【摘要】目的探讨激活内皮素-1(endothelin-1,ET1)导致腹膜血管新生是腹膜损伤的一种潜在的作用机制。方法培养人腹膜间皮细胞(human peritoneal mesothelial cells,HPMCs)(HMrSV5)，高糖刺激HPMCs，观察ET1 的变化；加入外源性ET1，Western blot 检测细胞外信号调节激酶1/2(ERK1/2)和E26 转录因子-1(Ets-1)磷酸化；分别使用siRNA 和封闭性抗体抑制Ets-1，通过BrdU 测定HPMC 的增殖；最后，通过小管形成实验，使用Image J 软件分析5 个随机选择的区域的总毛细管长度。结果高糖可诱导HMPC 高表达ET1(2.5%，F=5.561，P=0.036；4.25%，F=4.784，P=0.008)；外源性ET1 诱导下，HPMC 中ERK1/2 的磷酸化水平显著上调(F=66.347，P=0.026)；ERK1/2 下游的转录因子Ets- 1 表达显著升高(F=110.614，P=0.031)；阻断MEK-1 显著消除ET1 对ERK 1/2 的激活(F=56.361，P=0.006)；加入ETA和ETB受体抑制剂后，ETAR 抑制剂可明显降低Ets- 1 的表达(F=131.191，P=0.039)。ET1 可诱导HPMCs 增殖(F=4.671，P=0.046)，其可被ET1 拮抗剂减弱(F=5.329，P＜0.001；F=5.362，P＜0.001)；抗体和siRNA 介导的Ets-1 阻断具有相似的抗增殖作用(F=5.638，P＜0.001；F=4.611，P=0.007)。因此，ET1 通过ERK1/2-Ets-1 信号传导途径特异性地促进HPMCs 增殖。另外，ET1 处理的HPMsC 的条件培养基可以显著促进HPMCs 中血管内皮生长因子(vascular endothelial growth factor,VEGF)产生(F=11.614，P＜0.001)和内皮细胞血管生成。结论ET1 通过ERK1/2-Ets-1 信号通路促进HPMCs 增殖，并促进VEGF 产生和内皮细胞血管生成。
Abstract
【Abstract】Objective To investigate the mechanism of endothelin-1 (ET1) activation leading to the peritoneal mesothelial cell proliferation-induced peritoneal angiogenesis. Methods Human peritoneal mesothelial cells (HPMCs) (HMrSV5) were cultured and underwent high glucose stimulation. The secretion of ET1 from HPMCs was assayed. Exogenous ET1 was added in the medium and phosphorylated extracellular signalregulated kinase 1/2 (ERK1/2) and E26 transformation specific- 1 (Ets- 1) were detected by western blot. Knockdown and blockage of Ets-1 were carried out by siRNA interference and anti-Ets-1 antibody, respectively.
The proliferation of HPMCs was determined by BrdU method. Total capillary length of the five randomly selected regions was analyzed by capillary formation experiments and Image J software. Results High glucose stimulation induced the higher expression of ET1 in HMPCs (2.5% glucose, F=5.561, P=0.036; 4.25% glucose, F=4.784, P=0.008). Exogenous ET1 promoted the increases of phosphorylated ERK 1/2 (F=66.347, P=0.026) and Ets-1 (F=110.614, P=0.031), the downstream molecule of ERK1/2, in HPMCs. Blocking MEK-1 in HPMCs could significantly abolish the activation of ERK 1/2 induced by ET1 (F=56.361, P=0.006). After the addition of ETA and ETB receptor inhibitors to HPMCs, ETA receptor inhibitor was found to reduce the expression of Ets- 1 (F=131.191, P=0.039). ET1 was capable of inducing HPMC proliferation (F=4.671,P=0.046), which could be attenuated by ET1 antagonists (F=5.329, P＜0.001; F=5.362, P＜0.001). Knockdown and blockage of Ets- 1 had similar anti- proliferative effects (F=5.638, P<0.001; F=4.611, P=0.007).These findings suggest that ET1 specifically promotes HPMC proliferation through the ERK1/2-Ets-1 signaling pathway. In addition, HPMCs in conditional media and stimulated with ET1 significantly promoted VEGF production (F=11.614, P＜0.001) and endothelial cell angiogenesis. Conclusion ET1 promotes HPMCs proliferation, VEGF production and endothelial cell angiogenesis via the ERK1/2-Ets-1 signaling pathway.
关键词
腹膜透析 /
内皮素-1 /
细胞外信号调节激酶1/2 /
E26 转录因子-1 /
血管内皮生长因子 /
血管生成
Key words
Peritoneal dialysis /
Endothelin-1 /
ERK1/2-Ets-1 /
Angiogenesis
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参考文献
[1]Padwal M, Margetts PJ.Experimental systems to study the origin of the myofibroblast in peritoneal fibrosis.Kidney Res Clin Pract. 2016 Sep; 35(3):133-41.
[2]Lee HY, Park HC, Seo BJ, et al.Superior patient survival for continuous ambulatory peritoneal dialysis patients treated with a peritoneal dialysis fluid with neutral pH and low glucose degradation product concentration (Balance).Perit Dial Int. 2005 May-Jun; 25(3):248-55.
[3]Margetts PJ.Twist: a new player in the epithelial-mesenchymal transition of the peritoneal mesothelial cells.Nephrol Dial Transplant. 2012 Nov; 27(11):3978-81.
[4]Pernow J, Ahlborg G, Lundberg JM, et al.Long-lasting coronary vasoconstrictor effects and myocardial uptake of endothelin-1 in humans. Acta Physiol Scand. 1997; 159:147-153
[5]Fukuroda T, Fujikawa T, Ozaki S, et al.Clearance of circulating endothelin-1 by etb receptors in rats. Biochem Biophys Res Commun. 1994; 199:1461-1465
[6]Zhang J, Yang W, Hu B et al.Endothelin-1 activation of the endothelin B receptor modulates pulmonary endothelial CX3CL1 and contributes to pulmonary angiogenesis in experimental hepatopulmonary syndrome.Am J Pathol. 2014 Jun; 184(6):1706-14.
[7]Finley SD, Popel AS.Predicting the effects of anti-angiogenic agents targeting specific VEGF isoforms. AAPS J. 2012 Sep; 14(3):500-9.
[8]Szeto CC, Chow KM, Poon P, et al.Genetic polymorphism of VEGF: Impact on longitudinal change of peritoneal transport and survival of peritoneal dialysis patients. Kidney Int.2004; 65:1947-1955
[9]Zhan Y, Brown C, Maynard E, et al.Ets-1 is a critical regulator of Ang II-mediated vascular inflammation and remodeling. The Journal of clinical investigation. 2005 Sep; 115(9):2508-2516.
[10]Oettgen P.Regulation of vascular inflammation and remodeling by ETS factors. Circulation research. 2006 Nov 24; 99(11):1159-1166.
[11]Hynynen MM, Khalil RA.The vascular endothelin system in hypertension--recent patents and discoveries. Recent Pat Cardiovasc Drug Discov. 2006; 1:95-108
[12]Shimizu M, Ishibashi Y, Taki F, et al.Endothelin(B) receptor blocker inhibits high glucose-induced synthesis of fibronectin in human peritoneal mesothelial cells.Perit Dial Int. 2006 May-Jun; 26(3):393-401.
[13]Kandalaft LE, Facciabene A, Buckanovich RJ, et al.Endothelin b receptor, a new target in cancer immune therapy. Clinical Cancer Research. 2009; 15:4521-4528
[14]Pomonis JD, Rogers SD, Peters CM, et al.Expression and localization of endothelin receptors: Implications for the involvement of peripheral glia in nociception. The Journal of Neuroscience. 2001; 21:999-1006
[15]Simonson MS, Dunn MJ.Cellular signaling by peptides of the endothelin gene family. FASEB J. 1990; 4:2989-3000
[16]Adachi M, Yang YY, Furuichi Y, et al.Cloning and characterization of cdna encoding human a-type endothelin receptor. Biochem Biophys Res Commun. 1991; 180:1265-1272
[17]Mazzuca MQ, Khalil RA.Vascular endothelin receptor type b: Structure, function and dysregulation in vascular disease. Biochem Pharmacol. 2012; 84:147-162
[18]Ito H.Endothelins and cardiac hypertrophy. Life Sci 1997; 61:585-93.
[19]Sorokin A, Kohan DE.Physiology and pathology of endothelin-1 in renal mesangium. Am J Physiol Renal Physiol 2003; 285:F579–F589.
[20]Rochy DC, Chung JJ.Endothelin antagonism in experimental hepatic fibrosis. J Clin Invest 1996; 98:1381-8.
[21]Haines P, Samuel GH, Cohen H, et al.Caveolin-1 is a negative regulator of MMP-1 gene expression in human dermal fibroblasts via inhibition of Erk1/2/Ets1 signaling pathway. J Dermatol Sci. 2011 Dec; 64(3):210-6.
[22]Boesen EI.Endothelin etb receptor heterodimerization: Beyond the eta receptor. Kidney international.2008; 74:693-694
[23]Morgera S, Schlenstedt J, Hambach P, et al.Combined ETA/ETB receptor blockade of human peritoneal mesothelial cells inhibits collagen I RNA synthesis.Kidney Int. 2003 Dec; 64(6):2033-40.
[24]Stephens DC, Poon GM.Differential sensitivity to methylated DNA by ETS-family transcription factors is intrinsically encoded in their DNA-binding domains. Nucleic Acids Res. 2016 Jun 7. pii: gkw528. [Epub ahead of print]
[25]Zhu M, Li M, Zhang F, et al.Correction: FBI-1 Enhances ETS-1 Signaling Activity and Promotes Proliferation of Human Colorectal Carcinoma Cells.PLoS One. 2015 May 28; 10(5):e0129537.
[26]Li Q, Eppolito C, Odunsi K, et al.Antigen-induced Erk1/2 activation regulates Ets-1-mediated sensitization of CD8+ T cells for IL-12 responses.J Leukoc Biol. 2010 Feb; 87(2):257-63.
[27]Tsopanoglou NE, Maragoudakis ME.Thrombin's central role in angiogenesis and pathophysiological processes.Eur Cytokine Netw. 2009 Dec; 20(4):171-9.
[28]Arderiu G, Espinosa S, Pe?a E et al.PAR2-SMAD3 in microvascular endothelial cells is indispensable for vascular stability via tissue factor signaling.J Mol Cell Biol. 2016 Jun; 8(3):255-70.
[29]Kiselyov A, Balakin KV, Tkachenko SE.VEGF/VEGFR signalling as a target for inhibiting angiogenesis.Expert Opin Investig Drugs. 2007 Jan; 16(1):83-107.
基金
上海交通大学医工交叉基金(项目编号：YG2016QN27)；上海市卫计委青年项目(项目编号：20164Y0234)；
中华医学会临床医学科研专项资金-施维雅肾脏病青年研究与发展项目(项目编号：17010080677)；
国家自然科学基金(项目编号：81800676)
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