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Primary open-angle glaucoma (POAG), a leading cause of irreversible vision loss, is closely linked to increased intraocular pressure (IOP), with the trabecular meshwork (TM) playing a critical role in its regulation. The TM, located at the iridocorneal angle, acts as a sieve, filtering the aqueous humor from the eye into the collecting ducts, thus maintaining proper IOP levels. The transforming growth factor-beta 2 (TGF-β2) signaling pathway has been implicated in the pathophysiology of primary open-angle glaucoma POAG particularly, in the dysfunction of the TM. This study utilizes human TM explants to closely mimic in vivo conditions, thereby minimizing transcriptional changes that could arise from cell culture enabling an exploration of the transcriptomic impacts of TGF-β2. Through bulk RNA sequencing and immunohistological analysis, we identified distinct gene expression patterns and morphological changes induced by TGF-β2 exposure (5 ng/ml for 48 h). Bulk RNA sequencing identified significant upregulation in genes linked to extracellular matrix (ECM) regulation and fibrotic signaling. Immunohistological analysis further elucidated the morphological alterations, including cytoskeletal rearrangements and ECM deposition, providing a visual confirmation of the transcriptomic data. Notably, the enrichment analysis unveils TGF-β2's influence on both bone morphogenic protein (BMP) and Wnt signaling pathways, suggesting a complex interplay of molecular mechanisms contributing to TM dysfunction in glaucoma. This characterization of the transcriptomic modifications on an explant model of TM obtained under the effect of this profibrotic cytokine involved in glaucoma is crucial in order to develop and test new molecules that can block their signaling pathways., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Purpose: Posterior capsule opacification (PCO) is the major complication of visual impairment after cataract surgery. Circular RNAs (circRNAs) are involved in the development of many diseases. The purpose of this study was to explore the role and molecular mechanism of circ_0000099 in PCO., Methods: SRA01/04 cells were treated with TGF-β2 to establish a PCO cell model. The expression of circ_0000099, miR-223-3p, and connective tissue growth factor (CTGF) mRNA was determined by real-time quantitative polymerase chain reaction (qRT-PCR). Western blot assay was used to analyze the protein expression. Cell proliferation, migration, and invasion were analyzed by (4-5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT), 5-ethynyl-2 '-Deoxyuridine (EdU), transwell, and wound healing tests. The circ_0000099/miR-223-3p/CTGF relationship was verified by dual luciferase reporter gene and RNA binding protein immunoprecipitation (RIP) assays., Results: TGF-β2 treatment promoted SRA01/04 cell proliferation invasion, migration, and EMT. Circ_0000099 expression was increased in POC patients and TGF-β2-treated SRA01/04 cells.Knockdown of circ_0000099 suppressed TGF-β2-induced proliferation, invasion, migration, and EMT in SRA01/04 cells. miR-223-3p was identified as the target of circ_0000099, and miR-223-3p inhibitor might partly abolish the repression of circ_0000099 silencing on TGF-β2-triggered SRA01/04 cell disorders. MiR-223-3p directly targeted CTGF. Knockdown of CTGF suppressed TGF-β2-induced SRA01/04 cell injury. Circ_0000099 can regulate CTGF expression by targeting miR-223-3p., Conclusions: Circ_0000099 silencing might relieve TGF-2-induced SRA01/04 cell injury by the miR-223-3p/CTGF axis, providing new avenues for the prevention and treatment of PCO.

A cataract is a clinically common blinding disease closely related to the ageing of the eye cells, which has become a major health killer in the elderly. Our research seeks to analyze the primary targets linked to the pathogenesis of cataracts during the ageing process. We performed bioinformatics analyses on the GSE101727 dataset to discover genes linked with ageing and cataracts. To explore the impacts of Nucleophosmin 1 (NPM1) on cell apoptosis, proliferation, as well as epithelial-mesenchymal transition (EMT) processes, in vitro tests such as western blotting, flow cytometry, and MTT were carried out. Additionally, the study incorporated transforming growth factor β2 (TGF-β2) to examine its function in cellular responses, chloroquine (CQ) to regulate autophagic flow, and H2O2 therapy to mimic oxidative stress. Our study discovered seven ageing-related genes, including NPM1, that had substantial relationships with cataracts. NPM1 overexpression was shown to boost cell proliferation and prevent apoptosis in SRA01/04 cells. Notably, NPM1 modulated the TGF-β signalling pathway, influencing cell proliferation and EMT processes. miR-429 was shown to be adversely regulating NPM1 and autophagy-related proteins, as demonstrated by changes in their expression in response to TGF-β2 treatment. Furthermore, NPM1 knockdown restored autophagy activity suppressed by miR-429 mimics, indicating a complex interaction of miR-429, NPM1, and TGF-β2 pathways in regulating autophagy and EMT. Lens epithelial cell proliferation and apoptosis were largely regulated by NPM1, as well as autophagy and EMT, which were significantly mediated by TGF-β2 and the miR-429/NPM1 axis. These results imply new possible targets for prognosis and therapy of cataracts.

Fas ligand (FasL, CD178) belongs to classical apoptotic molecules, however, recent evidence expands the spectrum of FasL functions into non-apoptotic processes which also applies for the bone. Tgfb subfamily members (Tgfb1, Tgfb2, Tgfb3) represent major components in osteogenic pathways and extracellular matrix. Their possible association with FasL has not yet been investigated but can be postulated. To test such a hypothesis, FasL deficient (gld) calvaria-derived cells were examined with a focus on the expression of Tgfb receptor ligands. The qPCR analysis revealed significantly increased expression of Tgfb1, Tgfb2 and Tgfb3 in gld cells. To check the vice versa effect, the gld cells were stimulated by soluble FasL. As a consequence, a dramatic decrease in expression levels of all three ligands was observed. This phenomenon was also confirmed in IDG-SW3 (osteoblastic cells of endochondral origin). TFLink gateway identified Fosl2 as an exclusive candidate of FasL capable to impact expression of all three Tgfb ligands. However, Fosl2 siRNA did not cause any significant changes in expression of Tgfb ligands. Therefore, the upregulation of the three ligands is likely to occur separately. In this respect, we tested the only exclusive candidate transcription factor for Tgfb3, Prrx1. Additionally, an overlapping candidate for Tgfb1 and Tgfb2, Mef2c capable to modulate expression of sclerostin, was examined. Prrx1 as well as Mef2c were found upregulated in gld samples and their expression decreased after addition of FasL. The same effect of FasL treatment was observed in the IDG-SW3 model. Taken together, FasL deficiency causes an increase in the expression of Tgfb ligands and stimulation by FasL reduces Tgfb expression in osteoblastic cells. The candidates mediating the effect comprise Prrx1 for Tgfb3 and Mef2c for Tgfb1/2. These results indicate FasL as a novel cytokine interfering with Tgfb signaling and thus the complex osteogenic network. The emerging non-apoptotic functions of FasL in bone development and maintenance should also be considered in treatment strategies such as the anti-osteoporotic factor., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Purpose: The epithelial-mesenchymal transition of human lens epithelial cells plays a role in posterior capsule opacification, a fibrotic process that leads to a common type of cataract. Hyaluronic acid has been implicated in this fibrosis. Studies have investigated the role of transforming growth factor (TGF)-β2 in epithelial-mesenchymal transition. However, the role of TGF-β2 in hyaluronic acid-mediated fibrosis of lens epithelial cell remains unknown. We here examined the role of TGF-β2 in the hyaluronic acid-mediated epithelial-mesenchymal transition of lens epithelial cells., Methods: Cultured human lens epithelial cells (HLEB3) were infected with CD44-siRNA by using the Lipofectamine 3000 transfection reagent. The CCK-8 kit was used to measure cell viability, and the scratch assay was used to determine cell migration. Cell oxidative stress was analyzed in a dichloro-dihydro-fluorescein diacetate assay and by using a flow cytometer. The TGF-β2 level in HLEB3 cells was examined through immunohistochemical staining. The TGF-β2 protein level was determined through western blotting. mRNA expression levels were determined through quantitative real-time polymerase chain reaction., Results: Treatment with hyaluronic acid (1.0 μM, 24 h) increased the epithelial-mesenchymal transition of HLEB3 cells. The increase in TGF-β2 levels corresponded to an increase in CD44 levels in the culture medium. However, blocking the CD44 function significantly reduced the TGF-β2-mediated epithelial-mesenchymal transition response of HLEB3 cells., Conclusions: Our study showed that both CD44 and TGF-β2 are critical contributors to the hyaluronic acid-mediated epithelial-mesenchymal transition of lens epithelial cells, and that TGF-β2 in epithelial-mesenchymal transition is regulated by CD44. These results suggest that CD44 could be used as a target for preventing hyaluronic acid-induced posterior capsule opacification. Our findings suggest that CD44/TGF-β2 is crucial for the hyaluronic acid-induced epithelial-mesenchymal transition of lens epithelial cells.

Purpose: Primary open-angle glaucoma (POAG) is a leading cause of blindness, and its primary risk factor is elevated intraocular pressure (IOP) due to pathologic changes in the trabecular meshwork (TM). We previously showed that there is a cross-inhibition between TGFβ and Wnt signaling pathways in the TM. In this study, we determined if activation of the Wnt signaling pathway using small-molecule Wnt activators can inhibit TGFβ2-induced TM changes and ocular hypertension (OHT)., Methods: Primary human TM (pHTM) cells and transduced SBE-GTM3 cells were treated with or without Wnt and/or TGFβ signaling activators and used for luciferase assays; for the extraction of whole-cell lysate, conditioned medium, cytosolic proteins, and nuclear proteins for Western immunoblotting (WB); or for immunofluorescent staining. Human donor eyes were perfusion cultured to study the effect of Wnt activators on IOP., Results: We found that the small-molecule Wnt activators (GSK3β inhibitors) (BIO, SB216763, and CHIR99021) activated canonical Wnt signaling in pHTM cells without toxicity at tested concentrations. This activation inhibited TGFβ signaling as well as TGFβ2-induced extracellular matrix deposition and formation of cross-linked actin networks in pHTM cells or SBE-GTM3 cells. We also observed nuclear translocation of both Smad4 and β-catenin in pHTM cells, which suggested that the cross-inhibition between the TGFβ and Wnt signaling pathways may occur in the nucleus. Using our ex vivo model, we found that CHIR99021 inhibited TGFβ2-induced OHT in perfusion-cultured human eyes., Conclusions: Our results showed that small-molecule Wnt activators have the potential for treating TGFβ signaling-induced OHT in patients with POAG.

Transforming growth factor-β2 (TGF-β2) induced fibrogenic changes in human trabecular meshwork (HTM) cells have been implicated in trabecular meshwork (TM) damage and intraocular pressure (IOP) elevation in primary open-angle glaucoma (POAG) patients. Silibinin (SIL) exhibited anti-fibrotic properties in various organs and tissues. This study aimed to assess the effects of SIL on the TGF-β2-treated HTM cells and to elucidate the underlying mechanisms. Our study found that SIL effectively inhibited HTM cell proliferation, attenuated TGF-β2-induced cell migration, and mitigated TGF-β2-induced reorganization of both actin and vimentin filaments. Moreover, SIL suppressed the expressions of fibronectin (FN), collagen type I alpha 1 chain (COL1A1), and alpha-smooth muscle actin (α-SMA) in the TGF-β2-treated HTM cells. RNA sequencing indicated that SIL interfered with the phosphoinositide 3-kinase (PI3K)/protein kinase B (PKB, also known as AKT) signaling pathway, extracellular matrix (ECM)-receptor interaction, and focal adhesion in the TGF-β2-treated HTM cells. Western blotting demonstrated SIL inhibited the activation of Janus kinase 2 (JAK2)/signal transducers and activators of transcription 3 (STAT3) and the downstream PI3K/AKT signaling pathways induced by TGF-β2, potentially contributing to its inhibitory effects on ECM protein production in the TGF-β2-treated HTM cells. Our study demonstrated the ability of SIL to inhibit TGF-β2-induced fibrogenic changes in HTM cells. SIL could be a potential IOP-lowering agent by reducing the fibrotic changes in the TM tissue of POAG patients, which warrants further investigation through additional animal and clinical studies., Competing Interests: Declaration of competing interest The authors report no conflicts of interest., (Copyright © 2024 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Purpose: We sought to evaluate the efficacy of growth differentiation factor (GDF)-15 treatment for suppressing epithelial-mesenchymal transition (EMT) and alleviating transforming growth factor β2 (TGFβ2)-induced lens opacity., Methods: To test whether GDF-15 is a molecule that prevents EMT, we pretreated the culture with GDF-15 in neural progenitor cells, retinal pigment epithelial cells, and lens epithelial cells and then treated with factors that promote EMT, GDF-11, and TGFβ2, respectively. To further investigate the efficacy of GDF-15 on alleviating lens opacity, we used mouse lens explant culture to mimic secondary cataracts. We pretreated the lens culture with GDF-15 and then added TGFβ2 to develop lens opacity (n = 3 for each group). Western blot and quantitative reverse transcription polymerase chain reaction (qRT-PCR) were used to measure EMT protein and gene expression, respectively., Results: In cell culture, GDF-15 pretreatment significantly attenuated EMT marker expression in cultured cells induced by treatment with GDF-11 or TGFβ2. In the lens explant culture, GDF-15 pretreatment also reduced mouse lens opacity induced by exposure to TGFβ2., Conclusions: Our results indicate that GDF-15 could alleviate TGFβ2-induced EMT and is a potential therapeutic agent to slow or prevent posterior capsular opacification (PCO) progression after cataract surgery., Translational Relevance: Cataracts are the leading cause of blindness worldwide, with the only current treatment involving surgical removal of the lens and replacement with an artificial lens. However, PCO, also known as secondary cataract, is a common complication after cataract surgery. The development of an adjuvant that slows the progression of PCO will be beneficial to the field of anterior complications.

Tendons are frequently injured and have limited regenerative capacity. This motivates tissue engineering efforts aimed at restoring tendon function through strategies to direct functional tendon formation. Generation of a crosslinked collagen matrix is paramount to forming mechanically functional tendon. However, it is unknown how lysyl oxidase (LOX), the primary mediator of enzymatic collagen crosslinking, is regulated by stem cells. This study investigates how multiple factors previously identified to promote tendon formation and healing (transforming growth factor [TGF]β1 and TGFβ2, mechanical stimuli, and hypoxia-inducible factor [HIF]-1α) regulate LOX production in the murine C3H10T1/2 mesenchymal stem cell (MSC) line. We hypothesized that TGFβ signaling promotes LOX activity in C3H10T1/2 MSCs, which is regulated by both mechanical stimuli and HIF-1α activation. TGFβ1 and TGFβ2 increased LOX levels as a function of concentration and time. Inhibiting the TGFβ type I receptor (TGFβRI) decreased TGFβ2-induced LOX production by C3H10T1/2 MSCs. Low (5 mPa) and high (150 mPa) magnitudes of fluid shear stress were applied to test impacts of mechanical stimuli, but without TGFβ2, loading alone did not alter LOX levels. Low loading (5 mPa) with TGFβ2 increased LOX at 7 days greater than TGFβ2 treatment alone. Neither HIF-1α knockdown (siRNA) nor activation (CoCl 2 ) affected LOX levels. Ultimately, results suggest that TGFβ2 and appropriate loading magnitudes contribute to LOX production by C3H10T1/2 MSCs. Potential application of these findings includes treatment with TGFβ2 and appropriate mechanical stimuli to modulate LOX production by stem cells to ultimately control collagen matrix stiffening and support functional tendon formation.

Background: The multifunctional profibrotic cytokine transforming growth factor-beta2 (TGF-β2) is implicated in the pathophysiology of primary open angle glaucoma. Paeoniflorin (PAE) is a monoterpene glycoside with multiple pharmacological efficacies, such as antioxidant, anti-fibrotic, and anti-inflammatory properties. Studies have demonstrated that paeoniflorin protects human corneal epithelial cells, retinal pigment epithelial cells, and retinal microglia from damage. Here, the biological role of PAE in TGF-β2-dependent remodeling of the extracellular matrix (ECM) within the trabecular meshwork (TM) microenvironment., Methods: Primary or transformed (GTM3) human TM (HTM) cells conditioned in serum-free media were incubated with TGF-β2 (5 ng/mL). PAE (300 μM) was added to serum-starved confluent cultures of HTM cells for 2 h, followed by incubation with TGF-β2 for 22 h. SB-431542, a TGF-β receptor inhibitor (10 μM), was used as a positive control. The levels of intracellular ROS were evaluated by CellROX green dye. Western blotting was used to measure the levels of TGF-β2/Smad2/3 signaling-related molecules. Collagen 1α1, collagen 4α1, and connective tissue growth factor (CTGF) expression was evaluated by RT-qPCR. Immunofluorescence assay was conducted to measure collagen I/IV expression in HTM cells. Phalloidin staining assay was conducted for evaluating F-actin stress fiber formation in the cells., Results: PAE attenuated TGF-β2-induced oxidative stress and suppressed TGF-β2-induced Smad2/3 signaling in primary or transformed HTM cells. Additionally, PAE repressed TGF-β2-induced upregulation of collagen 1α1, collagen 4α1, and CTGF expression and reduced TGF-β2-mediated collagen I/IV expression and of F-actin stress fiber formation in primary or transformed HTM cells., Conclusion: PAE alleviates TGF-β2-induced ECM deposition and oxidative stress in HTM cells through inactivation of Smad2/3 signaling., (© 2024. The Author(s), under exclusive licence to Springer Nature B.V.)

Alterations in cell fate are often attributed to (epigenetic) regulation of gene expression. An emerging paradigm focuses on specialized ribosomes within a cell. However, little evidence exists for the dynamic regulation of ribosome composition and function. Here, we stimulated a chondrocytic cell line with transforming growth factor beta (TGF-β2) and mapped changes in ribosome function, composition and ribosomal RNA (rRNA) epitranscriptomics. 35S Met/Cys incorporation was used to evaluate ribosome activity. Dual luciferase reporter assays were used to assess ribosomal modus. Ribosomal RNA expression and processing were determined by RT-qPCR, while RiboMethSeq and HydraPsiSeq were used to determine rRNA modification profiles. Label-free protein quantification of total cell lysates, isolated ribosomes and secreted proteins was done by LC-MS/MS. A three-day TGF-β2 stimulation induced total protein synthesis in SW1353 chondrocytic cells and human articular chondrocytes. Specifically, TGF-β2 induced cap-mediated protein synthesis, while IRES-mediated translation was not (P53 IRES) or little affected (CrPv IGR and HCV IRES). Three rRNA post-transcriptional modifications (PTMs) were affected by TGF-β2 stimulation (18S-Gm1447 downregulated, 18S-ψ1177 and 28S-ψ4598 upregulated). Proteomic analysis of isolated ribosomes revealed increased interaction with eIF2 and tRNA ligases and decreased association of eIF4A3 and heterogeneous nuclear ribonucleoprotein (HNRNP)s. In addition, thirteen core ribosomal proteins were more present in ribosomes from TGF-β2 stimulated cells, albeit with a modest fold change. A prolonged stimulation of chondrocytic cells with TGF-β2 induced ribosome activity and changed the mode of translation. These functional changes could be coupled to alterations in accessory proteins in the ribosomal proteome.

Clear cell renal cell carcinoma (ccRCC) is a malignant tumor of kidney epithelial cells, one of the most common tumors in the world. Transforming growth factor beta (TGFβ)1 is a crucial factor that induces epithelial-mesenchymal transition (EMT) in cancer cells. microRNA-141-3p (miR-141-3p) is a microRNA that is considered a tumor suppressor. However, the role and mechanism of miR-141-3p in TGFβ1-induced ccRCC cells are not fully understood. This study investigated the roles of miR-141-3p and its target gene in regulating EMT in ccRCC development. 786-0 and Caki-1cells were treated with TGFβ1 to induce EMT. The levels of miR-141-3p and TGFβ2 were determined by quantitative real-time polymerase chain reaction and Western blotting. The progression of EMT was evaluated by E-cadherin detection by immunofluorescence, and E-cadherin, N-cadherin, and vimentin detection by Western blotting. Furthermore, migration and invasion capacities were assessed using a Transwell system. The direct binding of miR-141-3p with the target gene TGFβ2 was confirmed by dual luciferase reporter gene assay. Results indicated that TGFβ1 treatment decreased the protein abundance of E-cadherin while increasing the protein expression of N-cadherin and vimentin, indicating TGFβ1-induced EMT was constructed successfully. Moreover, TGFβ1 treatment repressed the expression of miR-141-3p . miR-141-3p mimics reversed the effect of TGFβ1 on the migration, invasion, and expression of E-cadherin, N-cadherin, and vimentin. The miR-141-3p directly binds with the 3' untranslated region of TGFβ2 mRNA and suppresses its expression. Furthermore, TGFβ2 overexpression abrogated the above changes regulated by miR-141-3p mimics. Taken together, miR-141-3p inhibited TGFβ1-induced EMT by suppressing the migration and invasion of ccRCC cells via directly targeting TGFβ2 gene expression.

Purpose: To investigate the effect of yes-associated protein (YAP) and transcriptional coactivator with PDZ-binding motif (TAZ) on connective tissue growth factor (CTGF) expression in adult retinal pigment epithelial (ARPE)-19 cells. We also studied the inhibitory effect of K-975, a new pan-transcriptional enhanced associate domain (TEAD) inhibitor, and luteolin, a plant-derived flavonoid on CTGF expression. Methods: ARPE-19 cells were transfected with either YAP or TAZ overexpression plasmid or treated with transforming growth factor (TGF)-β 2 . The cells were cultured either with or without K-975 or luteolin. The expression of YAP, TAZ, and CTGF was examined using real-time PCR. Results: ARPE-19 cells overexpressing YAP or TAZ exhibited significantly increased CTGF expression. This increase was attenuated by K-975 or luteolin alone. TGF-β 2 treatment significantly raised the expression of not just YAP and TAZ, but also CTGF in ARPE-19 cells. TGF-β 2 treatment-enhanced CTGF expression was considerably lowered by the addition of K-975 or luteolin. Conclusions: Overexpression of YAP or TAZ and treatment with TGF-β 2 led to an increase in the expression of CTGF in ARPE-19 cells. These increases were attenuated by treatment with K-975 and luteolin. These findings suggest that YAP and TAZ may be related to the expression of CTGF in ARPE-19 cells and that K-975 and luteolin can be explored as potential therapeutic agents for preventing CTGF production in vitreoretinal fibrosis.

Background: Posterior capsular opacification (PCO) is the main reason affecting the long-term postoperative result of cataract patient, and it is well accepted that fibrotic PCO is driven by transforming growth factor beta (TGFβ) signaling. Ferroptosis, closely related to various ocular diseases, but has not been explored in PCO., Methods: RNA sequencing (RNA-seq) was performed on both TGF-β2 treated and untreated primary lens epithelial cells (pLECs). Differentially expressed genes (DEGs) associated with ferroptosis were analyzed using Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) to investigate their biological function. Additionally, protein-to-protein interactions among selected ferroptosis-related genes by PPI network and the top 10 genes with the highest score (MCC algorithm) were selected as the hub genes. The top 20 genes with significant fold change values were validated using quantitative real-time polymerase chain reaction (qRT-PCR)., Results: Our analysis revealed 1253 DEGs between TGF-β2 treated and untreated pLECs, uncovering 38 ferroptosis-related genes between two groups. Among these 38 ferroptosis-related genes,the most prominent GO enrichment analysis process involved in the response to oxidative stress (BPs), apical part of cell (CCs),antioxidant activity (MFs). KEGG were mainly concentrated in fluid shear stress and atherosclerosis, IL-17 and TNF signaling pathways, and validation of top 20 genes with significant fold change value were consistent with RNA-seq., Conclusions: Our RNA-Seq data identified 38 ferroptosis-related genes in TGF-β2 treated and untreated pLECs, which is the first observation of ferroptosis related genes in primary human lens epithelial cells under TGF-β2 stimulation., (© 2024. The Author(s).)

N6-methyladenosine (m6A) is a major type of RNA modification implicated in various pathophysiological processes. Transforming growth factor β2 (TGF-β2) induces epithelial-mesenchymal transition (EMT) in retinal pigmental epithelial (RPE) cells and promotes the progression of proliferative vitreoretinopathy (PVR). However, the role of m6A methylation in the EMT of human telomerase reverse transcriptase (hTERT) retinal pigmental epithelium (RPE)-1 cells has not been clarified. Here, we extracted RNA from RPE cells subjected to 0 or 20 ng/mL TGF-β2 for 72 h and identified differentially methylated genes (DMGs) by m6A-Seq and differentially expressed genes (DEGs) by RNA-Seq. We selected the genes related to EMT by conjoint m6A-Seq/RNA-Seq analysis and verified them by qRT-PCR. We then confirmed the function of m6A methylation in the EMT of RPE cells by knocking down the methyltransferase METTL3 and the m6A reading protein YTHDF1. Sequencing yielded 5814 DMGs and 1607 DEGs. Conjoint analysis selected 467 genes altered at the m6A and RNA levels that are closely associated with the EMT-related TGF-β, AGE-RAGE, PI3K-Akt, P53, and Wnt signaling pathways. We also identified ten core EMT genes ACTG2, BMP6, CDH2, LOXL2, SNAIL1, SPARC, BMP4, EMP3, FOXM1, and MYC. Their RNA levels were evaluated by qRT-PCR and were consistent with the sequencing results. We observed that METTL3 knockdown enhanced RPE cell migration and significantly upregulated the EMT markers N-cadherin (encoded by CDH2), fibronectin (FN), Snail family transcription repressor (SLUG), and vimentin. However, YTHDF1 knockdown had the opposite effects and decreased both cell migration and the N-cadherin, FN, and SLUG expression levels. The present study clarified TGF-β2-induced m6A- and RNA-level differences in RPE cells, indicated that m6A methylation might regulate EMT marker expression, and showed that m6A could regulate TGF-β2-induced EMT., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

The formation of the epiretinal fibrotic membrane by retinal pigment epithelial (RPE) cells is a primary pathological change for proliferative vitreoretinopathy (PVR). Bone morphogenetic protein 6 (BMP6) is an antifibrogenic factor in various cells. To date, it is still unknown whether BMP6 can interfere with the fibrogenesis of RPE cells during the progression of PVR. This work aimed to address the relationship between BMP6 and transforming growth factor-β2 (TGF-β2)-elicited fibrogenesis of RPE cells, an experimental model for studying PVR in vitro. The BMP6 level was down-regulated, while the TGF-β2 level was up-regulated in the vitreous humor of PVR patients. The BMP6 level was down-regulated in human RPE cells challenged with TGF-β2. The treatment of RPE cells with TGF-β2 resulted in significant increases in proliferation, migration, epithelial-to-mesenchymal transition (EMT), and extracellular matrix (ECM) remodelling. These effects were found to be inhibited by the overexpression of BMP6 or exacerbated by the knockdown of BMP6. BMP6 overexpression reduced the phosphorylation of p38 and JNK in TGF-β2-stimulated RPE cells, while BMP6 knockdown showed the opposite effects. The inhibition of p38 or JNK partially reversed the BMP6-silencing-induced promoting effects on TGF-β2-elicited fibrogenesis in RPE cells. Taken together, BMP6 demonstrates the ability to counteract the proliferation, migration, EMT, and ECM remodelling of RPE cells induced by TGF-β2. This is achieved through the regulation of the p38 and JNK MAPK pathways. These findings imply a potential connection between BMP6 and PVR, and highlight the potential application of BMP6 in therapeutic interventions for PVR., (© 2024. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Purpose: To investigate the potential effects and mechanism of nicotinamide riboside (NR) on the oxidative stress and fibrosis model of human trabecular meshwork (HTM) cell line cells., Methods: HTM cells were pretreated with NR, followed by the induction of oxidative injury and fibrosis by hydrogen peroxide (H2O2) and TGF-β2, respectively. Cell viability was tested using Hoechst staining and MTT assays, cell proliferation was assessed by EdU assay, and cell apoptosis was detected by flow cytometry and western blotting. DCFH-DA and DHE probes were used to measure the level of reactive oxygen species (ROS), and MitoTracker staining was used to measure the mitochondrial membrane potential (MMP). Fibrotic responses, including cell migration and deposition of extracellular matrix (ECM) proteins, were detected via Transwell assays, qRT-PCR, and immunoblotting., Results: NR pretreatment improved the viability, proliferation, and MMP of H2O2-treated HTM cells. Compared to cells treated solely with H2O2, HTM cells treated with both NR and H2O2, exhibited a reduced rate of apoptosis and generation of ROS. Compared with H2O2 pretreatment, NR pretreatment upregulated expression of the JAK2/Stat3 pathway but inhibited mitogen-activated protein kinase (MAPK) pathway expression. Moreover, 10-ng/mL TGF-β2 promoted cell proliferation and migration, which were inhibited by NR pretreatment. Both qRT-PCR and immunoblotting showed that NR inhibited the expression of fibronectin in a TGF-β2-induced fibrosis model., Conclusions: NR has a protective effect on oxidative stress and fibrosis in HTM cells, which may be related to the JAK2/Stat3 pathway and MAPK pathway., Translational Relevance: Our research provides the ongoing data for potential therapy of NAD+ precursors in glaucoma.

Primary open-angle glaucoma (POAG) is the most common type of glaucoma leading to blindness. The search for ways to prevent/treat this entity is one of the main challenges of today's ophthalmology. One of such solution seems to be biologically active substances of natural origin, such as genistein (GEN), which can affect the function of isolated trabecular meshwork by the inhibition of protein tyrosine kinase. However, the role of GEN in viability as well as myofibroblastic transformation in human trabecular meshwork cells stimulated by TGF-β is unknown. Using human trabecular meshwork cells (HTMCs) we investigated the effect of genistein on cell viability and myofibroblastic transformation stimulated by TGF-β1 and TGF-β2. Using Real-Time PCR, western blot and immunofluorescence we determined the effect on the expression changes of αSMA, TIMP1, collagen 1 and 3 at mRNA and protein level. We found that genistein increases the viability of HTMCs (1, 2, 3 μg/ml; P < 0.05 and 4, 5, 10, 15, 20 μg/ml; P < 0.01). Moreover, we found that addition of 10, 15 and 20 μg/ml is able to prevent myofibroblastic transformation of HTMCs by decreasing αSMA, TIMP1, collagen 1 and 3 mRNA and protein expression (P < 0.01). Based on the obtained results, we can conclude that genistein is a potential factor that can prevent the myofibroblastic transformation of HTMCs accompanying glaucoma. Describing GEN influence on myofibroblastic transformation processes in HTMC allows us to conclude that it can be considered a potential therapeutic agent or a substance supporting treatment in patients with glaucoma., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Purpose: Trabecular meshwork (TM) fibrosis is a crucial pathophysiological process in the development of primary open-angle glaucoma. Pirfenidone (PFD) is a new, broad-spectrum antifibrotic agent approved for the treatment of idiopathic pulmonary fibrosis. This study investigated the inhibitory effect of PFD on TM fibrosis and evaluated its efficacy in lowering intraocular pressure (IOP)., Methods: Human TM cells were isolated, cultured, and characterized. Cell Counting Kit-8 was used to evaluate the proliferation and toxicity of different concentrations of PFD on normal or fibrotic TM cells. TM cells were treated with transforming growth factor beta-2 (TGF-β2) in the absence or presence of PFD. Western blotting and immunofluorescence analyses were used to analyze changes in the TM cell cytoskeleton and extracellular matrix (ECM) proteins, including alpha-smooth muscle actin (α-SMA), F-actin, collagen IV (COL IV), and fibronectin (FN). An ocular hypertension (OHT) mouse model was induced with Ad-TGF-β2C226/228S and then treated with PFD or latanoprost (LT) eye drops to confirm the efficacy of PFD in lowering IOP., Results: PFD inhibited the proliferation of fibrotic TM cells in a dose-dependent manner and inhibited TGF-β2-induced overexpression of α-SMA, COL IV, and FN in TM cells. PFD stabilized F-actin. In vivo, PFD eye drops reduced the IOP of the OHT models and showed no significant difference compared with LT eye drops., Conclusions: PFD inhibited TGF-β2-induced TM cell fibrosis by rearranging the disordered cytoskeleton and decreasing ECM deposition, thereby enhancing the aqueous outflow from the TM outflow pathway and lowering IOP, which provides a potential new approach to treating glaucoma., Translational Relevance: Our work with pirfenidone provides a new approach to treat glaucoma.

Purpose: This study aimed to explore the role of atorvastatin (ATO) in the prevention and treatment of the scarring of filtration channels after glaucoma surgery., Methods: Human Tenon's capsule fibroblasts (HTFs) were co-cultured with various concentrations of ATO. First, Cell Counting Kit-8 assay was performed to evaluate the effects of various concentrations of ATO on the viability of HTFs. Then, after the ATO stimulated the HTFs for 24 h, terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay was performed to evaluate the apoptosis of HTFs. Transwell assay was also performed to evaluate the migration of HTFs. Moreover, enzyme-linked immunosorbent assay (ELISA) was performed to detect the protein expression levels of transforming growth factor-β1 (TGF-β1) and TGF-β2 in the cell culture supernatant of HTFs. Western blot was carried out to detect the protein expression levels of smooth muscle actin (SMA), p38, Smad3, fibronectin, collagen I and collagen III in different groups., Results: The results revealed that ATO could inhibit the proliferation and migration of HTFs. Based on the TUNEL assay, 100 μM and 150 μM ATO could induce cell apoptosis. The ELISA results indicated that ATO could down-regulate the expression level of TGF-β2, and western blot analysis revealed that the protein expression levels of SMA, p38, Smad3, fibronectin, collagen I and collagen III in the TGF-β2 group were all up-regulated compared with the control group, whereas the addition of ATO could reverse this up-regulation., Conclusions: ATO could inhibit the proliferation and migration of HTFs and induce their apoptosis. It was preliminary proven that ATO could inhibit the signaling pathway induced by TGF-β. It is suggested that ATO could be a basis for the treatment of the scarring of filtration channels after glaucoma surgery., (© 2023. The Author(s), under exclusive licence to Springer Nature B.V.)

Objective: To explore whether Fork head box protein M1 (FOXM1) is involved in TGF-β2-induced injury of human lens epithelial cells and its related mechanism., Methods: Human lens epithelium samples from cataract patients and healthy controls were collected. A cellular epithelial injury model was established by treating HLE-B3 cells with TGF-β2. QPCR, immunoblot assays were performed to detect the levels of FOXM1 in human cataract samples and the lens epithelial injury cell model. FOXM1 siRNA and pcDNA3.1-FOXM1 plasmids were transfected into the cells to knockdown and overexpress FOXM1, respectively. MTT and wound closure and transwell assays were performed to analyze cell proliferation and migration in HLE-B3 cells. Immunoblot assays were also conducted to detect the effects of FOXM1 on EMT, VEGFA and MAPK/ERK signaling., Results: We found high expression of FOXM1 in lens tissues of cataract patients. Silencing of FOXM1 in TGF-β2-induced HLE-B3 cells suppressed cell proliferation, migration, and the EMT process. Mechanistically, we found that downregulation of FOXM1 inhibited the VEGFA/MAPK signaling pathway in TGF-β2-induced HLE-B3 cells., Conclusion: FOXM1 promoted TGF-β2-induced injury of human lens epithelial cells (hLECs) by promoting VEGFA expression. FOXM1 could be a potential drug target for the treatment of ocular diseases., (© 2023. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Retinal pigment epithelial (RPE) cells contribute to several clinical conditions resulting in retinal fibrotic scars. Myofibroblast trans-differentiation of RPE cells is a critical step in the process of retinal fibrosis. In this study, we investigated the effects of N-oleoyl dopamine (OLDA), a newer endocannabinoid with a structure distinct from classic endocannabinoids, on TGF-β2-induced myofibroblast trans-differentiation of porcine RPE cells. Using an in vitro collagen matrix contraction assay, OLDA was found to inhibit TGF-β2 induced contraction of collagen matrices by porcine RPE cells. This effect was concentration-dependent, with significant inhibition of contraction observed at 3 μM and 10 μM. OLDA did not affect the proliferation of porcine RPE cells. Immunocytochemistry showed that at 3 μM, OLDA decreased incorporation of α-SMA in the stress fibers of TGF-β2-treated RPE cells. In addition, western blot analysis showed that 3 μM OLDA significantly downregulated TGF-β2-induced α-SMA protein expression. Taken together these results demonstrate that OLDA inhibits TGF-β induced myofibroblast trans-differentiation of RPE cells. It has been established that classic endocannabinoid such as anandamide, by activating the CB1 cannabinoid receptor, promote fibrosis in multiple organ systems. In contrast, this study demonstrates that OLDA, an endocannabinoid with a chemical structure distinct from classic endocannabinoids, inhibits myofibroblast trans-differentiation, an important step in fibrosis. Unlike classic endocannabinoids, OLDA has weak affinity for the CB1 receptor. Instead, OLDA acts on non-classic cannabinoid receptors such as GPR119, GPR6, and TRPV1. Therefore, our study indicates that the newer endocannabinoid OLDA and its non-classic cannabinoid receptors could potentially be novel therapeutic targets for treating ocular diseases involving retinal fibrosis and fibrotic pathologies in other organ systems., Competing Interests: Declaration of competing interest All authors: Nothing to declare., (Copyright © 2023 Elsevier Inc. All rights reserved.)

Background: N6-methyladenosine (m6A) participates in diverse physiological processes and contributes to many pathological conditions. Epithelial-mesenchymal transition (EMT) of retinal pigmental epithelial (RPE) cells plays an essential role in retinal-related diseases, and transforming growth factor β2 (TGF-β2) is known to induce EMT in vitro . However, the effect of TGF-β2 on m6A methylation in RPE cells is not yet known., Methods: RNA-seq and MeRIP-seq were performed to analyze changes at the mRNA and m6A levels after TGF-β2 treatment of human ARPE-19 cells. mRNA levels and total m6A levels were subsequently validated., Results: Sequencing revealed 929 differentially expressed genes and 7328 differentially methylated genes after TGF-β2 treatment. Conjoint analysis identified 290 genes related to microtubule cytoskeleton, focal adhesion, ECM-receptor interaction, cell division, cell cycle, AGE-RAGE, PI3K-Akt and cGMP-PKG pathways. Further analysis revealed that 12 EMT-related genes were altered at the mRNA and m6A levels after TGF-β2 treatment ( CALD1 , CDH2 , FN1 , MMP2 , SPARC , KRT7 , CLDN3 , ELF3 , FGF1 , LOXL2 , SHROOM3 and TGFBI ). Moreover, the total m6A level was also reduced., Conclusions: This study revealed the transcriptional profiling of m6A modification induced by TGF-β2 in RPE cells. Novel connections were discovered between m6A modification and TGF-β2-induced EMT, suggesting that m6A may play crucial roles in the EMT process., Competing Interests: The authors declare no conflict of interest., (© 2023 The Author(s). Published by IMR Press.)

Purpose: To examine the effects of prostanoid FP and EP2 receptor agonists, PGF2α and Omidenepag (OMD), respectively, on the transforming growth factor beta (TGF-β2) induced conjunctival fibrogenesis. Methods: Two-dimension (2D) and three-dimension (3D) cultures of these fibroblasts were subjected to following analyses: (1) planar proliferation evaluated by transendothelial electron resistance (TEER) measurements, (2) real-time metabolic analyses, (3) subepithelial proliferation evaluated by 3D spheroid' size and stiffness measurements, and (4) the mRNA expression of extracellular matrix (ECM) molecules and their modulators. Results: TGF-β2 induced increase in the planar proliferation was significantly decreased or enhanced by PGF2α or OMD, respectively. The proportion of oxygen consumption required to drive ATP synthesis compared with that driving proton leakage was increased by PGF2α and OMD independently with TGF-β2. In contrast, maximal mitochondrial respiration was decreased by PGF2α and OMD, and the OMD-induced effect was further enhanced by the presence of TGF-β2. In addition, the TGF-β2 dependent increase in the glycolytic capacity was cancelled by PGF2α and/or OMD. Alternatively, subepithelial proliferation, as evidenced by the stiffness of the 3D spheroids, was substantially increased by both PGF2α and OMD, and these were differently modulated by TGF-β2. The expression of several related factors as above fluctuated among the conditions for both 2D and 3D and TGF-β2 untreated or treated cultures. Conclusion: The present findings indicate that the prostanoid FP or the EP2 receptor agonist may solely and differently induce the planar and subepithelial proliferation of HconF cells and these were also modulated by TGF-β2.

In the normal eye, most of the aqueous humor drains through the trabecular meshwork (TM) and Schlemm's canal (SC). The concentration of transforming growth factor beta 2 (TGF-β2) is increased in the aqueous humor of primary open angle glaucoma patients. TGF-β2 increases outflow resistance by affecting the TM and SC, and endothelial-mesenchymal transition (EndMT) of SC cells is involved in these changes. Here, we investigated the effect of a ROCK inhibitor on TGF-β2-induced EndMT in SC cells. The ROCK inhibitor Y-27632 suppressed the TGF-β2-induced increase in the trans-endothelial electrical resistance (TER) and proliferation of SC cells. Y-27632 suppressed the expression of α-SMA, N-cadherin, and Snail, which are upregulated by TGF-β2. Moreover, TGF-β2 decreased mRNA levels of bone morphogenetic protein (BMP) 4 and increased those of the BMP antagonist gremlin (GREM1), but Y-27632 significantly suppressed these changes. Y-27632 also inhibited TGF-β2-induced phosphorylation of p-38 mitogen-activated protein kinase (MAPK). BMP4 and the p-38 MAPK inhibitor SB203580 suppressed the TGF-β2-induced TER elevation in SC cells. Moreover, SB203580 suppressed TGF-β2-induced upregulation of fibronectin, Snail, and GREM1. These results indicate that a ROCK inhibitor inhibited the TGF-β2-induced EndMT in SC cells, implying the involvement of p38 MAPK and BMP4 signaling., (© 2023. The Author(s).)

Epithelial-mesenchymal transition (EMT) of retinal pigment epithelial (RPE) cells is a key fibrosis pathogenesis in proliferative vitreoretinopathy (PVR). However, few medicines can prevent proliferative membranes and cell proliferation in the clinic. Nintedanib, a tyrosine kinase inhibitor, has been shown to prevent fibrosis and be anti-inflammatory in multiple organ fibrosis. In our study, 0.1, 1, 10 μM nintedanib was added to 20 ng/mL transforming growth factor beta 2 (TGF-β2)-induced EMT in ARPE-19 cells. Western blot and immunofluorescence assay showed that 1 μM nintedanib suppressed TGF-β2-induced E-cadherin expression decreased and Fibronectin, N-cadherin, Vimentin, and α-SMA expression increased. Quantitative real-time PCR results showed that 1 μM nintedanib decreased TGF-β2-induced increase in SNAI1, Vimentin, and Fibronectin expression and increased TGF-β2-induced decrease in E-cadherin expression. In addition, the CCK-8 assay, wound healing assay, and collagen gel contraction assay also showed that 1 μM nintedanib ameliorated TGF-β2-induced cell proliferation, migration, and contraction, respectively. These results suggested that nintedanib inhibits TGF-β2-induced EMT in ARPE-19 cells, which may be a potential pharmacological treatment for PVR., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 The Authors. Published by Elsevier Masson SAS.. All rights reserved.)

Purpose: The proliferation, migration, and epithelial-mesenchymal transition (EMT) of lens epithelial cells (LECs) are believed to be the pathological mechanisms underlying anterior subcapsular cataract (ASC). Bone morphogenetic proteins (BMPs) inhibit transforming growth factor-beta (TGF-β)-induced fibrosis in the lens. Herein, we aimed to further clarify the roles of BMP-4/BMP-7 in the progression and the underlying mechanisms of fibrotic cataract., Methods: BMP-4/BMP-7, TGF-β2, jagged-1 peptide, or DAPT were applied in a mouse injury-induced ASC model and in the human LEC cell line SRA01/04. The volume of opacity was examined by a slit lamp and determined by lens anterior capsule whole-mount immunofluorescence. Global gene expression changes were assessed by RNA sequencing, and the levels of individual mRNAs were validated by real-time PCR. Protein expression was determined by the Simple Western sample dilution buffer. Cell proliferation was examined by CCK8 and EdU assays, and cell migration was measured by Transwell and wound healing assays., Results: Anterior chamber injection of BMP-4/BMP-7 significantly suppressed subcapsular opacification formation. RNA sequencing of the mouse ASC model identified the Notch pathway as a potential mechanism involved in BMP-mediated inhibition of ASC. Consistently, BMP-4/BMP-7 selectively suppressed Notch1 and Notch3 and their downstream genes, including Hes and Hey. BMP-4/BMP-7 or DAPT suppressed cell proliferation by inducing G1 cell cycle arrest. BMP-4/BMP-7 also inhibited TGF-β2-induced cell migration and EMT by modulating the Notch pathway., Conclusions: BMP-4/BMP-7 attenuated ASC by inhibiting proliferation, migration, and EMT of LECs via modulation of the Notch pathway, thereby providing a new avenue for ASC treatment.

Preclinical and clinical findings suggest sexual dimorphism in cardiotoxicity induced by a chemotherapeutic drug, doxorubicin (DOX). However, molecular alterations leading to sex-related differential vulnerability of heart to DOX toxicity are not fully explored. In the present study, RNA sequencing in hearts of B6C3F 1 mice indicated more differentially expressed genes in males than females (224 vs. 19; ≥1.5-fold, False Discovery Rate [FDR] < 0.05) at 1 week after receiving 24 mg/kg total cumulative DOX dose that induced cardiac lesions only in males. Pathway analysis further revealed probable inactivation of cardiac apelin fibroblast signaling pathway (p = 0.00004) only in DOX-treated male mice that showed ≥1.25-fold downregulation in the transcript and protein levels of the apelin receptor, APJ. In hearts of DOX-treated females, the transcript levels of apelin (1.24-fold) and APJ (1.47-fold) were significantly (p < 0.05) increased compared to saline-treated controls. Sex-related differential DOX effect was also observed on molecular targets downstream of the apelin-APJ pathway in cardiac fibroblasts and cardiomyocytes. In cardiac fibroblasts, upregulation of Tgf-β2, Ctgf, Sphk1, Serpine1, and Timp1 (fibrosis; FDR < 0.05) in DOX-treated males and upregulation of only Tgf-β2 and Timp1 (p < 0.05) in females suggested a greater DOX toxicity in hearts of males than females. Additionally, Ryr2 and Serca2 (calcium handling; FDR < 0.05) were downregulated in conjunction with 1.35-fold upregulation of Casp12 (sarcoplasmic reticulum-mediated apoptosis; FDR < 0.05) in DOX-treated male mice. Drug effect on the transcript level of these genes was less severe in female hearts. Collectively, these data suggest a likely role of the apelin-APJ axis in sex-related differential DOX-induced cardiotoxicity in our mouse model., (Published 2022. This article is a U.S. Government work and is in the public domain in the USA.)

Background: Gastrointestinal stromal tumors (GISTs) are the prevailing sarcomas of the gastrointestinal tract. Tyrosine kinase inhibitors (TKIs) therapy, exemplified by Imatinib mesylate (IM), constitutes the established adjuvant therapy for GISTs. Nevertheless, post-treatment resistance poses a challenge that all patients must confront. The presence of tumor heterogeneity and secondary mutation mechanisms fail to account for some instances of acquired drug resistance. Certain investigations suggest a strong association between tumor drug resistance and mesenchymal stromal cells (MSC) in the tumor microenvironment, but the underlying mechanism remains obscure. Scarce research has explored the connection between GIST drug resistance and the tumor microenvironment, as well as the corresponding mechanism., Methods: Immunofluorescence and fluorescence-activated cell sorting (FACS) methodologies were employed to detect the presence of MSC in GIST samples. The investigation encompassed the examination of MSC migration towards tumor tissue and the impact of MSC on the survival of GIST cells under IM treatment. Through ELISA, western blotting, and flow cytometry analyses, it was confirmed that Transforming Growth Factor Beta 2 (TGF-β2) triggers the activation of the PI3K-AKT pathway by MSC, thereby facilitating drug resistance in GIST., Results: Our findings revealed a positive correlation between a high proportion of MSC and both GIST resistance and a poor prognosis. In vitro studies demonstrated the ability of MSC to migrate towards GIST. Additionally, MSC were observed to secrete TGF-β2, consequently activating the PI3K-AKT pathway and augmenting GIST resistance., Conclusions: Our investigation has revealed that MSC within GISTs possess the capacity to augment drug resistance, thereby highlighting their novel mechanism and offering a promising target for intervention in GIST therapy., (© 2023. The Author(s).)

Fibroblast growth factor (FGF) and transforming growth factor-beta (TGF-β) can regulate and/or dysregulate lens epithelial cell (LEC) behaviour, including proliferation, fibre differentiation, and epithelial-mesenchymal transition (EMT). Earlier studies have investigated the crosstalk between FGF and TGF-β in dictating lens cell fate, that appears to be dose dependent. Here, we tested the hypothesis that a fibre-differentiating dose of FGF differentially regulates the behaviour of lens epithelial cells undergoing TGF-β-induced EMT. Postnatal 21-day-old rat lens epithelial explants were treated with a fibre-differentiating dose of FGF-2 (200 ng/mL) and/or TGF-β2 (50 pg/mL) over a 7-day culture period. We compared central LECs (CLECs) and peripheral LECs (PLECs) using immunolabelling for changes in markers for EMT (α-SMA), lens fibre differentiation (β-crystallin), epithelial cell adhesion (β-catenin), and the cytoskeleton (alpha-tropomyosin), as well as Smad2/3- and MAPK/ERK1/2-signalling. Lens epithelial explants cotreated with FGF-2 and TGF-β2 exhibited a differential response, with CLECs undergoing EMT while PLECs favoured more of a lens fibre differentiation response, compared to the TGF-β-only-treated explants where all cells in the explants underwent EMT. The CLECs cotreated with FGF and TGF-β immunolabelled for α-SMA, with minimal β-crystallin, whereas the PLECs demonstrated strong β-crystallin reactivity and little α-SMA. Interestingly, compared to the TGF-β-only-treated explants, α-SMA was significantly decreased in the CLECs cotreated with FGF/TGF-β. Smad-dependent and independent signalling was increased in the FGF-2/TGF-β2 co-treated CLECs, that had a heightened number of cells with nuclear localisation of Smad2/3 compared to the PLECs, that in contrast had more pronounced ERK1/2-signalling over Smad2/3 activation. The current study has confirmed that FGF-2 is influential in differentially regulating the behaviour of LECs during TGF-β-induced EMT, leading to a heterogenous cell population, typical of that observed in the development of post-surgical, posterior capsular opacification (PCO). This highlights the cooperative relationship between FGF and TGF-β leading to lens pathology, providing a different perspective when considering preventative measures for controlling PCO.

Pancreatic ductal adenocarcinoma (PDAC) is one of the most aggressive cancers, with high mortality and recurrence rate. In this study, we generated a human immune system mouse model by transplanting human peripheral blood mononuclear cells into NSG-B2m mice followed by xenografting AsPC-1 cells, after which we assessed the role of transforming growth factor-β2 (TGF-β2) in T-cell-mediated anti-tumor immunity. We observed that inhibiting the TGF-β2 production by TGF-β2 antisense oligonucleotide (TASO) combined with IL-2 delays pancreatic cancer growth. Co-treatment of TASO and IL-2 had little effect on the SMAD-dependent pathway, but significantly inhibited the Akt phosphorylation and sequentially activated GSK-3β. Activation of GSK-3β by TASO subsequently suppressed β-catenin and α-SMA expression and resulted in attenuated fibrotic reactions, facilitating the infiltration of CD8 + cytotoxic T lymphocytes (CTLs) into the tumor. TGF-β2 inhibition suppressed the Foxp3 + regulatory T-cells in peripheral blood and tumors, thereby enhancing the tumoricidal effects of CTLs associated with increased granzyme B and cleaved caspase-3. Moreover, changes in the T-cell composition in peripheral blood and at the tumor site by TASO and IL-2 induced the increase of pro-inflammatory cytokines such as IFN-γ and TNF-α and the decrease of anti-inflammatory cytokines such as TGF-βs. These results indicate that the TGF-β2 inhibition by TASO combined with IL-2 enhances the T-cell mediated anti-tumor immunity against SMAD4-mutated PDAC by modulating the tumor-associated fibrosis, suggesting that TASO in combination with IL-2 may be a promising immunotherapeutic intervention for PDAC., Competing Interests: Conflict of interest statement The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 The Authors. Published by Elsevier Masson SAS.. All rights reserved.)

Purpose: To determine the role of calcipotriol, a vitamin D3 analogue, in myopia development and altering the expression of scleral α1 chain of type I collagen (Col1α1) in mice. We also aimed to identify if the signaling pathway mediating the above changes is different from the one involved in transforming growth factor β2 (TGF-β2)-mediated increases of COL1A1 in cultured human scleral fibroblasts (HSFs)., Methods: C57BL/6J mice were either intraperitoneally injected with calcipotriol and subjected to form deprivation (FD) or exposed to normal refractive development for 4 weeks. Scleral vitamin D receptor (Vdr) expression was knocked down using a Sub-Tenon's capsule injection of an adeno-associated virus-packaged short hairpin RNA (AAV8-shRNA). Refraction and biometric measurements evaluated myopia development. A combination of knockdown and induction strategies determined the relative contributions of the vitamin D3 and the TGF-β2 signaling pathways in modulating COL1A1 expression in HSFs., Results: Calcipotriol injections suppressed FD-induced myopia (FDM), but it had no significant effect on normal refractive development. AAV8-shRNA injection reduced Vdr mRNA expression by 42% and shifted the refraction toward myopia (-3.15 ± 0.99D, means ± SEM) in normal eyes. In HSFs, VDR knockdown reduced calcipotriol-induced rises in COL1A1 expression, but it did not alter TGF-β2-induced increases in COL1A1 expression. Additionally, TGF-β2 augmented calcipotriol-induced rises in COL1A1 expression. TGF-β receptor (TGFBRI/II) knockdown blunted TGF-β2-induced increases in COL1A1 expression, whereas calcipotriol-induced increases in VDR and COL1A1 expression levels were unaltered., Conclusions: Scleral vitamin D3 inhibits myopia development in mice, potentially by activating a VDR-dependent signaling pathway and increasing scleral COL1A1 expression levels.

Epithelial mesenchymal transition (EMT) plays a vital role in a variety of human diseases including proliferative vitreoretinopathy (PVR), in which retinal pigment epithelial (RPE) cells play a key part. Transcriptomic analysis showed that the phosphoinositide 3-kinase (PI3K)/Akt signaling pathway was up-regulated in human RPE cells upon treatment with transforming growth factor (TGF)-β2, a multifunctional cytokine associated with clinical PVR. Stimulation of human RPE cells with TGF-β2 induced expression of p110δ (the catalytic subunit of PI3Kδ) and activation of NFκB/p65. CRISPR-Cas9-mediated depletion of p110δ or NFκB/p65 suppressed TGF-β2-induced fibronectin expression and activation of Akt as well as migration of these cells. Intriguingly, abrogating expression of NFκB/p65 also blocked TGF-β2-induced expression of p110δ, and luciferase reporter assay indicated that TGF-β2 induced NFκB/p65 binding to the promoter of the PIK3CD that encodes p110δ. These data reveal that NFκB/p65-mediated expression of PI3Kδ is essential in human RPE cells for TGF-β2-induced EMT, uncovering hindrance of TGF-β2-induced expression of p110δ as a novel approach to inhibit PVR.

α-Synuclein (α-Syn) is implicated in Parkinson's disease (PD), a neuromotor disorder with prominent visual symptoms. The underlying cause of motor dysfunction has been studied extensively, and is attributed to the death of dopaminergic neurons mediated in part by intracellular aggregation of α-Syn. The cause of visual symptoms, however, is less clear. Neuroretinal degeneration due to the presence of aggregated α-Syn has been reported, but the evidence is controversial. Other symptoms including those arising from primary open angle glaucoma (POAG) are believed to be the side-effects of medications prescribed for PD. Here, we explored the alternative hypothesis that dysfunction of α-Syn in the anterior eye alters the interaction between the actin cytoskeleton of trabecular meshwork (TM) cells with the extracellular matrix (ECM), impairing their ability to respond to physiological changes in intraocular pressure (IOP). A similar dysfunction in neurons is responsible for impaired neuritogenesis, a characteristic feature of PD. Using cadaveric human and bovine TM tissue and primary human TM cells as models, we report two main observations: 1) α-Syn is expressed in human and bovine TM cells, and significant amounts of monomeric and oligomeric α-Syn are present in the AH, and 2) primary human TM cells and human and bovine TM tissue endocytose extracellular recombinant monomeric and oligomeric α-Syn via the prion protein (PrP C ), and upregulate fibronectin (FN) and α-smooth muscle actin (α-SMA), fibrogenic proteins implicated in POAG. Transforming growth factor β2 (TGFβ2), a fibrogenic cytokine implicated in ∼50% cases of POAG, is also increased, and so is RhoA-associated coiled-coil-containing protein kinase 1 (ROCK-1). However, silencing of α-Syn in primary human TM cells reduces FN, α-SMA, and ROCK-1 in the absence or presence of over-expressed active TGFβ2, suggesting modulation of FN and ROCK-1 independent of, or upstream of TGFβ2. These observations suggest that extracellular α-Syn modulates ECM proteins in the TM independently or via PrP C by activating the RhoA-ROCK pathway. These observations reveal a novel function of α-Syn in the anterior eye, and offer new therapeutic options., Competing Interests: Declaration and competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

Background: Pathogenesis of posterior capsular opacification (PCO) was related to pathological epithelial-mesenchymal transition (EMT) of lens epithelial cells (LECs). It has been reported that blue light could have an effect on EMT. This study aims to elucidate the role and potential mechanism of autophagy in EMT after blue light exposure in LECs., Methods: HLE-B3 cells were treated with TGF-β2 with different concentration and time to induce EMT as a model of PCO in vitro. Cells were exposed to blue light with or without TGF-β2. The expression levels of EMT-associated markers were analyzed by qRT-PCR, western blotting and cell migration ability was determined by transwell migration assay and wound healing assay. The expressions of autophagy-related proteins were analyzed by western blotting, immunofluorescence and transmission electron microscopy. Rapamycin and chloroquine were utilized in cells for autophagy activation and inhibition., Results: TGF-β2 induced autophagy activation during EMT progression in HLE-B3 cells in a dose- and time-dependent manner. Blue light exposure inhibited TGF-β2-induced EMT characterized by inhibited expression of EMT related markers and reduced migration capacity. Meanwhile, blue light exposure impaired autophagy activated by TGF-β2. Furthermore, Autophagy activation with rapamycin rescued EMT attenuated by blue light. Autophagy inhibition with chloroquine reduced TGF-β2-induced EMT in HLE-B3 cells., Conclusion: Blue light exposure had inhibited effects on TGF-β2-induced EMT in LECs through autophagy impairment, which provides a new insight on prevention and treatment of PCO., (© 2022. The Author(s).)

Purpose: Elevated transforming growth factor beta2 (TGFβ2) levels in the aqueous humor have been linked to glaucomatous outflow tissue dysfunction. Potential mediators of dysfunction are the transcriptional coactivators, Yes-associated protein (YAP) and transcriptional coactivator with PDZ binding motif (TAZ). However, the molecular underpinnings of YAP/TAZ modulation in Schlemm's canal (SC) cells under glaucomatous conditions are not well understood. Here, we investigate how TGFβ2 regulates YAP/TAZ activity in human SC (HSC) cells using biomimetic extracellular matrix hydrogels, and examine whether pharmacological YAP/TAZ inhibition would attenuate TGFβ2-induced HSC cell dysfunction., Methods: Primary HSC cells were seeded atop photo-cross-linked extracellular matrix hydrogels, made of collagen type I, elastin-like polypeptide and hyaluronic acid, or encapsulated within the hydrogels. HSC cells were induced with TGFβ2 in the absence or presence of concurrent actin destabilization or pharmacological YAP/TAZ inhibition. Changes in actin cytoskeletal organization, YAP/TAZ activity, extracellular matrix production, phospho-myosin light chain levels, and hydrogel contraction were assessed., Results: TGFβ2 significantly increased YAP/TAZ nuclear localization in HSC cells, which was prevented by either filamentous-actin relaxation or depolymerization. Pharmacological YAP/TAZ inhibition using verteporfin without light stimulation decreased fibronectin expression and actomyosin cytoskeletal rearrangement in HSC cells induced by TGFβ2. Similarly, verteporfin significantly attenuated TGFβ2-induced HSC cell-encapsulated hydrogel contraction., Conclusions: Our data provide evidence for a pathologic role of aberrant YAP/TAZ signaling in HSC cells under simulated glaucomatous conditions and suggest that pharmacological YAP/TAZ inhibition has promising potential to improve outflow tissue dysfunction.

Purpose: Transforming growth factor-beta (TGF-β) is known to influence many cell functions. In the corneal endothelium, TGF-β1 exerts contextual effects, promoting endothelial-mesenchymal transition in proliferating cells and enhancing barrier integrity in early confluent maturing cells. Herein, we studied how TGF-β isoforms participate in the formation of corneal endothelial intercellular junctions., Methods: Corneal endothelial cells (CECs) were cultured using a two-phase media approach. When CECs reached confluence, the proliferation medium was replaced with maturation medium, which was supplemented or not with TGF-β isoforms. The cell morphology (circularity index), intercellular junction protein expression, trans-endothelial electrical resistance (TEER), and permeability of 7-day postconfluent CECs were assessed. Gene transcription and signaling pathways that were activated following maturation in the presence of TGF-β2 were also studied. The beneficial effect of TGF-β2 on CEC maturation was evaluated using ex vivo corneas mounted on a corneal bioreactor., Results: The results showed increases in circularity index, membrane localization of junction-related proteins, and TEER when TGF-β isoforms were individually added during the maturation phase, and TGF-β2 was the most effective isoform. Gene profiling revealed an increase in extracellular matrix-related gene expression. In ex vivo cell adhesion experiments, CECs that were matured in the presence of TGF-β2 had a higher circularity index and cell density and exhibited cell membrane-localized junction-related protein expression at earlier time points., Conclusions: These results suggest that TGF-β2 can strengthen cell-cell and cell-substrate adhesion, which accelerates barrier integrity establishment and thus enhances CEC functionality.

The objective of the current study was to examine the effects of fibroblast growth factor-2 (FGF-2) on conjunctival fibrogenesis that was induced by the presence of transforming growth factor-β2 (TGF-β2). Two-dimension (2D) and three-dimension (3D) cultured human conjunctival fibroblasts (HconF) were used for this purpose. The 2D and 3D cultured HconF were characterized by transendothelial electrical resistance (TEER) and FITC dextran permeability measurements (2D), real-time metabolic analyses (2D), size and stiffness measurements (3D), and the mRNA expression of extracellular matrix molecules, their modulators, Tissue inhibitor of metalloproteinases and matrix metalloproteinases and ER-stress related genes (2D and 3D). FGF-2 significantly increased planar proliferation, as evidenced by TEER values and FITC dextran permeability, and shifted glucose metabolism to the energetic phenotype of 2D HconF cells, and the stiffness of the 3D spheroids, and these effects were further enhanced in the presence of TGF-β2. Analyses of the expression of possible candidate molecules involved in cell architecture and stress indicated that some additive effects caused by both factors were also recognized in some of these molecules. The findings reported herein indicate that the FGF-2, either along or additively with TGF- β2 increased the fibrogenetic changes on the plane as well as in the spatial space of HconF cells., (© 2022. The Author(s).)

Cardiac aging is characterized by progressive fibrosis. Epidemiological studies have found that advanced paternal age is associated with an increased risk of heart failure in the next generation. This study is aimed at evaluating the effect of paternal age, in the young male rat progeny, on cardiac phenotype under circulatory stress conditions. Offspring rats were obtained by mating old males (24 months old) with young females (two months old) and by mating young males (two months old) with the same young females. Hypertension was induced in old father offspring (OFO) rats and young old father (YFO) offspring rats using L-NAME (N( ω )-nitro-L-arginine methyl ester). The OFO L-NAME rats showed a high blood pressure phenotype associated with substantial cardiac hypertrophy and an exacerbation of cardiac fibrosis compared to the YFO L-NAME rats. Histological analysis of heart tissue showed an expansion of the extracellular matrix, with fibroblasts displaying markers of epicardial origin (Tcf21, Tbx18, and Wt1) in the OFO group. Moreover, western blot and protein phosphorylation antibody array identified the TGF- β 2 receptor pathway as preferentially activated in aged hearts as well as in OFO cardiac tissue treated with L-NAME. In addition, old father offspring rats (OFO+OFO L-NAME) had increased cardiac DNA methylation. In young hypertensive progeny, advanced paternal age at conception may be a risk factor for early progression towards cardiac fibrosis. An intergenerational transmission may be behind the paternal age-related cardiac remodeling in the young offspring., Competing Interests: The authors declare that there is no conflict of interest., (Copyright © 2022 A. Ismail et al.)

Posterior capsular opacification (PCO) is the major complication after cataract surgery and can result in secondary vision loss. Circular RNAs (circRNAs) are reported to play critical regulatory roles in multiple cell biological processes. The most common working mechanism of circRNAs is by acting as microRNA sponges. Here, we analyzed the role and mechanism of circRNA RNA polymerase III subunit A (POLR3A) in PCO. Cell viability was analyzed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. Cell motility was assessed by transwell and wound healing assays. Dual-luciferase reporter and RNA-pull-down assays were performed to verify the interaction between microRNA-31 (miR-31) and circ-POLR3A or thioredoxin interacting protein (TXNIP). PCO cell model was established by treating SRA01/04 cells with transforming growth factor-β2 (TGF-β2). We found that TGF-β2 enhanced SRA01/04 cell viability, migration, and invasion abilities. Circ-POLR3A expression was upregulated in PCO tissues and TGF-β2-induced SRA01/04 cells. TGF-β2 promoted the viability and motility of SRA01/04 cells largely by upregulating circ-POLR3A. Circ-POLR3A negatively regulated the miR-31 level by directly interacting with it. Circ-POLR3A absence-induced influences in TGF-β2-induced SRA01/04 cells were partly reversed by silencing miR-31. miR-31 is directly bound to the 3'-untranslated region of TXNIP. TXNIP overexpression largely attenuated miR-31 overexpression-mediated effects in TGF-β2-induced SRA01/04 cells. Circ-POLR3A could elevate the protein expression of TXNIP by sponging miR-31. Exosomes were involved in mediating the delivery of circ-POLR3A in SRA01/04 cells. In conclusion, circ-POLR3A contributed to TGF-β2-induced promotion of cell viability, migration, and invasion of SRA01/04 cells by targeting miR-31/TXNIP axis., (© 2022 Wiley Periodicals LLC.)

We report herein on the effects of all- trans retinoic acid (ATRA) on two-dimensional (2D) and three-dimensional (3D) cultures of human trabecular meshwork (HTM) cells that were treated with transforming growth factor β2 (TGF-β2). In the presence of 5 ng/mL TGF-β2, the effects of ATRA on the following were observed: (1) the barrier function of the 2D HTM monolayers, as determined by trans -endothelial electrical resistance (TEER) and fluorescein isothiocyanate (FITC) dextran permeability measurements; (2) a Seahorse cellular bio-metabolism analysis; (3) physical properties, including the size and stiffness, of 3D spheroids; (4) the gene expression of extracellular matrix (ECM) molecules, ECM modulators including tissue inhibitor of metalloproteinases (TIMPs), matrix metalloproteinases (MMPs), tight junction (TJ)-related molecules, and endoplasmic reticulum (ER)-stress-related factors. ATRA significantly inhibited the TGF-β2-induced increase in the TEER values and FITC dextran permeability of the 2D monolayers, while an ATRA monotreatment induced similar effects as TGF-β2. A real-time metabolic analysis revealed that ATRA significantly inhibited the TGF-β2-induced shift in metabolic reserve from mitochondrial oxidative phosphorylation to glycolysis in 2D HTM cells, whereas ATRA alone did not induce significant metabolic changes. In contrast, ATRA induced the formation of substantially downsized and softer 3D spheroids in the absence and presence of TGF-β2. The different effects induced by ATRA toward 2D and 3D HTM cells were also supported by the qPCR analysis of several proteins as above. The findings reported here indicate that ATRA may induce synergistic and beneficial effects on TGF-β2-treated 2D- and 3D-cultured HTM cells; those effects varied significantly between the 2D and 3D cultures.

Increased trabecular meshwork (TM) cell and tissue contractility is a driver of the reduced outflow facility and elevation of intraocular pressure (IOP) associated with primary open-angle glaucoma (POAG). Connective tissue growth factor (CTGF) is an established mediator of TM cell contractility, and its expression is increased in POAG due to transforming growth factor β 2 (TGFβ2) signalling. Inhibiting CTGF upregulation using microRNA (miRNA) mimetics could represent a new treatment option for POAG. A combination of in silico predictive tools and a literature review identified a panel of putative CTGF-targeting miRNAs. Treatment of primary human TM cells with 5 ng/mL TGFβ2 for 24 h identified miR-18a-5p as a consistent responder, being upregulated in cells from five different human donors. Transfection of primary donor TM cells with 20 nM synthetic miR-18a-5p mimic reduced TGFβ2-induced CTGF protein expression, and stable lentiviral-mediated overexpression of this miRNA reduced TGFβ2-induced contraction of collagen gels. Together, these findings identify miR-18a-5p as a mediator of the TGFβ2 response and a candidate therapeutic agent for glaucoma via its ability to inhibit CTGF-associated increased TM contractility., Competing Interests: The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses or interpretation of data; in the writing of the manuscript; or in the decision to publish the results.

Purpose: Transforming growth factor-beta 2 (TGFβ2) is a major contributor to the pathologic changes occurring in human trabecular meshwork (HTM) cells in primary open-angle glaucoma (POAG). TGFβ2 activates extracellular-signal-regulated kinase (ERK) and Rho-associated kinase (ROCK) signaling pathways, both affecting HTM cell behavior. However, exactly how these signaling pathways converge to regulate HTM cell contractility is unclear. Here, we investigated the molecular mechanism underlying TGFβ2-induced pathologic HTM cell contractility, and the crosstalk between ERK and ROCK signaling pathways with different culture substrates., Methods: Hydrogels were engineered by mixing collagen type I, elastin-like polypeptide, and hyaluronic acid, each containing photoactive functional groups, followed by UV crosslinking. Primary HTM cells were seeded atop pre-formed hydrogels for comparisons with glass, or encapsulated within the hydrogels. Changes in actin cytoskeleton, extracellular matrix (ECM) production, phospho-myosin light chain (p-MLC) levels, and hydrogel contraction were assessed., Results: HTM cell morphology and filamentous (F)-actin organization were affected by the underlying culture substrates. TGFβ2 increased HTM cell contractility via ERK and ROCK signaling pathways by differentially regulating F-actin, α-smooth muscle actin (αSMA), fibronectin (FN), and p-MLC in HTM cells. ERK inhibition, even as short as 4 h, further increased TGFβ2-induced p-MLC in HTM cells on hydrogels, but not on glass. This translated into hypercontractility of HTM cell-laden hydrogels. ROCK inhibition had precisely the opposite effects and potently relaxed the TGFβ2-induced hydrogels., Conclusions: Our data suggest that ERK signaling negatively regulates ROCK-mediated HTM cell contractility. These findings emphasize the critical importance of using tissue-mimetic ECM substrates for investigating HTM cell physiology and glaucomatous pathophysiology in vitro .

Purpose: Both epidermal growth factor receptor (EGFR) and the Yes-associated protein (YAP) signaling pathway are implicated in cell proliferation and differentiation. In this study, we explored whether the formation of proliferative vitreoretinopathy (PVR) depends on the interaction of the EGFR receptor and YAP pathway., Methods: We studied the effects of EGFR and YAP activation on retinal fibrosis in a PVR mouse model as well as in knockout mice (conditional deletion of EGFR or YAP specifically in RPE cells). Reversal and knockdown experiments were performed to induce a model of ARPE-19 cells treated with TGF-β2 in vitro. The effect of EGFR/YAP signaling blockade on the PVR-induced cell cycle and TGF-β2-induced ARPE-19 cell activation was determined., Results: The EGFR inhibitor erlotinib or conditional deletion of EGFR attenuated YAP activation and decreased the expression of YAP and its downstream target Cyr61 and of connective tissue growth factor in vivo and in vitro. EGFR-PI3K-PDK1 signaling induced by PVR promoted YAP activation and cell cycle progression. Furthermore, activated EGFR signaling bypassed RhoA to increase the protein levels of YAP, C-Myc, CyclinD1, and Bcl-xl., Conclusions: Our work highlights that EGFR-PI3K-PDK1-dependent YAP activation plays a crucial role in the formation of PVR. Targeting EGFR and the YAP pathway provides promising therapeutic treatments for PVR.

This study provides comprehensive mechanistic evidence for the role of clusterin, a stress-response secretory chaperone protein, in the modulation of intraocular pressure (IOP) by regulating the trabecular meshwork (TM) actin cytoskeleton and the extracellular matrix (ECM). The pathological stressors on TM known to elevate IOP significantly lowered clusterin protein levels indicating stress-related clusterin function loss. Small interfering RNA-mediated clusterin loss in human TM cells in vitro induced actin polymerization and stabilization via protein kinase D1, serine/threonine-protein kinase N2 (PRK2), and LIM kinase 1 (LIMK1), and the recruitment and activation of adhesome proteins including paxillin, vinculin, and integrin αV and β5. A complete loss of clusterin as seen in clusterin knockout mice (Clu -/- ) led to significant IOP elevation at postnatal Day 70. Contrarily, constitutive clusterin expression using adenovirus (AdCLU) in HTM cells resulted in the loss of actin polymerization via decreased PRK2, and LIMK1 and negative regulation of integrin αV and β5. Furthermore, we found that AdCLU treatment in HTM cells significantly decreased the ECM protein expression and distribution by significantly increasing matrix metalloprotease 2 (MMP2) activity and lowering the levels of pro-fibrotic proteins such as transforming growth factor-β2 (TGFβ2), thrombospondin-1 (TSP-1), and plasminogen activator inhibitor-1 (PAI-1). Finally, we found that HTM cells supplemented with recombinant human clusterin attenuated the pro-fibrotic effects of TGFβ2. For the first time this study demonstrates the importance of clusterin in the regulation of TM actin cytoskeleton - ECM interactions and the maintenance of IOP, thus making clusterin an interesting target to reverse elevated IOP., (© 2022 The Authors. Journal of Cellular Physiology published by Wiley Periodicals LLC.)

Transforming growth factor beta 2 (TGFβ2) is a pleiotropic growth factor that plays a vital role in smooth muscle cell (SMC) function. Our prior in vitro work has shown that SMC response can be modulated with TGFβ2 stimulation in a dose dependent manner. In particular, we have shown that increasing concentrations of TGFβ2 shift SMCs from a migratory to a synthetic behavior. In this work, electrospun compliance-matched and hypocompliant TGFβ2-eluting tissue engineered vascular grafts (TEVGs) were implanted into Sprague Dawley rats for 5 days to observe SMC population and collagen production. TEVGs were fabricated using a combined computational and experimental approach that varied the ratio of gelatin:polycaprolactone to be either compliance matched or twice as stiff as rat aorta (hypocompliant). TGFβ2 concentrations of 0, 10, 100 ng/mg were added to both graft types ( n  = 3 in each group) and imaged in vivo using ultrasound. Histological markers (SMC, macrophage, collagen, and elastin) were evaluated following explanation at 5 days. In vivo ultrasound showed that compliance-matched TEVGs became stiffer as TGFβ2 increased (100 ng/mg TEVGs compared to rat aorta, p  < 0.01), while all hypocompliant grafts remained stiffer than control rat aorta. In vivo velocity and diameter were also not significantly different than control vessels. The compliance-matched 10 ng/mg group had an elevated SMC signal (myosin heavy chain) compared to the 0 and 100 ng/mg grafts ( p  = 0.0009 and 0.0006). Compliance-matched TEVGs containing 100 ng/mg TGFβ2 had an increase in collagen production ( p  < 0.01), general immune response ( p  < 0.05), and a decrease in SMC population to the 0 and 10 ng/mg groups. All hypocompliant groups were found to be similar, suggesting a lower rate of TGFβ2 release in these TEVGs. Our results suggest that TGFβ2 can modulate in vivo SMC phenotype over an acute implantation period, which is consistent with our prior in vitro work. To the author's knowledge, this is the first in vivo rat study that evaluates a TGFβ2-eluting TEVG. Impact statement TGFβ2 affects the SMCs in a vascular graft.

Valproic acid (VPA), widely used for the treatment of neurological disorders, has anti-fibrotic activity by reducing collagen production in the postoperative conjunctiva. In this study, we investigated the capacity of VPA to modulate the postoperative collagen architecture. Histochemical examination revealed that VPA treatment was associated with the formation of thinner collagen fibers in the postoperative days 7 and 14 scars. At the micrometer scale, measurements by quantitative multiphoton microscopy indicated that VPA reduced mean collagen fiber thickness by 1.25-fold. At the nanometer scale, collagen fibril thickness and diameter measured by transmission electron microscopy were decreased by 1.08- and 1.20-fold, respectively. Moreover, delicate filamentous structures in random aggregates or closely associated with collagen fibrils were frequently observed in VPA-treated tissue. At the molecular level, VPA reduced Col1a1 but induced Matn2, Matn3, and Matn4 in the postoperative day 7 conjunctival tissue. Elevation of matrilin protein expression induced by VPA was sustained till at least postoperative day 14. In primary conjunctival fibroblasts, Matn2 expression was resistant to both VPA and TGF-β2, Matn3 was sensitive to both VPA and TGF-β2 individually and synergistically, while Matn4 was modulable by VPA but not TGF-β2. MATN2, MATN3, and MATN4 localized in close association with COL1A1 in the postoperative conjunctiva. These data indicate that VPA has the capacity to reduce collagen fiber thickness and potentially collagen assembly, in association with matrilin upregulation. These properties suggest potential VPA application for the prevention of fibrotic progression in the postoperative conjunctiva. KEY MESSAGES: VPA reduces collagen fiber and fibril thickness in the postoperative scar. VPA disrupts collagen fiber assembly in conjunctival wound healing. VPA induces MATN2, MATN3, and MATN4 in the postoperative scar., (© 2022. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

To explore the role of atorvastatin in regulating intraocular pressure (IOP) in glaucoma in vivo , and to investigate its related molecular pathway in vitro , an ocular hypertension model was generated by intravitreal injection of an adenoviral vector encoding transforming growth factor (TGF)‑β2 in the right eye of BALB/cJ mice, while the left was treated with an empty control adenovirus. To determine its anti‑intraocular hypertension role, these induced hyper‑IOP mice were gavaged with atorvastatin (20 mg/kg/day). Furthermore, extracellular matrix (ECM) factors were examined in the primary human trabecular meshwork (HTM) cells followed atorvastatin (0~200 µM) treatment in vitro . Whole genome microarray was employed to identify potential therapeutic target molecules associated with ECM regulation. Unilateral murine ocular hypertension was induced, via intravitreal injection of the adenoviral vector carrying the human TGF‑β2 gene (Ad.hTGF‑β2 226/228 ), raising IOP from 12±1.6 to 32.3±0.7 mmHg (n=6, P<0.05) at day 15, which plateaued from day 15 to 30. Atorvastatin administration from day 15 to 30 decreased IOP from 32.3±0.7 to 15.4±1.1 mmHg (n=6, P<0.05) at day 30. Additionally, atorvastatin administration changed the morphology of cultured HTM cells from an elongated and adherent morphology into rounded, less elongated and less adherent cells, accompanied with suppressed expression of ECM. Gene Ontology and Genome analysis revealed that FGD4 (FYVE, RhoGEF and PH domain containing 4) might be a key factor contributing to these changes. Our data demonstrated that atorvastatin reduced TGF‑β2‑induced ocular hypertension in vivo , perhaps via modifying cellular structure and decreasing ECM, using the FGD4 signaling pathway, as demonstrated in HTM cells. Our findings provide some useful information for the management of glaucoma, with statin therapy revealing a potential novel therapeutic pathway for glaucoma treatment.

Epithelial-melancholy transition (EMT) is the main cause of organ fibrosis and a common pathogenetic mechanism in most cataracts. This study aimed to explore the molecular mechanism of Toll-like receptor (TLR)-3 in the occurrence and development of post-cataract EMT and to provide new ideas for the prevention and treatment of posterior capsule opacification (PCO). In the presence or absence of TLR3, the human lens epithelial cell (LEC) line, SRA01/04, was treated with the transforming growth factor (TGF)-β2. Cell counting kit-8 (CCK-8) and Transwell assays were used to analyze the cell proliferation, migration, and invasion. The expression levels of proteins and RNAs were detected by western blotting and quantitative polymerase chain reaction (qPCR) experiments. Functional gain and loss studies showed that TLR3 regulates the proliferation, migration, and invasion of LECs and EMT induced by TGF-β2. Moreover, TLR3 regulates the expression of Jagged-1, Notch-1, and Notch-3 These findings indicate that TLR3 prevents the progression of lens fibrosis by targeting the Jagged-1/Notch signaling pathway to regulate the proliferation, migration, and invasion of LECs, and TGF-β2-induced EMT. Therefore, the TLR3-Jagged-1/Notch signaling axis may be a potential therapeutic target for the treatment of fibrotic cataracts.

Glaucoma is a leading cause of blindness worldwide. The loss of vision in glaucoma patients is due to optic nerve damage. The most important risk factor of glaucoma is elevated intraocular pressure (IOP) which is due to glaucomatous changes in the trabecular meshwork. Animal models, especially mouse models for ocular hypertension (OHT), are important for studying glaucoma. Published studies showed that 2.5 × 10 7  PFU adenoviral vectors expressing the biologically active form of human TGFβ2 elevate IOP in female C57BL/6J mice when they are intravitreally delivered. In this study, we found that 2.5 × 10 7  PFU adenoviral TGFβ2 vector did not elevate IOP in 3- or 5-month old male C57BL/6J mice. In contrast, 5 × 10 7  PFU of the same viral vectors elevated IOP in both 3- and 5-month old male C57BL/6J mice. Also, 5-month old mice showed earlier OHT and higher IOP compared to 3-month old mice. In summary, our data showed that age and sex play roles in adenoviral vector-mediated TGFβ2-induced OHT in C57BL/6J mice., (Copyright © 2022 Elsevier Ltd. All rights reserved.)