| Despite increasing the use of arterial grafts in recent years, saphenous veins remain the most widely used bypass conduit in coronary artery bypass procedure. Unfortunately, vein grafts develop accelerated atherosclerosis as a result of inti-mal hyperplasia(IH) , which is responsible for the grafts failure. Even though the many technological advances in vascular intervention, IH remains an expensive , morbid and unsolved problem. The development of IH is characterized by uncontrolled proliferation and migration of media smooth muscle cell into the in-tima and massive deposition of interstitial collagen. Mitogen - activated protein kinases (MAPK) , composed of extracellular signal regulated kinases ( ERKs ) ,c -jun NH2 -terminal kinases(JNKs) ,and p38 - MAP kinases,are protein ser-ine/threonine kinases and play a critical role in cell differentiation, growth and apoptosis, and the regulation of various transcription factors and gene expressions. The results of the in vivo experiments have revealed that three MAPKs are all involved in the development of vascular remodeling and inhibition of MAPKs could prevent intimal hyperplasia after balloon injury, associated with the inhibition of smooth muscle cell proliferation and migration. The recent studies reported that MAPK signaling pathways were activated during the preparation and ar-terialization of vein grafts and may play as potential mediators of vein graft remodeling and subsequent IH. Green tea polyphenols have been shown to cause growth arrest of rat, rabbit and human vascular smooth muscle cells (SMCs) in culture. Hofmann and his colleagues demonstrated that treatment of aortic SMCs with epigallocatechin -3 - gallate(EGCG) , a major tea polyphenol, at a high dose could increase expression of p53 and cause apoptosis, while lower dosecould arrest proliferation. In vivo studies, green tea catechins could inhibit IH in rat balloon - injury models either applied by intake with drinking water or by local delivery. In the present study, we investigated whether the administration of EGCG during the preparation of vein could suppress IH in a rabbit model of interposition vein grafts. In addition, we also examined the effects of EGCG on MAPK activation in vitro and in vivo.MethodsRabbit jugular vein segments were incubated in saline containing EGCG 0. lmg/ml or 1. Omg/ml. HPLC and FITC - labelled EGCG were used to assess the absorption characteristics of EGCG in vitro. Autogenous vein graft model was established in 30 rabbits which were divided into thee groups ;EGCG 0. lmg/ nu\l. Omg/ml and control group( n = 10,10,10). Vein grafts were incubated with EGCG or saline before arterial interposition grafting. Animals were sacrificed at 21 d after operation and intimal thickening[Intimal thickness (I) ,medial thickness, intima - to - media ratio (I/M) , intimal area, medial area and intima -to -media area ratio] was assessed by computerized digital morphometry. In EGCG 0. lmg/ml group and control group at lw and 3w (n =5) , intimal thickness was analyzed and calculated using a Scion Image System (version 4. 02) , the proliferation and apoptosis of neointimal cells were determined by Ki67 staining and terminal deoxynucleotidyl transferase biotin nick end - labeling (TUNEL) method, respectively. At 3h,24h, lw and 3w,five grafts in EGCG 0. lmg/ml group and control group, were individually frozen, and total protein was extracted. Phosphorylation of extracellular signal - regulated kinase ( ERK) 1/2 was determined with Western blot analysis.ResultsResults After incubation with FITC - labelled EGCG for lh, a distinct green fluorescent signals were observed in intimal cells and adventitial cells of vein segments. The extent and intensity of staining was much greater in EGCG1. Omg/ml group than 0. 1 mg/ml group. The positive staining was observed with either dose of FITC - labelled EGCG at 24h after transplantation in a dose dependent manner in vivo. At a dose of 0. 1mg/ml, EGCG was absorbed at levels of2.9±0.9μg/mg,5.8±2. 1 μg/mg,6.1 ±2.1μg/mg and 8.0 ±2.3μg/mg at 1h,2h,3h and 4h after incubation, respectively. While in 1.0mg/ml group, 13.0±2.2μg/mg,19.9 ±3.6μg/mg,26. 8 ±4.5μg/mg and 27. 8 ±4.1μg/mg of EGCG was absorbed. Compared with the control group, In EGCG 0.1mg/ ml group, intimal hyperplasia development was prevented at 3w[ I:36. 8(11. 5-58.1)vs67.9(41.3-214.7)μm,Z= -2.87,p <0.01; I/M:0. 79(0. 50 -1.31)vs0. 31(0.20-0.68) ,Z = -2. 88, p < 0.01; intimal area:0.45(0.10 -0. 80)vs0. 69(0. 33 -2. 66)mm2,Z= -2. 08, p < 0. 05; intima - to -medial area ratio: 0.31(0.10-0.52) vs 0.68(0.30-1. 19), Z= -3.07,p<0. 01]. A significant reduction was showed in neointimal formation of EGCG 1. Omg/ml group at 3w [ I:48.0( 12. 7-71.4) vs 67.9(41.3 -214.7)μm,Z =-2.27 ,p <0. 05; intima - to - media area ratio: [0. 68 (0. 30 - 1. 19) vsO. 27 (0.07-0.80),Z= -1.82,p<0.05] either. In EGCG 0.1mg/ml group, The intimal thickness was inhibited at lw after operation than control group(I;26. 6± 12.2 vs 52.0 ± 14.9μm, t =2.95,p <0.05) . Immunohistochemic -al analysis of Ki67 indicated decreased amount of positive smooth muscle cells in the neointimaat lw (1139.2 ± 117. 8 vs 1921. 1 ±448.8/mm2 p<0.05) and 3w (171.9 ±85.5 vs 690.9 ± 350. 5/mm2, p <0.05) too, whereas cell apoptosis was not different between two groups. In control group, ERK demonstrated activation compared with preincubation level. However, when compared with the saline solution, the EGCG incubation showed greater than 21. 8% inhibition of ERK(t=3.08,p<0.05). EGCG 0. lmg/ml group showed 24.6% and 31.9% inhibition by 3h and day 7 (t =3. 04,p <0.05和t =3.46,p <0.01) than control group, but no significant difference by 3h and day 21 (p>0.05).ConclusionThese results indicate that the local delivery of EGCG prevents intimal hyperplasia in a rabbit vein bypass grafting model through the downregulation of... |
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