Effect of atorvastatin on advanced glycation end products induced monocyte chemoattractant protein-1 expression in cultured human endothelial cells / 中华心血管病杂志

<p><b>OBJECTIVE</b>To investigate the effects of atorvastatin on advanced glycation end products (AGE) induced monocyte chemoattractant protein-1 (MCP-1) expression in human umbilical vein endothelial cells (HUVECs) and whether this effect could be linked to peroxisome proliferator-activated receptor-γ (PPAR-γ) and nuclear factor-κB (NF-κB).</p><p><b>METHODS</b>Grouping: (1) Blank control group; (2) BSA group; (3) AGE group: cells were incubated with different concentrations of AGE (10(-4), 10(-3), 10(-2) and 10(-1) g/L) for 24 hours; (4) AGE + Atorvastatin group: cells were incubated with different concentrations of atorvastatin (0.1, 1, 10 µmol/L) for 1 hour, then incubated with AGE (10(-1) g/L) for 24 hours; (5) PPAR-γ agonist (15 d-PGJ2) group: cells were incubated with 15 d-PGJ2 (10 µmol/L) for 1 hour, then incubated with AGE (10(-1) g/L) for 24 hours; (6) PPAR-γ inhibitor (GW9662) group: cells were incubated with GW9662 (5000 nmol/L) for 1 hour, then incubated with atorvastatin (1 µmol/L) and AGE (10(-1) g/L) for 24 hours. Collagenase was used to isolate the endothelial cell from human umbilical vein; RT-PCR was performed to examine the mRNA expression of MCP-1 and PPAR-γ; Western blot was performed to detect NF-κB p65 protein.</p><p><b>RESULTS</b>(1) The expression of MCP-1 mRNA was increased in proportion with increasing concentrations of AGEs which could be blocked by atorvastatin in a dose-dependent manner. (2) AGE (10(-1) g/L) significantly downregulated the expression of PPAR-γ mRNA (0.22 ± 0.08 vs. 0.69 ± 0.09, P < 0.01) while upregulated the expression of phospho-NF-κB p65 protein (0.78 ± 0.06 vs. 0.31 ± 0.01, P < 0.01) and nonphospho-NF-κB p65 protein (1.61 ± 0.16 vs. 0.59 ± 0.14, P < 0.01) compared with the control group which could be significantly attenuated by atorvastatin. (3) PPAR-γ agonist decreased the expression of phospho-NF-κB p65 protein (0.21 ± 0.01 vs. 0.78 ± 0.06, P < 0.01), nonphospho-NF-κB p65 protein (0.67 ± 0.14 vs. 1.61 ± 0.16, P < 0.01) and MCP-1 mRNA (0.17 ± 0.02 vs. 0.93 ± 0.12, P < 0.01) compared with AGE (10(-1) g/L) group. (4) PPAR-γ inhibitor antagonized the effect of atorvastatin on the expression of phospho-NF-κB p65 protein, nonphospho-NF-κB p65 protein and MCP-1 mRNA stimulated by AGE in HUVECs (P < 0.01).</p><p><b>CONCLUSION</b>The anti-inflammatory properties of atorvastatin in AGE stimulated HUVECs may partly be attributed to the effect on upregulation of PPAR-γ and downregulation of NF-κB signaling pathway.</p>

Effects of human tissue kallikrein 1 gene delivery on carotid artery neointima formation after balloon angioplasty in spontaneously hypertensive rats / 中华心血管病杂志

<p><b>OBJECTIVE</b>To investigate the effects of human tissue kallikrein 1(Ad-hKLK1) gene delivery on the neointima formation in carotid arteries of spontaneously hypertensive rats (SHRs).</p><p><b>METHODS</b>Carotid artery restenosis was induced in male SHR rats by balloon-injury. Rats were randomly assigned into 4 groups: Sham-operated (n = 6); Angioplasty (phosphate buffered solution 50 microl, n = 8); Vector virus (control virus, 1 x 10(9) IU in 50 microl, n = 8) and Ad-hKLK1(Ad-hKLK1, 1 x 10(9) IU in 50 microl, n = 8). Rats were sacrificed 4 weeks later. The wall-to-lumen area ratio and intima/media ratio in carotid artery were assessed by image analysis in HE stained sections. The mRNA bradykinin receptor (B1R and B2R) expressions were detected by RT-PCR. The protein expression of the cycle-independent kinase inhibitors p27Kip1 and p2lCip1 were determined by Western blot analysis.</p><p><b>RESULTS</b>Wall-to-lumen area ratio reduced 35.6% and intima/media ratio reduced 38.8%in Ad-hKLK1 treated SHRs compared to angioplasty group (all P < 0.001). The expression of p27Kip1 and p2lCip1 increased significantly in Ad-hKLK1 treated SHRs compared with angioplasty rats (all P < 0.001). The mRNA expression of B2R was significantly upregulated in angioplasty rats compared with sham-operated rats (P < 0.05) while mRNA expression of B1R was similar between the 2 groups.</p><p><b>CONCLUSION</b>hKLK1 gene delivery may effectively reduce neointimal formation via downregulating bradykinin B2R and up-regulating the expressions of p27Kip1, p2lCip1 signaling pathways in carotid arteries of SHRs after balloon injury.</p>

Effects of human tissue kallikrein gene transfer on the migration of vascular smooth muscule cells / 南方医科大学学报

<p><b>OBJECTIVE</b>To investigate the effects of adenovirus-mediated human tissue kallikrein (Ad-hKLK1) gene transfer on platelet-derived growth factor-BB (PDGF-BB)-induced migration of vascular smooth muscle cells from spontaneously hypertensive rats (VSMC(SHR)).</p><p><b>METHODS</b>A bicistronic recombinant adenovirus vector (Ad-hKLK1) carrying the target hKLK1 gene and the reporter gene EGFP was constructed. VSMCs isolated from the thoracic aorta of male SHR were passaged, and the quiescent VSMC(SHR) in passages 3-6 seeded in 6-well plates were treated with Ad-hKLK1 and control virus. Human PDGF-BB or icatibant Hoe140, a BK B2 antagonistat, was used as the chemoattractant and placed in the bottom chamber of the Boyden chamber. The mRNA expressions of bradykinin B1 receptor and B2 receptor were detected by RT-PCR in VSMC(SHR).</p><p><b>RESULTS</b>hKLK1 gene transfer significantly inhibited PDGF-BB-induced migration of VSMC(SHR), with the peak inhibition rate of 34.6% (P<0.001). PDGF-BB significantly increased the mRNA expression of B2 receptor but not B1 receptor in VSMC(SHR).</p><p><b>CONCLUSIONS</b>hKLK1 gene transfer can inhibit the migration of VSMC(SHR) induced by PDGF-BB, and the inhibitory effects may be not mediated by bradykinin B2 receptor.</p>

Effects of human tissue kallikrein gene delivery on the proliferation of vascular smooth muscle cells / 中华心血管病杂志

<p><b>OBJECTIVE</b>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).</p><p><b>METHODS</b>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).</p><p><b>RESULTS</b>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).</p><p><b>CONCLUSION</b>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).</p>