LY-3200882

Recurrent spontaneous miscarriage is one of the complications during pregnancy. However, the pathogenesis of recurrent spontaneous miscarriage is far from fully elucidated.

Since the endocytic pathway is crucial for cellular homeostasis, our study aimed to explore the roles of endocytic recycling, especially EH domain containing 1, a member of the endocytic recycling compartment, in recurrent spontaneous miscarriage.

We first investigated the expression of the endocytic pathway member EH domain containing 1 in villi from the normal and recurrent spontaneous miscarriage groups. Then, we performed ribonucleic acid sequencing and experiments in villi, HTR8 cells and BeWo cells to determine the mechanisms by which EH domain containing 1–induced recurrent spontaneous miscarriage. Finally, placenta–specific EH domain containing 1–overexpressing mice were generated to investigate the recurrent spontaneous miscarriage phenotype in vivo.

EH domain containing 1 was expressed in extravillous trophoblasts and syncytiotrophoblast in the villi. Compared with the control group, recurrent spontaneous miscarriage patients expressed higher EH domain containing 1. A high level of EH domain containing 1 decreased proliferation, promoted apoptosis, and reduced the migration and invasion of HTR8 cells by activating the TGFβ receptor 1-SMAD2/3 signaling pathway. The TGFβ receptor 1 antagonist LY3200882 partially reversed the EH domain containing 1 overexpression-induced changes in the cell phenotype. Besides, a high level of EH domain containing 1 also induced abnormal syncytialization, which disturbed maternal–fetal material exchanges. In a mouse model, placenta-specific overexpression of EH domain containing 1 led to the failure of spiral artery remodeling, excessive syncytialization, and miscarriage.

Increased expression of EH domain containing 1 impaired the invasion of extravillous trophoblasts mediated by the TGFβ receptor 1-SMAD2/3 signaling pathway and induced abnormal syncytialization of syncytiotrophoblast, which is at least partially responsible for recurrent spontaneous miscarriage.

Atrial fibrosis is associated with the occurrence of atrial fibrillation (AF) and regulated by the transforming growth factor-β1 (TGF-β1)/Smad2/3 signalling pathway. Unfortunately, the mechanisms of regulation of TGF-β1/Smad2/3-induced atrial fibrosis and vulnerability to AF remain still unknown. Previous studies have shown that sirtuin3 (SIRT3) sulfhydration has strong anti-fibrotic effects. We hypothesised that SIRT3 sulfhydration inhibits angiotensin II (Ang-II)-induced atrial fibrosis via blocking the TGF-β1/Smad2/3 signalling pathway. In this study, we found that SIRT3 expression was decreased in the left atrium of patients with AF compared to that in those with sinus rhythm (SR). In vitro, SIRT3 knockdown by small interfering RNA significantly expanded Ang-II-induced atrial fibrosis and TGF-β1/Smad2/3 signalling pathway activation, whereas supplementation with Sodium Hydrosulfide (NaHS, exogenous hydrogen sulfide donor and sulfhydration agonist) and SIRT3 overexpression using adenovirus ameliorated Ang-II-induced atrial fibrosis. Moreover, we observed suppression of the TGF-β1/Smad2/3 pathway when Ang-II was combined with NaHS treatment, and the effect of this co-treatment was consistent with that of Ang-II combined with LY3200882 (Smad pathway inhibitor) on reducing atrial fibroblast proliferation and cell migration in vitro. Supplementation with dithiothreitol (DTT, a sulfhydration inhibitor) and adenovirus SIRT3 shRNA blocked the ameliorating effect of NaHS and AngII co-treatment on atrial fibrosis in vitro. Finally, continued treatment with NaHS in rats ameliorated atrial fibrosis and remodelling, and further improved AF vulnerability induced by Ang-II, which was reversed by DTT and adenovirus SIRT3 shRNA, suggesting that SIRT3 sulfhydration might be a potential therapeutic target in atrial fibrosis and AF.