血管紧张素Ⅱ1型受体相关蛋白抑制血管平滑肌细胞增殖和血管内膜增生机制的研究

Inhibitory effect of angiotensin Ⅱ type 1 receptor - associated protein on vascular smooth muscle cells proliferation and neointimal formation

目的探讨血管紧张素Ⅱ（AngⅡ）1型（ATI）受体相关蛋白（ATRAP）通过AngⅡ1型受体（AT1R）调节血管平滑肌细胞（VSMCs）增殖及血管内膜增生的作用机制。方法将ATRAPcDNA转至pSV2neo载体，然后转染至来源于成年sD大鼠胸主动脉的VSMCs。VSMCs内DNA合成，细胞外信号渊控激酶（ERK）及ERK磷酸化的表达分别通过。H胸腺嘧啶核苷的合成和Westernblot方法检测。利用球囊损伤血管的模型，观察转染和未转染ATRAPcDNA的12周雄性SD大鼠的血管形态学变化。结果（1）在AngⅡ刺激VSMCs48h后，ATRAP过表达可以抑制AngⅡ诱导的3H胸腺嘧啶核苷的合成（P〈0．05）。ATRAP的过表达使磷酸化ERK的活性显著降低（P〈0．05），并明显抑制了损伤动脉的新生内膜增生。（2）术后4周阴性对照组的内膜厚度和中膜厚度为（99．43±10．27）μm和（146．82±12．61）μm，术后8周为（158．62±15．93）μm和（201．04±17．92）μm，术后4周ATRAP转染组的内膜厚度和中膜厚度为（54．27±7．71）μm和（99．62±9．82）μm，术后8周为（84．82±9．16）μm和（137．93±13．94）μm。术后4周阴性对照组的内膜面积和中膜面积为（203．38±29．57）μm和（374．63±38．27）μm，术后8周为（347．16±27．05）μm和（439．59±35．52）μm，术后4周ATRAP转染组的内膜面积和中膜面积为（126．18±19．26）μm和（258．42±23．92）μm，术后8周为（161．46±22．49）μm和（160．28±22．91）μm。术后4、8周各组实验动物颈总动脉壁内膜和中膜均有所增厚，面积也有所增大，pSV2neo转染组较对照组内膜和中膜厚度增加均明显减轻（P〈0．01）；管腔面积在各时间段pSV2neo转染组均大干阳性对照组和阴性对照组（P〈0．01）、（3）定量图像分析显示：与pSV2neo转染组比较，ATRAP转染组的动脉内膜面积儿乎减半，其差异有统计学意义（P〈0．05）。ATRAP转染的损伤动脉内膜形成受到了显著抑制，但ATRAP过发表迅评术影响损伤动脉的中膜面积。结论AT1受体介导的ERK活性在AngⅡ调节的VSMCs增殖中起重要作用。ATRAP对VSMCs增殖和新生内膜形成的抑制作用可能是由于其干扰AT1受体介导的ERK活性。

Objective To investigate the effect of angiotensin Ⅱ （Ang Ⅱ） type 1 （AT1） recep- tor - associated protein （ATRAP） on vascular smooth muscle cells （VSMCs） proliferation and neointimal formation mediated by ATI receptor. Methods ATRAP cDNA was subcloned into pcDNA3 vector, and transfected into VSMCs isolated from thoracic aorta of adult Sprague -Dawley （SD） rats. DNA synthesis, extracellular signal- regulated kinase （ERK） and phospho- ERK expression in VSMCs were analyzed by measurement of 3H thymidine synthesis and Western blotting respectively. Morphological changes were ob- served in the balloon injured artery of 12 - week male SD rats with or without transfection of ATRAP cDNA. Results （ 1 ） ATRAP overexpression in VSMCs inhibited Aug Ⅱ - induced 3H thymidine synthe- sis after 48 h stimulation （ P 〈 0. 05 ）. ATRAP overexpression in injuried arteries significantly decreased the activation of phospho -ERK and inhibited neointimal formation （P 〈 0. 05 ）. （2） At 4th and 8th week postoperation, intima thickness and media thickness were （99. 43 ± 10. 27 ） μm and （146. 82 ± 12.61 ） μm, and （158.6 ±15.93） μm and （201.04 ±17.92） μm in negative control group, （54. 27 ± 7. 71） μm and （99.62±982） μm, and （84. 82 ±9.16） μm and （137.93 ± 13.94） μm in ATRAPtransfection group, respectively. On the postoperative week 4 and 8, the intimal area and the membrane ar- ea were （203.38 ±29.57）μm and （374. 63 ±38.27） μm, and （347.16 ±27.05）μm and （439. 59 ± 35.52） μm in negative control group, and （126. 18 ± 19.26） Irm and （258.42 ±23.92） μm, and （ 161.46 ± 22.49 ） μm and （ 160. 28 ± 22. 91 ） txm ATRAP transfection group, respectively. At 4th and 8th week postoperation, the intima and media thickness of the carotid artery on the postoperative week 4 and 8 were increased to varying degrees in all groups, and the increase of the intima and media thickness in psv2neo transfection group was significantly alleviated as compared with control groups （ P 〈 0. 01 ）. The lumen area in each time period of psv2neo transfection group was greater than that in positive control group and negative control group （P 〈 0. 01 ）. （3） Quantitative image analysis showed that the arterial intima ar- ea in the ATRAP group was almost halved as compared with the pSV2neo transfection group （ P 〈 0. 05 ）. The intimal formation of injured arteries was significantly inhibited by ATRAP transfection, but the expres- sion of ATRAP was not affected by the membrane area. Conclusion The AT1 receptor - mediated activa- tion of ERK plays an essential role in Ang Ⅱ- regulated VSMCs proliferation. The inhibitory effects of AT- RAP on VSMCs proliferation and neointimal formation might be due to the interference of AT1 receptor - mediated activation of ERK.