| BackgroundPercutaneous transluminal coronary angioplasty (PTCA) is an important method of treating coronary heart disease, but about 20% to 40% of patients suffer from vascular restenosis within 6 months after angioplasty. Neointimal formation plays an important role in the pathogenesis of restenosis after angioplasty. Cathepsin K can degrade extracellular matrix, promote VSMC and EC migration, and have an closely relation to intimal hyperplasia. Paclitaxel inhibits vascular smooth muscle cell and endothelial cells proliferation and migration and can prevent in-stent restenosis. It is not clear whether or not Paclitaxel can reduce neointimal formation through decrease Cat K expressionObjectives:The aim of this study was to evaluate the effect of paclitaxel on the neointimal formation, Cat K and NF-KB expression in rabbit iliac artery after balloon injury, to explore the potential mechanisms of paclitaxel for diminishing the neointimal formation.Methods:40 male New Zealand white rabbits were randomly divided into normal control group (n= 8), operation group (n= 16) and the paclitaxel group (n= 16), feeding with normal diet. The rabbits of operation group and the paclitaxel group were performed with balloon injury to the right iliac artery. Paclitaxel group were given paclitaxel 2mg/kg.d from intraperitoneal injection until to 7th day or to 14th day after operation. Control group and operation group were injected daily with the same volume of saline.The serum levels of TNF-a were measured before operation,first day,7th day and 28th day after operation using enzyme-link-immunosorbent assay(ELISA).The right iliac artery were excised 7th day and 28th day while they were killed after operation and then were taken to pathological test.We used RT-PCR, Western-blotting and immunohistochemistry to measure the expression of Cat K of the artery; we also used Western-blotting and immunohistochemistry to detect the expression of NF-KB of the artery.Result1. There was no significantly difference in the level of serum TNF-a between three group before operation.The first day,7th day,28th day after operation level of serum TNF-a in operation group were17.92±3.71, 23.65±4.51 and 19.73±3.83ng/l respectively, all of them were significantly higher than preoperative 12.97±2.24 ng/1, P< 0.01.but There was no significantly difference in the level of serum TNF-a between operation group and paclitaxel group at the same time. the level of serum TNF-a is highest 7th day after operation.2. Compared with the control group, operation group and the paclitaxel group intimal area and intima/media area (I/M) significantly increased, The intimal area and intimal and medial area ratio of 7th day and 28th day after operation in operation group were 0.19±0.05 mm2,0.35±0.11 and 0.69±0.11 mm2,1.39±0.13 respectively,and they comparing, P< 0.01.with the postoperative time being longer, the operation group and the paclitaxel group intimal area and intima/media area ratio (I/M) significantly increased.intimal area and intima/media area (I/M) of the paclitaxel group were less than the operation group at the same time.The medial area had no significant difference in the three groups at the two time points.3. There was little Cat K mRNA expression in the control group operation group and the paclitaxel group Cat K mRNA expression significantly increased,compared with the control group. The expression of Cat K mRNA of 7th day and 28th day after operation in paclitaxel group were 0.559±0.055 and 0.786±0.025, comparing with the two, P< 0.01.with the postoperative time prolonged, Cat K mRNA expression significantly increased in the operation group and the paclitaxel group. Cat K mRNA expression in operation group increased comparing with the paclitaxel group at the same time, P< 0.05.4. There was no obvious Cat K expression in the control group, compared with the control group, operation group and the paclitaxel group Cat K expression significantly increased. The 7th day and 28th day after operation in operation group Cat K expression were 0.421±0.05 and 0.819±0.10, Comparing with them, P< 0.01.As postoperative time moved on, Cat K expression significantly increased in the operation group and the paclitaxel group. Cat K expression(0.632±0.06) in paclitaxel group were less than the operation group (0.819±0.10) at the same time.5. Immunohistochemical analysis, there was no obvious Cat K protein expression in normal controls group, operation group and the paclitaxel group Cat K expression significantly increased.With the postoperative time being longer, The Cat K immunohistochemistry scores significantly increased in the operation group and the paclitaxel group. Compared with the operation group(16.13±1.27),the Cat K immunohisto-chemistry scores in paclitaxel group(12.46±1.53)significantly decreased at the same time, P< 0.05.6. There was little NF-KB expression in the normal control group, operation group and the paclitaxel group NF-KB expression significantly increased.As the postoperative time moved on, NF-KB expression significantly increased in the operation group and the paclitaxel group. Compared with the operation group(0.369±0.04),NF-KB expression in paclitaxel group(0.241±0.03) was less than the operation group at the same time, P< 0.01.7. Immunohistochemical analysis, there was no distinct NF-KB protein expression in normal controls group, the Cat K score of 7th day and 28th day after operation in operation group were 4.75±0.79 and 14.29±1.36 respectively,comparing between them, P< 0.01. with the postoperative time prolonged, The NF-KB immunohistochemistry scores significantly increased in the operation group and the paclitaxel group. The NF-KB immunohistochemistry scores in paclitaxel group(11.37±1.45) were significantly less than the operation group (14.29±1.36)at the same time, P< 0.05.Conclusion:Ballon injury obviously induced New Zealand white rabbits iliac artery neointimal formation, Cat K and NF-KB expression growth in intima, and they increased as postoperative time moved on. Paclitaxel maybe reduce Cat K expression through inhibiting NF-KB to decrease iliac artery neointimal hyperplasia after ballon angioplasty.The levels of serum TNF-a increased after ballon angioplasty,and it maybe has not direct relation with Cat K expression in rabbit vessel wall. BackgroundTumor necrosis factor-a (TNF-a) is an important pro-inflammatory factors, can regulate inflammatory cytokine and growth factor expression. The expression of TNF-a in the vascular neointima after balloon injury increased, Vascular injury leading to multiple inflammatory factors and cytokines being activated, the interaction of these factors increase the inflammatory reaction in the vascular wall, promotes the formation of neointima. Cathepsin K can degrade extracellular matrix, promote VSMC and EC migration, and lead to intimal hyperplasia.It is reported that TNF-a can stimulate vascular endothelial cell expression of Cat K, but the time-response and dose-response effect on TNF-a induced Cat K expression is not complete understood. It is not very clear about the potential signaling pathway mechanism for the effect.Objective:This study was aimed to study the dose-response and time-response effect of TNF-a on the expression of Cat K in human umbilical vein endothelial cells and the effects of different signaling pathway inhibitors on Cat K expression induced by TNF-a, elucidating the underlying signaling pathway mechanism for the expression of Cat K expression induced by TNF-a.Methods:HUVECs in the third to sixth generation were used in the present study.The cells were treated with different concentrations of TNF-a for 24 hours or with lOng/ml TNF-a for different times. The cells were also treated with the P38MAPK pathway inhibitor SB203580,the PI3K pathway inhibitor LY294002 and the NF-kB pathway inhibitor PDTC for 24 hours in the presence of 1Ong/ml TNF-a. We used the RT-PCR method to measure Cathepsin K mRNA levels and used the western-blotting method to measure Cathepsin K protein levels of these cells.Result1.Normal human umbilical vein endothelial cell had very little Cat K mRNA expression. With the increasing concentration of TNF-a, Cat K mRNA expression increased in endothelial cell.The Cat K mRNA expression were 0.27±0.03,0.39±0.04 and 0.50±0.04 respectively in the TNF-a concentration of 0.1ng/ml, 1ng/ml and 10ng/ml, comparing with any two of them,P<0.05. The level of lOng/ml TNF-a group Cat K mRNA expression was the highest,100ng/ml TNF-a group Cat K mRNA expression was 0.44±0.06 and it is less than lOng/ml TNF-a group, P <0.05. 2.With the increasing concentration of TNF-a, Cat K expression is growing, The Cat K expression of 0.1ng/ml, 1ng/ml and 10ng/ml TNF-a concentration group were 1.12±0.13,1.55±0.11 and 1.94±0.10 respectively, comparing with any two of them, P<0.05.The level of lOng/ml TNF-a group Cat K protein expression was the highest, 100ng/ml TNF-a group Cat K protein expression(1.36±0.56) is less than lOng/ml TNF-a group, P< 0.05.3.Compared with control group, the cells with lOng/ml TNF-a for different time had higher Cat K mRNA expression. Cat K mRNA expression were 0.37±0.04,0.59±0.06 and 0.69±0.04 respectively in the TNF-a intervention time for 6h,12h and 24h, comparing with any two of them, P<0.01.The time of induced being longer, the expression of Cat K mRNA became higher from 6 hours to 24 hours. Cat K mRNA expression(0.61±0.07) stimulated by TNF-a for 48 hour was significantly less than stimulated by TNF-a for 24 hour, P<0.05.4.The Cat K expression of TNF-a intervention time for 6h,12h and 24h were 0.36±0.04,0.54±0.05和0.84±0.07 respectively, comparing with any two of them, P<0.05.As the time of induced moved on, the expression of Cat K become higher during 6 hours to 24 hours. Cat K expression stimulated by TNF-a for 48 hour was 0.60±0.07, which was significantly less than stimulated by TNF-a for 24 hour, P<0.05.5.Compared with l0ng/ml TNF-a stimulation group(0.65±0.04), the expression of Cat K mRNA significantly decreased to 0.55±0.04, 0.57±0.01 and 0.52±0.04 respectively in the cells that were treated with SB203580,LY294002 and PDTC for 24 hours with lOng/ml TNF-a stimulation,all P<0.05.6.The expression of Cat K were 0.70±0.08,0.67±0.11 and 0.62±0.09 in the cells that were treated with SB203580,LY294002 and PDTC for 24 hours with lOng/ml TNF-a stimulation, they all were obviously less than the lOng/ml TNF-a stimulation group(0.91±0.15), all P<0.01.Conclusion:TNF-a could induce the expression of Cathepsin K in human umbilical vein endothelial cells in a dose-dependent and time-dependent manner. TNF-a may induce Cat K expression through the activation of signaling pathway of P38,PI3K AND NF-Kb. BackgroundIntegrity of intimal play an important role in preventing thrombosis and restenosis, late thrombosis and restenosis maybe have relations to the inhibition of regeneration of endothelial cells by Paclitaxel. Cathepsin K which could degrade various ECM components and promote VSMC and EC migration is closely associated with intimal hyperplasia. NF-kB is an important transcription factor and could regulates the expression of multiple gene products. Paclitaxel inhibited cell migration, but the effect of paclitaxel on the expression of cathepsin K is still unknown. It is not clear whether or not Paclitaxel can reduce Cat K expression through inhibiting NF-kB in endothelial cells.Objective:The present study was aimed to investigate the effect of Paclitaxel on TNF-a induced human umbilical vein endothelial cells expression of Cat K and NF-kB, trying to elucidate the of mechanism of paclitaxel inhibiting intimal hyperplasia.Methods:HUVECs in the third to sixth generation were used in the present study.The cells were treated with 10ng/ml TNF-a and then different concentrations of paclitaxel were added into it for 24hours.We used the RT-PCR method to measure Cathepsin K mRNA levels and used the western-blotting method to measure Cathepsin K and NF-kB levels of these cells.Results:1. The expression of Cat K mRNA were 0.85±0.06,0.68±0.04,0.59±0.02 and 0.49±0.03 respectively in the paclitaxel treated group of 1nmol/l,10nmol/l, 100nmol/l and 1μmol/1, comparing with any two of them,P<0.05. comparing with TNF-a group(1.02±0.07), their expressions decreased, P<0.05. paclitaxel could inhibit TNF-a induced Cathepsin K mRNA expression in a dose-dependent manner. With the higher concentration of paclitaxel, inhibition of the expression of Cat K mRNA became stronger.2. The Cat K expression of 1nmol/l paclitaxel group and TNF-a group were 1.08±0.07 and 0.87±0.03, both comparing, P<0.05。paclitaxel could inhibit TNF-a induced Cathepsin K protein expression in a dose-dependent manner. The higher concentration of paclitaxel, the less Cat K expression of the cells.3. Normal human umbilical vein endothelial cell had very little NF-kB expression.The NF-kB expression of 1nmol/l paclitaxel group and TNF-a group were 0.93±0.04 and 0.87±0.07, the two comparing, P <0.05. paclitaxel could inhibit TNF-a induced NF-kB protein expression in a dose-dependent manner in endothelial cells. The higher concentration of paclitaxel, the less NF-kB expression of endothelial cells.Conclusion:Paclitaxel could decrease the expression of Cathepsin K and NF-kB stimulated with TNF-a in a dose-dependent manner. paclitaxel maybe inhibit the activation of NF-kB to reduced Cat K expression in endothelial cells.
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