title: 5-HT(2B) antagonism arrests non-canonical TGF-β1-induced valvular myofibroblast differentiation.
abstract: Transforming growth factor-β1 (TGF-β1) induces myofibroblast activation of quiescent aortic valve interstitial cells (AVICs), a differentiation process implicated in calcific aortic valve disease (CAVD). The ubiquity of TGF-β1 signaling makes it difficult to target in a tissue specific manner; however, the serotonin 2B receptor (5-HT(2B)) is highly localized to cardiopulmonary tissues and agonism of this receptor displays pro-fibrotic effects in a TGF-β1-dependent manner. Therefore, we hypothesized that antagonism of 5-HT(2B) opposes TGF-β1-induced pathologic differentiation of AVICs and may offer a druggable target to prevent CAVD. To test this hypothesis, we assessed the interaction of 5-HT(2B) antagonism with canonical and non-canonical TGF-β1 pathways to inhibit TGF-β1-induced activation of isolated porcine AVICs in vitro. Here we show that AVIC activation and subsequent calcific nodule formation is completely mitigated by 5-HT(2B) antagonism. Interestingly, 5-HT(2B) antagonism does not inhibit canonical TGF-β1 signaling as identified by Smad3 phosphorylation and activation of a partial plasminogen activator inhibitor-1 promoter (PAI-1, a transcriptional target of Smad3), but prevents non-canonical p38 MAPK phosphorylation. It was initially suspected that 5-HT(2B) antagonism prevents Src tyrosine kinase phosphorylation; however, we found that this is not the case and time-lapse microscopy indicates that 5-HT(2B) antagonism prevents non-canonical TGF-β1 signaling by physically arresting Src tyrosine kinase. This study demonstrates the necessity of non-canonical TGF-β1 signaling in leading to pathologic AVIC differentiation. Moreover, we believe that the results of this study suggest 5-HT(2B) antagonism as a novel therapeutic approach for CAVD that merits further investigation.
authors.affiliation: Department of Biomedical Engineering, Vanderbilt University, Nashville, TN 37232-0493, United States.
authors.forename: Joshua D
authors.initials: JD
authors.lastname: Hutcheson
authors.forename: Larisa M
authors.initials: LM
authors.lastname: Ryzhova
authors.forename: Vincent
authors.initials: V
authors.lastname: Setola
authors.forename: W David
authors.initials: WD
authors.lastname: Merryman
mesh_terms: D000199:Actins / Q000235:genetics / Q000378:metabolism; D000818:Animals; D001018:Aortic Diseases / Q000473:pathology; D001021:Aortic Valve / Q000473:pathology; D002114:Calcinosis / Q000473:pathology; D002454:Cell Differentiation / Q000187:drug effects*; D002478:Cells, Cultured; D015870:Gene Expression; D007211:Indoles / Q000494:pharmacology*; D008840:Microfilament Proteins / Q000235:genetics / Q000378:metabolism; D009124:Muscle Proteins / Q000235:genetics / Q000378:metabolism; D058628:Myofibroblasts / Q000502:physiology*; D010766:Phosphorylation; D017395:Plasminogen Activator Inhibitor 1 / Q000235:genetics / Q000378:metabolism; D011401:Promoter Regions, Genetic; D011499:Protein Processing, Post-Translational; D021381:Protein Transport; D011725:Pyridines / Q000494:pharmacology*; D044403:Receptor, Serotonin, 5-HT2B / Q000378:metabolism*; D058830:Serotonin 5-HT2 Receptor Antagonists / Q000494:pharmacology*; D015398:Signal Transduction; D051900:Smad3 Protein / Q000378:metabolism; D034421:Sus scrofa; D015533:Transcriptional Activation; D053773:Transforming Growth Factor beta1 / Q000502:physiology*; D014508:Urea / Q000031:analogs & derivatives* / Q000494:pharmacology; D048051:p38 Mitogen-Activated Protein Kinases / Q000378:metabolism; D019061:src-Family Kinases / Q000378:metabolism / Q000502:physiology
chemical_list: D000199:Actins; D007211:Indoles; D008840:Microfilament Proteins; D009124:Muscle Proteins; C092756:N-(1-methyl-5-indolyl)-N'-(3-methyl-5-isothiazolyl)urea; D017395:Plasminogen Activator Inhibitor 1; D011725:Pyridines; D044403:Receptor, Serotonin, 5-HT2B; D058830:Serotonin 5-HT2 Receptor Antagonists; D051900:Smad3 Protein; D053773:Transforming Growth Factor beta1; C051615:transgelin; C118947:SB 228357; D014508:Urea; D019061:src-Family Kinases; D048051:p38 Mitogen-Activated Protein Kinases