RAGE与肺动脉高压.docx

晚期糖基化终末产物受体在肺动脉高压形成中的重要作用背景：肺动脉高压是一种以促进肺动脉血管平滑肌细胞增殖并抑制其凋亡为特点的血管病变。这种病变使肺动脉压和肺血管的阻力增高。最近调查已经表明了信号转导与转录激活因子（STAT3）/骨形成蛋白2型受体（BMPR2）/ 过氧化酶增殖因子活化受体（PPARy）/在肺动脉高压中的影响。STAT3激活导致BMPR2下调，并减少PPARy，这有助于在PAH中出现细胞增殖及抑制细胞凋亡。在软骨细胞中，这条轴的激活已经被证实其归功于RAGE，由于RAGE在PAH的病人中是最好的上调蛋白之一并且具有强大的STAT3催化剂，因此，我们假定这是通过激活STAT3，RAGE诱导BMPR2和PPARy的下调，促进肺动脉高压-肺动脉血管平滑肌（PAH-PASMC）的增殖和抑制其凋亡而实现。方法与结果实验组在试管内，分别取PAH病人和健康人的PASMc，我们观察的到RAGE在PAH-PASMC过度表达（增高6倍），包括STAT3的激活（阳性细胞从10%增加到40%）和BMPR2与PPARy水平 的降低（减少超过50%）。在体内建立PAH模型（用S100A4 recapitulates）：通过控制细胞内的RAGE药理活性（即增加RAGE到6倍，包括激活STAT3并减低BMPR3和PPARy。）在以上两种情形下，这种表型的功能都因RAGE的抑制而被翻转。在试管内，通过抑制在野百合碱（MCT）和Sugen诱导的PAH的RAGE后，观察到以肺动脉压和右心室肥厚减轻为特点的效应。（对照组大鼠平均动脉压设置在15mmhg作用，PAH模型组设置在平均动脉压超过40mmhg,然后将RAGE抑制，结果用MCT和Sugen处理后的两种模型大的大鼠平均动脉压分别降低至20和28mmhg）。这与改善肺灌注压和血管重建导致的细胞增殖受抑制（抑制超过50%）和BMPR2/PPARy轴的恢复有密切联系。结论：我们证实了RAGE在PAH形成过程中的作用。由此，提出RAGE构成了一种治疗PAH新的，具有应用前景的作用靶点。结果1：晚期糖基化终末产物受体（RAGE）增加并激活了在体肺动脉高压（PAH）A, RAGE expression measured by quantitative reverse-transcription polymerase chain reaction (qRT-PCR) normalized to 18S is significantly increasedin both whole lung tissue (n=8 patients per group) and in PA smooth-muscle cells (PASMCs) isolated from PAH patients compared with controls. Thiswas confirmed by immunoblots showing a 6-fold increase in PAH-PASMCs compared with controls (n=3 PAH and n=5 control cell lines, P0.01).通过逆转录聚合酶链反应的定量检测，RAGE在肺动脉高压（PAH）患者的在体的整个肺组织和离体的肺动脉平滑肌细胞（PASMCs）的表达均增高。RAGE的表达与PAH严重程度的关系: 通过免疫荧光测定，RAGE蛋白在终末肺动脉平滑肌细胞中的表达与PAH患者的严重程度呈密切的相关性。注：DAPI(4,6-二脒基-2-苯基吲哚)是一种能够与DNA强力结合的荧光材料。除了肺部以外，RAGE在其他器官及组织没有明显增高。图2：RAGE的表达可以被改变A, Control PASMCs treated with S100A4 had a significant increasein RAGE expression measured by immunoblot (n=3 to 5 cell lines per group, P0.05). It is also shown that scrambled siRNA (negative control,siSCRM) transfection had no additional effect on PAH-PASMCs.通过免疫印迹法定量检测我们发现：对照组中加入了S100A4蛋白之后RAGE 的表达明显上升，与PAH患者的肺动脉平滑肌细胞内 RAGE表达相当，而在此基础上加入了siRAGE之后RAGE的表达显著下降，同时也发发现在转染了干扰siRNA(siSCRM)之后的PAH-PASMCs其RAGE的表达没有明显变化。而加入了siRAGE之后RAGE的表达显著下降。B, This increase in RAGE expression and activation correlates with STAT3activation measured in the exact same conditions by nuclear translocation assay (percentage of cells with PY705-STAT3 nuclear localization) andimmunoblot (PY705-STAT3/STAT3 ratio normalized to amidoblack) (n_50 cells/patient in 3 to 5 patients, P0.05).在相同的条件下通过核易位检测与免疫印迹法检测我们发现这种RAGE表达和激活的增加与STAT3蛋白的活化相关在人体PASMC中RAGE能促进STAT3的活化。图3：我们已经知道，BMPR2和 PPARc是PAH的致病因子。研究表明，RAGE是这些致病因子的上游致病因子。A, BMPR2mRNA and protein expression levels were measured in S100A-treated cells and in PAH-PASMCs with and without siRAGE. We demonstrated thatBMPR2 is modulated with RAGE expression, as it was decreased in S100A4-treated (100 ng/mL for 48 hours) and PAH cells and was restored onRAGE inhibition in both groups with both techniques (n=3 to 5 cell lines per group, P0.05).S100A4处理后的细胞与PAH-PASMCs分别加入和不加siRAGE处理，可以观察到，BMPR2的变化与RAGE的表达密切相关，与对照组相比。经上述处理后的两组实验，MPR2的表达明显降低，而在加入RAGE抑制剂之后，两组实验MPR2的表达量恢复从前。B, Effect on PPARc expression was measured byimmunofluorescence (percentage of cells expressing PPARc of total number of cells measured by DAPI; n_50 cells/patient in 3 to 5 patients,P0.05). These results were confirmed by the PPARc luciferase assay (relative luminescence units; n=3 to 5 cell lines per group, P0.05).Furthermore, we demonstrated that BMPR2 is upstream of PPARc, as its inhibition (by siBMPR2) decreased PPARc activity. In both stimulatedcontrol cells and in PAH-PASMCs, RAGE inhibition increased PPARc activity.用免疫印迹法测定PPARc的表达，用荧光素酶定量检测PPARc的表达。结果表明:BMPR2在PPARc的上游表达，即它被抑制将减少PPARc的活化。在对照组与实验组中，RAGE的抑制都使PPARc的活化增多。为了探究RAGE对在试管内PASMC的增殖与凋亡的作用，我们设计如下实验：图4：RAGE对PASMC的增殖与凋亡均有调节作用。S100A4-treated cells had increased proliferation rates (percentage of Ki67-positive cells; *P0.001 vs control cells). This was reversed on RAGE(siRAGE) or STAT3 (siSTAT3) inhibition or PPARc activation (rosiglitazone; dP0.05 vs S100A4). Decrease in BMPR2 (siBMPR2) or PPARc (W9662)showed increased proliferation levels, like what was found with S100A4 (*P0.01 vs control cells). Right, PAH-PASMCs had increasedproliferation, which was reversed on RAGE (siRAGE) or STAT3 (siSTAT3) inhibition (dP0.05 vs PAH+siSCRM) or PPARc activation rosiglitazone;*P0.01 vs PAH). The beneficial effect of RAGE inhibition was bypassed with BMPR2 (siBMPR2) or PPARc (W9662) dowregulation (#P0.05 vsPAH+siRAGE), demonstrating that they are downstream of RAGE (n_50 cells/patient in 3 to 5 patients, P0.05).由图可知：左图S100A4处理过的细胞促进增殖，该作用可以被RAGE或STAT3抑制作用或PPARc的激活所翻转；而降低MBPR2或PPARc则可以促进细胞的增殖。右图中，PAH-PASM细胞增殖作用可以被RAGE或STAT3的抑制剂或PPARc活化剂所翻转。RAGE抑制剂的效应与BMPR2或PPARy的下调作用相当，由此表明：后两种者在RAGE的下游。B, S100A4-treated and PAHPASMCshad decreased apoptosis (percentage of terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling TUNEL) and RAGEinhibition (siRAGE) significantly promoted apoptosis in both groups (n_50 cells/patient in 3 to 5 patients, P0.05).为了探究RAGE在正常和PAH患者肺部的表达，我们进行以下实验“图5：在用注射MCT-和Sugen制备的大鼠PAH模型中，RAGE的表达增多，而抑制RAGE之后可以翻转PAH。Heavy RAGE mRNA and protein expression can be seen in lungs of rats with severe PAH在严重的PAH大鼠肺内有大量RAGE的mRNA和蛋白质的表达RAGE inhibition (1 nebulization of 1 nmol of siRAGEon day 15 for the MCT model and weekly nebulization of 1 nmol starting at week 7 in the Sugen model) decreased mean PA pressure. SugeninducedPAH was more severe than in the MCT-induced model, as shown by mean PA pressure (n=8 rats per group, P0.05)抑制RAGE降低平均动脉压；Sugen诱导 的PAH比MCT诱导的PAH模型更严重。C, The Sugen modelalso had increased right ventricular (RV) hypertrophy compared with the MCT model.Sugen模型也比MCT模型在增大右心室肥厚方面更严重。图6：在PAH中抑制RAGE可以翻转血管重塑。 A, Lung perfusion was measured by CT scan, and decreased lungperfusion was found in both PAH models. RAGE inhibition increased lung perfusion in both models通过CT扫描测定肺灌注，实验发现在MCT和Sugen模型中肺灌注都降低，而抑制RAGE之后可增加两者的肺灌注。、图7：As with in vitro, proliferation was mediated by STAT3, BMPR2, and PPARc, and RAGE inhibition restored whole signaling pathway.Thus, RAGE inhibition decreased STAT3 activation (PY705/STAT3 ratio by immunoblot and PY705-STAT3 nuclear translocation byimmunofluorescence), increased BMPR2 and PPARc mRNA and protein expression (qRT-PCR and immunofluorescence IF) in total lung extractsand in distal PA, respectively (for IF: n=5 arteries at random/rat with 8 rats per group; for qRT-PCR and immunoblot: n=5 rats per group, P0.05在试管内细胞的增殖可以被STAT3，BMPR2,PPARc调节，RAGE抑制剂可以使全部信号通路恢复。因此，RAGE抑制剂可抑制STAT3的激活，促进BMPR2和PPARc的表达。图8：由此可以提出机制图在PAH中，RAGE/STAT3/BMPR2/PPARc轴的 发病机制图Increased S100A4 triggersRAGE activation, resulting in STAT3 activation, decreased BMPR2expression and PPARc activation. Once this axis is activated, itincreases smooth muscle cell proliferation and resistance toapoptosis, characteristic of PAH.讨论：现在，我们已经证明了RAGE与PAH的发展过程有密切联系，我们确信其将成为一种新的研究PAH治疗措施的途径。RAGE在PAH患者肺内的过度表达及其被S100A4激活，这又增加了人的PAH。我们注意到RAGE导致STAT3激活，同时引起BMPR2和PPARy的下调（图8）。 我们提供了直接在体外和体内证据显示：RAGE的抑制翻转PAH的机制涉及抑制PASMC增殖通过重建肺动脉和恢复BMPR2及PPARy的表达并在血管壁激活。由于我们的目的的确定一种翻转已经建立起的PAH的新的方式，所以我们更关注PASM而并非内皮细胞，这似乎更能影响PAH的发作。尽管如此，RAGE在Sugen诱导的PAH模型中的有益影响表明RAGE也可能在内皮细胞相关的血管损伤如在PAH患者及实验模型中所见的从状的病灶，有重要作用。未来的研究将重点评估RAGE在PAH的内皮功能紊乱方面的作用。由于在PAH发病过程中的作用，RAGE是一个有趣的治疗靶点。目前已经证实，STAT3在PAH发病前被激活，因为RAGE引起STAT3的激活，所以RAGE可能在PAH发生发展中起重要作用。事实上,在兔软骨细胞,RAGE通过丝裂素活化的蛋白激酶(MAPKs)和JNK下调PPARc表达。这种机制也可以发生在PAH因为MAPK在肺动脉高压症中可以影响细胞迁移。最近有研究显示微小RNA可能在信号通路中有潜在的作用。此外，噻唑烷二酮类如罗格列酮已经表明可以降低糖尿病话患者内皮细胞RAGE的表达，这意味着一种可能的反馈环正如我们模型中提及的RAGE是PPARy的上游调节器的存在。由于在肺循环和体循环有许多不同的机制，所以更多的调查研究急需证实以上假说。最后,肺循环在PAH中是选择性的病变也是一个主要治疗的挑战。大多数的药物针对脉管系统，如果的系统地给药,势必会影响健康的正常循环,从而限制疗效。在PAH,RAGE的下游靶点如STAT3,即使其影响血管重塑，在许多组织结构性表达以及影响免疫反应等但在治疗上可能不会成为治疗靶点。最近研究显示，恢复BMPR2