Probes for TGF-β

Human gastric development has not been well studied. The generation of human pluripotent stem cell-derived gastric organoids (hGOs) comprising gastric marker-expressing epithelium without an apparent smooth muscle (SM) structure has been reported. We modified previously reported protocols to generate hGOs with muscularis mucosa (MM) from hiPSCs. Time course analyses revealed that epithelium development occurred prior to MM formation. Sonic hedgehog (SHH) and TGF-β1 were secreted by the epithelium. HH and TGF-β signal inhibition prevented subepithelial MM formation. A mechanical property of the substrate promoted SM differentiation around hGOs in the presence of TGF-β. TGF-β signaling was shown to influence the HH signaling and mechanical properties. In addition, clinical specimen findings suggested the involvement of TGF-β signaling in MM formation in recovering gastric ulcers. HH and TGF-β signaling from the epithelium to the stroma and the mechanical properties of the subepithelial environment may influence the emergence of MM in human stomach tissue.

Whereas genomic aberrations in the SLIT-ROBO pathway are frequent in pancreatic ductal adenocarcinoma (PDAC), their function in the pancreas is unclear. Here we report that in pancreatitis and PDAC mouse models, epithelial Robo2 expression is lost while Robo1 expression becomes most prominent in the stroma. Cell cultures of mice with loss of epithelial Robo2 (Pdx1Cre;Robo2F/F) show increased activation of Robo1+ myofibroblasts and induction of TGF-β and Wnt pathways. During pancreatitis, Pdx1Cre;Robo2F/F mice present enhanced myofibroblast activation, collagen crosslinking, T-cell infiltration and tumorigenic immune markers. The TGF-β inhibitor galunisertib suppresses these effects. In PDAC patients, ROBO2 expression is overall low while ROBO1 is variably expressed in epithelium and high in stroma. ROBO2low;ROBO1high patients present the poorest survival. In conclusion, Robo2 acts non-autonomously as a stroma suppressor gene by restraining myofibroblast activation and T-cell infiltration. ROBO1/2 expression in PDAC patients may guide therapy with TGF-β inhibitors or other stroma /immune modulating agents.

The aortic root is the predominant site for development of aneurysm caused by heterozygous loss-of-function mutations in positive effectors of the transforming growth factor-β (TGF-β) pathway. Using a mouse model of Loeys-Dietz syndrome (LDS) that carries a heterozygous kinase-inactivating mutation in TGF-β receptor I, we found that the effects of this mutation depend on the lineage of origin of vascular smooth muscle cells (VSMCs). Secondary heart field-derived (SHF-derived), but not neighboring cardiac neural crest-derived (CNC-derived), VSMCs showed impaired Smad2/3 activation in response to TGF-β, increased expression of angiotensin II (AngII) type 1 receptor (Agtr1a), enhanced responsiveness to AngII, and higher expression of TGF-β ligands. The preserved TGF-β signaling potential in CNC-derived VSMCs associated, in vivo, with increased Smad2/3 phosphorylation. CNC-, but not SHF-specific, deletion of Smad2 preserved aortic wall architecture and reduced aortic dilation in this mouse model of LDS. Taken together, these data suggest that aortic root aneurysm predisposition in this LDS mouse model depends both on defective Smad signaling in SHF-derived VSMCs and excessive Smad signaling in CNC-derived VSMCs. This work highlights the importance of considering the regional microenvironment and specifically lineage-dependent variation in the vulnerability to mutations in the development and testing of pathogenic models for aortic aneurysm.

Elevated transforming growth factor (TGF)-β2 in aqueous humor (AH) has been suggested to contribute to trabecular meshwork (TM) fibrosis and intraocular pressure (IOP) regulation in primary open-angle glaucoma (POAG), but TGF-β2 is downregulated in secondary open-angle glaucoma (SOAG). Because autotaxin (ATX) is upregulated in SOAG, we investigated the relationships and trans-signaling interactions of these mediators.The level of ATX in AH was determined using a two-site immunoenzymetric assay, and TGF-β levels were measured using the Bio-Plex Pro TGF-β Assay. RNA scope was used to assess the expression of ATX and TGF-β2 in human's eye specimen. And in vitro studies were performed using hTM cells to explore if trans-signaling of TGF-β2 regulates ATX expressions.TGF-β2/ATX ratio was significantly high in AH of control or POAG compared with SOAG, and negatively correlated with IOP. RNA scope revelated positive expressions of both TGF-β2 and ATX in ciliary body (CB) and TM in control, but ATX expressions was significantly enhanced in SOAG. In hTM cells, ATX expressions were regulated by TGF-β2 with concentration-dependent manner. In counter, ATX also induced TGF-β1, TGF-β2 and TGFBI upregulations and activation of the Smad-sensitive promoter, as well as upregulation of fibrotic markers, and these upregulation was significantly suppressed by both TGF-β and ATX inhibition.Trans-signaling of TGF-β2 regulates ATX expressions and thereby induced upregulations of TGF-βs or fibrosis of hTM. TGF-β2 trans-signaling potently regulate ATX transcription and signaling in hTM cells, which may reflect different profile of these mediators in glaucoma subtypes. Trial Registration This prospective observational study was approved by the Institutional Review Board of the University of Tokyo and was registered with the University Hospital Medical Information Network Clinical Trials Registry of Japan (ID: UMIN000027137). All study procedures conformed to the Declaration of Helsinki. Written informed consent was obtained from each patient.

Although sympathetic blockade is clinically used to treat pain, the underlying mechanisms remain unclear. We developed a localized microsympathectomy (mSYMPX), by cutting the grey rami entering the spinal nerves near the rodent lumbar dorsal root ganglia (DRG). In a chemotherapy-induced peripheral neuropathy model, mSYMPX attenuated pain behaviors via DRG macrophages and the anti-inflammatory actions of transforming growth factor-β (TGF-β) and its receptor TGF-βR1. Here, we examined the role of TGF-β in sympathetic-mediated radiculopathy produced by local inflammation of the DRG (LID). Mice showed mechanical hypersensitivity and transcriptional and protein upregulation of TGF-β1 and TGF-βR1 three days after LID. Microsympathectomy prevented mechanical hypersensitivity and further upregulated Tgfb1 and Tgfbr1. Intrathecal delivery of TGF-β1 rapidly relieved the LID-induced mechanical hypersensitivity, and TGF-βR1 antagonists rapidly unmasked the mechanical hypersensitivity after LID+mSYMPX. In situ hybridization showed that Tgfb1 was largely expressed in DRG macrophages, and Tgfbr1 in neurons. We suggest that TGF-β signaling is a general underlying mechanism of local sympathetic blockade.