| ObjectiveOver-expression of tissue kallikrein 1(TK-1) gene or tissue inhibitor of matrix metalloproteinase 1(TIMP-1) gene has previously been shown to partially inhibit the intimal proliferation in rat vascular restenosis models. However, the synergistic effects of combination with TK-1 and TIMP-1 on improving vascular restenosis remain to be determined. In present study, we firstly transfected adenoviral vectors with respectively human TK-1(Ad-h TK-1) or human TIMP-1(Ad-h TIMP-1) gene and dual gene co-expression vector(Ad-h TK-1-h TIMP-1) into Sprague-Dawley rats’ carotid artery balloon angioplasty segment, investigate the effects of Ad-h TK-1 and/or Ad-h TIMP-1 gene delivery on the neointima formation. MethodsAdult male Sprague-Dawley rats weighing 250-350 g underwent carotid artery balloon angioplasty and local delivery of adenoviruses. Rats were randomly assigned into 6 groups:(1) Sham-operated(n=6),(2) Angioplasty(saline buffered solution 30μl, n=8),(3) Vector virus(control virus, 1×109pfu in 30μl, n=8),(4) Ad-h TK-1(1×109pfu in 30μl, n=8),(5) Ad-h TIMP-1(1×109pfu in 30μl, n=8), and(6) Ad-h TK-1-h TIMP-1(1×109pfu in 30μl, n=8). Rats were sacrificed 2 weeks later. Morphometric assessment for neointimal hyperplasia and immunohistochemical staining for PCNA were performed. The levels of h TK-1, h TIMP-1, MMP-2, MMP-9 protein expression were detected by western blot assay. Results1. The rat carotid artery balloon angioplasty model was established successfully. 7, 14 or 28 days after balloon angioplasty, intimal area(IA) and intima/media area ratio(I/M) are gradually increased(trend test, all P<0.01); IA, I/M of experimental groups were time-dependent(7-28 days) increased(P<0.01).2. Compared with sham-operated rats, balloon angioplasty resulted in increases in IA and I / M ratio(all P<0.01). No significant difference was found between rats underwent angioplasty or vector virus delivery(all P>0.05). Compared with rats delivered vector virus, morphometric analysis revealed that Ad-h TK-1, Ad-h TIMP-1 or Ad-h TK-1-h TIMP-1 gene transfer reduced IA( 0.110±0.015 vs. 0.160±0.010;0.121±0.016 vs. 0.160±0.010;0.081±0.008 vs. 0.160±0.010;all P<0.01) and I/M ratio(1.443±0.097 vs. 2.035±0.117; 1.522±0.078 vs. 2.035±0.117; 0.972±0.072 vs. 2.035±0.117;all P<0.01), reduced neointima formation by 29.1%, 25.2% and 52.2% respectively. Compared with rats delivered Ad-h TK-1 or Ad-h TIMP-1 respectively, dual gene delivery further reduced the IA and I / M ratio(all P<0.01), resulted in a significant inhibition of neointima thickening.3. Compared with sham-operated rats, balloon angioplasty resulted in increases in PCNA, MMP-2 and MMP-9 levels(all P<0.01). No significant difference was found between rats underwent angioplasty or vector virus delivery(all P>0.05). Ad-h TK-1, Ad-h TIMP-1 or Ad-h TK-1-h TIMP-1 gene transfer also down regulated PCNA, MMP-2 and MMP-9 levels(all P<0.01). Compared with Ad-h TK-1 or Ad-h TIMP-1 gene transfer, Ad-h TK-1-h TIMP-1 dual gene transfer significantly reinforced down regulation of PCNA, MMP-2 and MMP-9 levels(all P<0.01)(P<0.01). Conclusions1. The rat carotid artery balloon angioplasty model was established successfully. With local application of recombined adenoviruses, the target gene TK-1 and(or) TIMP-1 can be successfully transferred to the carotid artery and effectively expressed.2. In the rat carotid artery balloon angioplasty restenosis model, TK-1 or TIMP-1 over-expression can inhibit neointimal formation after balloon angioplasty, while dual gene delivery further reduced neointimal hyperplasia, which may via down regulating the expressions of PCNA, MMP-2 and MMP-9.3. Our results suggest co-expression of TK-1 gene and TIMP-1 gene as an experimental improvement of rat carotid artery neointima formation after balloon angioplasty. These results provide new insight into the role of several genes therapy. |
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