Table_1_TGF-β phospho antibody array identifies altered SMAD2, PI3K/AKT/SMAD, and RAC signaling contribute to the pathogenesis of myxomatous mitral valve disease.docx

BackgroundTGFβ signaling appears to contribute to the pathogenesis of myxomatous mitral valve disease (MMVD) in both dogs and humans. However, little is known about the extent of the downstream signaling changes that will then affect cell phenotype and function in both species.ObjectiveIdentify changes in downstream signals in the TGFβ pathway in canine MMVD and examine the effects of antagonism of one significant signal (SMAD2 was selected).Materials and methodsCanine cultures of normal quiescent valve interstitial cells (qVICs) and disease-derived activated myofibroblasts (aVICs) (n = 6) were examined for TGFβ signaling protein expression using a commercial antibody array. Significant changes were confirmed, and additional proteins of interest downstream in the TGFβ signaling pathway and markers of cell phenotype were examined (PRAS40, S6K, elF4E IRS-1, αSMA, and VIM), using protein immunoblotting. RT-PCR examined expression of gene markers of VIC activation (ACTA2, TAGLN, and MYH10; encoding the proteins αSMA, SM22, and Smemb, respectively). Attenuation of pSMAD2 in aVICs was examined using a combination of RNA interference technology (siRNA) and the SMAD7 (antagonizes SMAD2) agonist asiaticoside.ResultsThe antibody array identified significant changes (P < 0.05) in 19 proteins, of which six were phosphorylated (p). There was increased expression of pSMAD2 and pRAC1 and decreased expression of pmTOR, pERK1/2, and pAKT1. Expression of pPRAS40 and pIRS-1 was increased, as was the mTOR downstream transcription factor pS6K, with increased expression of peIF4E in aVICs, indicating negative feedback control of the PI3K/AKT/mTOR pathway. SMAD2 antagonism by siRNA and the SMAD7 agonist asiaticoside decreased detection of pSMAD by at least 50%, significantly decreased expression of the aVIC gene markers ACTA2, TAGLN, and MYH10, and pαSMA, pAKT2, and pERK1, but had no effect on pS6K, pERK2, or pVIM expression in aVICs. SMAD2 antagonism transitioned diseased aVICs to normal qVICs, while maintaining a mesenchymal phenotype (VIM+) while concurrently affecting non-canonical TGFβ signaling.ConclusionMMVD is associated with changes in both the canonical and non-canonical TGFβ signaling pathway. Antagonism of SMAD2 transitions diseased-activated myofibroblasts back to a normal phenotype, providing data that will inform studies on developing novel therapeutics to treat MMVD in dogs and humans.

背景：TGFβ信号通路似乎在犬类和人类心肌黏液性二尖瓣疾病（MMVD）的发病机制中发挥重要作用。然而，关于影响两种物种细胞表型和功能的下游信号变化程度知之甚少。目标：识别犬类MMVD中TGFβ通路下游信号的变化，并考察一种显著信号（选择SMAD2）的拮抗作用。材料与方法：对正常静息瓣膜间质细胞（qVICs）和疾病来源的活化肌成纤维细胞（aVICs）（n = 6）进行检测，以商业抗体阵列分析TGFβ信号蛋白的表达。确认显著变化后，进一步考察TGFβ信号通路下游感兴趣蛋白和细胞表型标记物（PRAS40、S6K、eIF4E、IRS-1、αSMA和VIM），使用蛋白免疫印迹技术。RT-PCR检测VIC激活的基因标记物（ACTA2、TAGLN、MYH10；分别编码αSMA、SM22和Smemb蛋白）的表达。通过RNA干扰技术（siRNA）和SMAD7（拮抗SMAD2）激动剂asiaticoside的组合考察aVICs中pSMAD2的减弱。结果：抗体阵列鉴定出19种蛋白质的显著变化（P < 0.05），其中6种为磷酸化（p）。pSMAD2和pRAC1表达增加，而pmTOR、pERK1/2和pAKT1表达减少。pPRAS40和pIRS-1表达增加，以及mTOR下游转录因子pS6K的表达增加，同时aVICs中eIF4E的表达增加，表明PI3K/AKT/mTOR通路的负反馈调控。通过siRNA和SMAD7激动剂asiaticoside拮抗SMAD2，pSMAD的检测至少降低50%，显著降低aVIC基因标记物ACTA2、TAGLN和MYH10以及pαSMA、pAKT2和pERK1的表达，但对pS6K、pERK2或pVIM表达在aVICs中无影响。SMAD2拮抗将疾病活化肌成纤维细胞转变为正常qVICs，同时保持间质表型（VIM+），并影响非典型TGFβ信号。结论：MMVD与典型和非典型TGFβ信号通路的变化相关。SMAD2拮抗将疾病活化肌成纤维细胞转变为正常表型，为开发治疗犬类和人类MMVD的新疗法提供了数据。