effects of adenovirus-mediated hKLK1 gene delivery on the proliferation, migration of VSMCS induced by platelet derived growth factor-BB(PDGF-BB) and underling mechanism, and thirdly to test the effects of hKLK1 gene delivery on the neointima formation in balloon-injured SHR carotid arteries.Methods:1 The hKLK1 gene in plasmid pBluescritII KS was digested with endonuclease and inserted into shuttle plasmid with enhanced green fluorescent protein(EGFP ) as reportor gene to construct recombinant shuttle plasmid,which was packaged in 293A cells to obtain bicistronic recombinant adenovirus vector.2 The expression of EGFP in VSMCS and carotid artery after were examined using by fluorensce microscopy . Flow cytometry was used to examine the EGFP expression and VSMCs cell-cycle. The mRNA expressions levels of hKLK1 , bradykinin B1 receptor and B2 receptor was determined by RT-PCR in VSMCs and in carotid artery . Western-blotting was used to determine the expression of hKLK1 , the cycle-independent kinase inhibitors p27Kip1 and p2lCip13 The VSMCs proliferation induced by PDGF-BB was accessed by cell counting and methyl thiazolyl tetrazoliuin (MTT) , the migration was assessed by modified Boyden chamber assay.4 30 SHR(350-450g) were randomly assigned into 4 groups: Sham-operated(normal rat carotid artery) ; Angioplasty( rat carotid artery after balloon-injured); Vector virus( balloon-injured plus infusion of control virus); Ad-hKLK1( balloon-injured plus delivery of Ad-hKLK1). After balloon-injured of the left common carotid artery , Ad-hKLK1 or control virus(1×109IU in 50μl) were infused into the injured segment and incubated for 30-45 minutes at room temperature .After incubation, the cannula was removed and blood flow to the common carotid artery was restored. The SHRs were sacrified 4 weeks after hKLK1 gene transfer .The wall-to-lumen area ratio and intima/media ratio in carotid artery were assessed by HE staining and image analysis.Results:1 A correct and funtional recombinant shuttle plasmid pDC316-hKLK1-IRES-EGFP was constructed,which was verified by PCR, restriction enzyme digesting, and DNA sequence analysis. Two vectors were transfected successfully into 293A packaging cells, amplified and then concentrated to detect recombinant adenovirus Ad-hKLK1-IRES-EGFP or control virus Ad- IRES-EGFP respectively. The expression of EGFP in VSMCS reached up to 90% by fluorensce microscope and flow cytometry after VSMCs were transfected Ad-hKLK1-IRES-EGFP in a MOI-dependent(20-100MOI) and time-dependent(1d-5d) manner. The expression of hKLK1 gene detected by RT-PCR and WB were in a MOI-dependent and time-dependent manner in VSMCs . The high expression of hKLK1 were consistent with the expression of EGFP in VSMCs.2 Proliferation of VSMCs induced by PDGF-BB were inhibited after the transfection of Ad-hKLK1 (20-100MOI) in a MOI-dependent manner by cell counting. The peak inhibition titer of Ad-hKLK1 fell on 100 MOI , with peak inhibition rate of 39.3%(cell coounting, n=3,P<0.01), 30.2% (MTT, n=3,P<0.01), peak stunning rate of cell-cycle in phase G0/G1 at 36.4%. The inhibitory effects of proliferation and cell-cycle caused by hKLK1 gene delivery were significantly abolished by Hoe140 , a bradykinin B2 receptor antagonist. Migration of VSMCs induced by PDGF-BB were inhibited after hKLK1 gene delivery, with peak inhibition rate of 34.6% (n=6,p<0.001). However the inhibition effects of migration was not blocked by Hoe140. The protein expression of p27Kip1 and p2lCip1 increased significantly after the hKLK1 gene delivery, whereas Hoe140 nearly completely blocked these effects (n=3,P<0.001,respectively). PDGF-BB was significantly increased the mRNA expression of B2 receptor not B1 receptor in VSMCs .3 The protein expression of hKLK1 in carotid artery was observed 4 weeks after hKLK1 gene delivery, with non-expression in sham-operated , angioplasty and vector virus groups. In SHR that received hKLK1 gene delivery , 35.6% reduction in wall-to-lumen area ratio and 38.8% inhibition in intima/media ratio were noted at the ballon angioplasty vessels compared with that of angioplasty SHR (n=8, P<0.001,respectively). The protein expression of p27Kip1 and p2lCip1 increased significantly after the hKLK1 gene delivery on rats and sham-operated rats compared with angioplasty rats (n=8, P<0.001, respectively) .The mRNA expression of B2 recetpor were significantly increased in rats balloon-injured carotid arteries compared with sham-operated rats. However there was no difference of mRNA expression of B1 receptor in all rats.Conclusion:1 We successfully constructed recombinant adenovirus vector of Ad-hKLK1-IRES-EGFP . The functional gene hKLK1 and reportor gene EGFP may coexpress in VSMCS ,which may provide a direct and highly efficient expression vector in the study of hKLK1 gene therapy .2 The hKLK1 gene delivery may inhibits the proliferation and migration of VSMCS induced by PDGF-BB. Bradykinin B2 receptor probably mediated the up-regulating expression of p27Kip1 and p2lCip1 that contributed to the inhibitory effects of proliferation of hKLK1 . However, the inhibitory effects of migration of hKLK1 gene delivery may be mediated by bradykinin B1 receptor.3 In the animal model, the reduction neointimal formation after hKLK1 gene delivery may also be the involvement of bradykinin B2 receptor and up-regulating the expression of p27Kip1 , p2lCip1 signaling pathways in the carotid arteries of SHR after balloon injury.4 The study suggest that the hKLK1 gene delivery may effectively contribute to the reduction neointimal formation via inhibition of VSMCS proliferation and migration through bradykinin B2 receptor and up-regulating the expression of p27Kip1, p2lCip1signaling pathways in the carotid arteries of SHR after balloon injury.
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