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AIM: To evaluate the effect of bone morphogenetic protein-6 (BMP-6) on transforming growth factor (TGF)-β2-induced epithelial-mesenchymal transition (EMT) in retinal pigment epithelium (RPE). METHODS: Adult retinal pigment epithelial cell line (ARPE-19) were randomly divided into control, TGF-β2 (5 µg/L), and BMP-6 small interfering RNA (siRNA) group. The cell morphology was observed by microscopy, and the cell migration ability were detected by Transwell chamber. The EMT-related indexes and BMP-6 protein levels were detected by Western blotting. Furthermore, a BMP-6 overexpression plasmid was constructed and RPE cells were divided into the control group, TGF-β2+empty plasmid group, BMP-6 overexpression group, and TGF-β2+BMP-6 overexpression group. The EMT-related indexes and extracellular regulated protein kinases (ERK) protein levels were detected. RESULTS: Compared with the control group, the migration of RPE cells in the TGF-β2 group was significantly enhanced. TGF-β2 increased the protein expression levels of α-smooth muscle actin (α-SMA), fibronectin and vimentin but significantly decreased the protein levels of E-cadherin and BMP-6 (P

AIM: To determine the in vitro protective effect of recombinant prominin-1 (Prominin-1)+microRNA-29b (P1M29) on N-methyl-D-aspartate (NMDA)-induced excitotoxicity in retinal ganglion cells (RGCs). METHODS: RGC-5 cells were cultured, and NMDA-induced excitotoxicity at the range of 100–800 μmol/L was assessed using the MTT assay. NMDA (800 μmol/L) was selected as the appropriate concentration for preparing the cell model. To evaluate the protective effect of P1M29 on the cell model, Prominin-1 was added at the concentration of 1–6 ng/mL for 48h, and the cell survival was investigated with/without microRNA-29b. After obtaining the appropriate concentration and time of P1M29 at 48h, real-time polymerase chain reaction (PCR) was utilized to detect the relative mRNA expression of vascular endothelial growth factor (VEGF) and transforming growth factor (TGF)-β2. Western blot detection was applied to measure the phosphorylation levels of protein kinase B (AKT) and extracellular regulated protein kinases (ERK) in RGC-5 cells after treatment with Prominin-1. Apoptosis study of the cell model was conducted by flow cytometry for estimating the anti-apoptotic effect of P1M29. Immunofluorescence analysis was used to analyze the expression levels of VEGF and TGF-β2. RESULTS: MTT cytotoxicity assays demonstrated that P1M29 group had significantly higher cell survival rate than Prominin-1 group (P

AIM: To investigate the effect of all-trans retinoic acid (ATRA) on retinol dehydrogenase 5 (RDH5), matrix metalloproteinase-2 (MMP-2) and transforming growth factor-β2 (TGF-β2) transcription levels, and the effect of RDH5 on MMP-2 and TGF-β2 in retinal pigment epithelium (RPE) cells. METHODS: After adult RPE cell line-19 (ARPE-19 cells) intervened with gradient concentrations of ATRA (0-20 μmol/L) for 24h, flow cytometry was used to detect the proliferation and apoptosis of cells in each group, and quantitative real-time polymerase chain reaction (qRT-PCR) was used to detect RDH5, MMP-2 and TGF-β2 mRNA expression. Then, after ARPE-19 cells transfected with three different siRNA targets for 48h, the RDH5 knockdown efficiency of each group and expression of MMP-2 and TGF-β2 mRNA within them was detected by qRT-PCR. RESULTS: Flow cytometry results showed that ATRA could inhibit the proliferation of RPE cells and promote the apoptosis of RPE cells, and the difference of apoptosis was statistically significant when the ATRA concentration exceeded 5 μmol/L and compared with the normal control group (P=0.027 and P=0.031, respectively). qRT-PCR results showed that ATRA could significantly inhibit the expression level of RDH5 mRNA (P

Chondrocytes rely heavily on glycolysis to maintain the metabolic homeostasis and cartilage matrix turnover. Glycolysis in chondrocytes is remodeled by diverse biochemical and biomechanical factors due to the sporty joint microenvironment. Transforming growth factor-β2 (TGF-β2), one of the most abundant TGF-β superfamily members in chondrocytes, has increasingly attracted attention in cartilage physiology and pathology. Although previous studies have emphasized the importance of TGF-β superfamily members on cell metabolism, whether and how TGF-β2 modulates glycolysis in chondrocytes remains elusive. In the current study, we investigated the effects of TGF-β2 on glycolysis in chondrocytes and explored the underlying biomechanisms. The results showed that TGF-β2 could enhance glycolysis in chondrocytes by increasing glucose consumption, up-regulating liver-type ATP-dependent 6-phosphofructokinase (Pfkl) expression, and boosting lactate production. The TGF-β2 signal entered chondrocytes via TGF-β receptor type I (TβRI), and activated p-Smad3 signaling to regulate the glycolytic pathway. Subsequent experiments employing specific inhibitors of TβRI and p-Smad3 further substantiated the role of TGF-β2 in enhancement of glycolysis via TβRI/p-Smad3 axis in chondrocytes. The results provide new understanding of the metabolic homeostasis in chondrocytes induced by TGF-β superfamily and might shed light on the prevention and treatment of related osteoarticular diseases. • TGF-β2 enhances glycolysis in chondrocytes. • TGF-β2 enhances glycolysis in chondrocytes by increasing glucose consumption, up-regulating Pfkl expression, and boosting lactate production. • TGF-β2 activates the TβRI/p-Smad3 axis to enhance glycolysis in chondrocytes. [ABSTRACT FROM AUTHOR]

Objective: To explore the changes of Pigment Epithelium-Derived Factor (PEDF), Matrix Metalloproteinase-2 (MMP-2), and Transforming Growth Factor-β2 (TGF-β2) levels in the aqueous humor of cataract patients and their correlation with disease severity., Methods: 93 cataract patients and 56 healthy subjects were study objects. PEDF, MMP-2, and TGF-β2 levels of aqueous humor were compared, and the correlation between each index and Lens Opacity Classification System (LOCS) III classification was analyzed. ROC curve was used to analyze the evaluation value of the combined detection of each index on cataract development, and logistic regression to analyze the influence of the changes of each index on cataract development., Results: PEDF levels were lower and MMP-2 and TGF-β2 levels were higher in the aqueous humor of cataract patients than in healthy subjects. PEDF levels in the aqueous humor were negatively correlated with LOCS III classification, while MMP-2 and TGF-β2 levels were positively correlated with LOCS III classification. The AUC value of combined detection was higher than that of PEDF, MMP-2, and TGF-β2 in the aqueous humor alone. MMP-2 ≥ 15.13 pg/mL, TGF-β2 ≥ 385.91 pg/mL and PEDF < 198.85 ng/mL were risk factors for cataract development., Conclusion: The changes in PEDF, MMP-2, and TGF-β2 levels in the aqueous humor of cataract patients are related to LOCS III classification. The combined detection is valuable in evaluating cataract development., Competing Interests: Declaration of competing interest The authors declare no conflicts of interest., (Copyright © 2024 HCFMUSP. Published by Elsevier España, S.L.U. All rights reserved.)

AIM: To investigate the effects of sulforaphane (SFN) on transforming growth factor (TGF)-β2 stimulated migration and epithelial-mesenchymal transition (EMT) in ARPE-19 cells. METHODS: ARPE-19 cells were cultured in the presence or absence of SFN or TGF-β2. SFN toxicity was assessed by performing a lactate dehydrogenase assay (LDH) and 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) assays, and cell migration was evaluated by Transwell migration assay. Actin stress fiber formation in ARPE-19 cells was determined using immunofluorescence analysis. Immunoblotting analysis was used to determine fibronectin and α-smooth muscle actin expressions along with the degree of Smad and Akt phosphorylation. RESULTS: SFN inhibited ARPE-19 migration. Additionally, SFN attenuated TGF-β2-induced appearance of actin stress fibers as well as fibronectin and α-smooth muscle actin expressions in these cells. SFN also hindered the TGF-β2-stimulated phosphorylation of Smad2, Smad3, and Akt. SFN showed no cytotoxicity towards ARPE-19 cells. CONCLUSION: SFN inhibits TGF-β2-stimulated migration and EMT in ARPE-19 cells, probably by preventing the establishment of actin stress fibers and Akt and Smad2/3 signaling.

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. • Silibinin counteracts TGF-β2-induced fibrotic changes in HTM cells. • Silibinin inhibits key fibrotic signaling pathways. • Silibinin as a potential drug for POAG treatment. [ABSTRACT FROM AUTHOR]

AIM: To evaluate the expression level of transforming growth factor β2(TGF-β2)in the aqueous humor and trabecular tissue of patients with primary angle-closure glaucoma(PACG)of Han and Kazakh ethnic groups and the formation of filter bleb after trabeculectomy.METHODS: Prospective research. Between July 2018 to April 2019, 46 PAGG patients(49 eyes)underwent trabeculectomy in our hospital, including 25 Han nationality(26 eyes)and 21 Kazak nationality(23 eyes). Aqueous humor and trabecular tissue were obtained through trabeculectomy. ELISA method was used to detect the content of total aqueous TGF-β2(tTGF-β2)and activated TGF-β2(aTGF-β2). And the expression of TGF-β2 in trabecular tissue was detected by immunohistochemistry and immunofluorescence.RESULTS:Immunohistochemical staining and immunofluorescence staining showed that the expression of TGF-β2 in the trabecular meshwork of Han patients was significantly higher than that of Kazakh patients. ELISA quantitative analysis showed that the aTGF-β2 content of Han and Kazak patients were 172.015±79.367pg/mL and 83.436±41.743pg/mL, respectively, the difference was statistically significant(t=4.794, Pt=2.421, P=0.026). In the comparison between preoperative and postoperative, there were time differences and interaction effects in intraocular pressure between Han and Kazak patients. At 6mo postoperatively, the formation of type Ⅰ and Ⅱ filter blebs in Han and Kazak patients was different, and the difference was statistically significant(50% vs 78%; χ2=4.841, P=0.028).CONCLUSION: The expression of TGF-β2 in the aqueous humor and trabecular meshwork of patients with PACG in the two ethnic groups is different. The expression of TGF-β2 in the aqueous humor and trabecular meshwork of Kazakh patients is significantly lower, which reduces the promotion of postoperative filtering scar Functional filtration filtration bleb.

AIM: To explore the roles of microRNA-let7c (miR-let7c) and transforming growth factor-β2 (TGF-β2) and cellular signaling during epithelial-to-mesenchymal transition (EMT) of retinal pigment epithelial cells. METHODS: Retinal pigment epithelial (ARPE-19) cells were cultured with no serum for 12h, and then with recombinant human TGF-β2 for different lengths of time. ARPE-19 cells were transfected with 1×106 TU/mL miR-let7c mimcs (miR-let7cM), miR-let7c mimcs negative control (miR-let7cMNC) and miR-let7c inhibitor (miR-let7cI) using the transfection reagent. The expression of keratin-18, vimentin, N-cadherin, IKB alpha, p65 were detected by Western blot, quantitative polymerase chain reaction and immunofluorescence. RESULTS: The expression of miR-let7c was dramatically reduced and the nuclear factor-kappa B (NF-κB) signaling pathway was activated after induction by TGF-β2 (P

Objective: To investigate the mechanism by which conbercept reverses transforming growth factor-β 2 (TGF-β 2 )-induced epithelial-mesenchymal transition (EMT) in human lens epithelial cells (HLECs)., Methods: Cultured HLEC SRA01/04 cells were treated with TGF-β 2 , conbercept, or both, and the changes in cell proliferation, apoptosis, and migration were observed using MTT assay, flow cytometry, scratch assay, and Transwell assay. Western blotting and qRT-PCR were used to detect the changes in the expression of EMT-related epithelial cell markers (E-Cadherin, α-SMA, and Snail), extracellular matrix components, and genes related to the TGF-β/Smad signaling pathway., Results: Conbercept significantly reduced TGF-β 2 -induced EMT of SRA01/04 cells, decreased the expression levels of mesenchymal and extracellular matrix markers α-SMA, Snail, collagen I, collagen IV, and FN1, and upregulated the protein and mRNA expressions of E-cadherin ( P <0.05). Transwell assay showed significantly lower cell migration ability in TGF-β 2 +conbercept group than in TGF-β 2 group ( P <0.05). Conbercept also inhibited the increase in Smad2/3 phosphorylation levels in HLEC-SRA01/04 cells with TGF-β 2 -induced EMT ( P <0.01)., Conclusion: Conbercept inhibits TGF-β 2 induced EMT by downregulating the expression of pSmad2/3 in TGF-β/Smad signaling pathway, indicating a potential therapeutic strategy against visual loss induced by posterior capsule opacification.

AIM: To investigate the mechanism of celastrol in inhibiting lens epithelial cells (LECs) fibrosis, which is the pathological basis of cataract. METHODS: Human LEC line SRA01/04 was treated with celastrol and transforming growth factor-β2 (TGF-β2). Wound-healing assay, proliferation assay, flow cytometry, real-time polymerase chain reaction (PCR), Western blot and immunocytochemical staining were used to detect the pathological changes of celastrol on LECs. Then, we cultured Sprague-Dawley rat lens in medium as a semi-in vivo model to find the function of celastrol further. RESULTS: We found that celastrol inhibited the migration of LECs, as well as proliferation (P

Abstract Background To assess bioactive transforming growth factor-β2 (TGFβ2) and secreted frizzled-related protein-1 (SFRP1) levels in aqueous humor (AH) of different types of glaucoma. Methods AH samples were obtained immediately before ophthalmic surgery with a 27-gauge needle attached to a microsyringe from 126 eyes (105 patients) divided into five groups: cataract (control), primary open-angle glaucoma (POAG), chronic angle-closure glaucoma (CACG), primary angle-closure suspects (PACS), and acute angle-closure glaucoma (AACG). Bioactive TGFβ2 and SFRP1 levels were assayed by ELISA. Results The concentration of TGFβ2 in AH of POAG patients, but not CACG, PACS, or AACG patients, was significantly higher than control eyes. However, within the AACG group, although the TGFβ2 levels in AH did not differ significantly from the control level when all AACG patients were grouped together, there were differences when the AACG patients were divided into high and normal intraocular pressure (IOP); TGFβ2 of AACG patients with high IOP (> 21 mmHg) was significantly higher than those with normal IOP. AH levels of SFRP1 were not significantly different among the groups. However, a statistical significant, negative correlation between SFRP1 and IOP existed in the POAG group. POAG patients with high IOP had lower levels of SFRP1 than those with normal IOP. In contrast, a significant, positive correlation between SFRP1 level and IOP was detected in the AACG group. AACG patients with high IOP had a higher level of SFRP1 than those with normal IOP. Concentrations of TGFβ2 and SFRP1 did not correlate significantly with each other, or with age. Conclusions These results indicate that AH levels of TGFβ2 and SFRP1 showed different profiles in different types of glaucomas.

Abstract Background By investigating that (i) all-trans retinoic acid (ATRA) affects human retinal pigment epithelium (RPE) in expressing and secreting transforming growth factor (TGF)-β2 and (ii) U73122 (phospholipase C inhibitor) and SQ22536 (adenylyl cyclase inhibitor) regulate the ATRA-induced secretion of TGF-β2 in human RPE, we sought to interpret the signaling pathway of ATRA in promoting the development of myopia. Methods The RPE cell line (D407) was treated with (i) ATRA (10 μM), (ii) U73122 (5–40 μM) and ATRA (10 μM), or (iii) SQ22536 (5–40 μM) and ATRA (10 μM). The control group was no-treated. After stimulated at 2, 4, 8, 16, 24, and 48 h, The expression and secretion of TGF-β2 was detected. Results TGF-β2 in the cytoplasm was time-dependent increased by ATRA (p 0.05). Conclusions In RPE cells, ATRA stimulates the secretion of TGF-β2 via the phospholipase C signaling pathway but not the adenylyl cyclase signaling pathway. U73122 may inhibit the promotion of ATRA in the development of myopia.

Endothelial-to-mesenchymal transition (EndMT) plays an important role in embryonic development and disease progression. Yet, how different members of the transforming growth factor-β (TGF-β) family regulate EndMT is not well understood. In the current study, we report that TGF-β2, but not bone morphogenetic protein (BMP)9, triggers EndMT in murine endothelial MS-1 and 2H11 cells. TGF-β2 strongly upregulates the transcription factor SNAIL, and the depletion of Snail is sufficient to abrogate TGF-β2-triggered mesenchymal-like cell morphology acquisition and EndMT-related molecular changes. Although SLUG is not regulated by TGF-β2, knocking out Slug also partly inhibits TGF-β2-induced EndMT in 2H11 cells. Interestingly, in addition to SNAIL and SLUG, BMP9 stimulates inhibitor of DNA binding (ID) proteins. The suppression of Id1, Id2, or Id3 expression facilitated BMP9 in inducing EndMT and, in contrast, ectopic expression of ID1, ID2, or ID3 abrogated TGF-β2-mediated EndMT. Altogether, our results show that SNAIL is critical and indispensable for TGF-β2-mediated EndMT. Although SLUG is also involved in the EndMT process, it plays less of a crucial role in it. In contrast, ID proteins are essential for maintaining endothelial traits and repressing the function of SNAIL and SLUG during the EndMT process. These data suggest that the control over endothelial vs. mesenchymal cell states is determined, at least in part, by a balance between the expression of SNAIL/SLUG and ID proteins.

Purpose: To investigate whether TGF-β2 had a different effect on the expression levels of low-density lipoprotein receptor (LDLr) in the subconjunctival fibroblasts from glaucoma patients who underwent a reoperation (RGSFs) compared with those from glaucoma patients who underwent first filtering surgery (GSFs) and control patients with cataracts (HSFs). Methods: Human subconjunctival fibroblasts were obtained from the three groups of patients. Different concentrations of TGF-β2 were added to the fibroblasts for 1, 3, and 5 days. The proliferation of the fibroblasts was determined by CCK-8 assays. Real-time PCR and western blotting were performed to analyze the mRNA and protein levels of LDLr. The uptake of DiI-labeled LDL was determined by confocal microscopy. Results: The results revealed that under TGF-β2 exposure, fibroblast proliferation was positively correlated with LDLr expression (all p <.001). The LDLr mRNA and protein levels were affected by TGF-β2 in a concentration-dependent and time-dependent manner in the RGSFs, GSFs and HSFs. The maximal expression of LDLr after TGF-β2 stimulation was consistent with the peak uptake of DiI-LDL, which was obviously highest in the RGSFs, followed by the GSFs, and then the HSFs (all p <.05). All 3 groups took up DiI-LDL in a similar time-dependent manner, with maximal uptake at 6 h following DiI-LDL incubation (all p <.05). In addition, there were significant differences in the LDLr protein levels in the subconjunctival tissues isolated from the glaucoma patients during reoperation, the glaucoma patients during first filtering surgery and the control patients at day 3 (p <.05). The highest protein expression of LDLr was observed in the RG group. Conclusion: These data suggested that the RGSFs had the highest LDLr expression and the highest peak uptake of LDL among three groups. The LDLr-drug-LDL delivery system could potentially be used for targeted delivery of antimetabolite agents in anti-scarring therapy for glaucoma patients after filtering surgery. [ABSTRACT FROM AUTHOR]

Aim: To evaluate the effect of bone morphogenetic protein-6 (BMP-6) on transforming growth factor (TGF)-β 2 -induced epithelial-mesenchymal transition (EMT) in retinal pigment epithelium (RPE)., Methods: Adult retinal pigment epithelial cell line (ARPE-19) were randomly divided into control, TGF-β 2 (5 µg/L), and BMP-6 small interfering RNA (siRNA) group. The cell morphology was observed by microscopy, and the cell migration ability were detected by Transwell chamber. The EMT-related indexes and BMP-6 protein levels were detected by Western blotting. Furthermore, a BMP-6 overexpression plasmid was constructed and RPE cells were divided into the control group, TGF-β 2 +empty plasmid group, BMP-6 overexpression group, and TGF-β 2 +BMP-6 overexpression group. The EMT-related indexes and extracellular regulated protein kinases (ERK) protein levels were detected., Results: Compared with the control group, the migration of RPE cells in the TGF-β 2 group was significantly enhanced. TGF-β 2 increased the protein expression levels of α-smooth muscle actin (α-SMA), fibronectin and vimentin but significantly decreased the protein levels of E-cadherin and BMP-6 ( P <0.05) in RPE. Similarly, the migration of RPE cells in the BMP-6 siRNA group was also significantly enhanced. BMP-6 siRNA increased the protein expression levels of α-SMA, fibronectin and vimentin but significantly decreased the protein expression levels of E-cadherin ( P <0.05). Overexpression of BMP-6 inhibited the migration of RPE cells induced by TGF-β 2 and prevented TGF-β 2 from affecting EMT-related biomarkers ( P <0.05)., Conclusion: BMP-6 prevents the EMT in RPE cells induced by TGF-β 2 , which may provide a theoretical basis for the prevention and treatment of proliferative vitreoretinopathy., Competing Interests: Conflicts of Interest: Liu X, None; Liu M, None; Ji M, None; Ma B, None; Hou YC, None; Yao XY, None; Cheng QC, None; Chen L, None., (International Journal of Ophthalmology Press.)

AIM: To investigate the role of small interference RNA interference targeted Integrin-linked kinase(ILK SiRNA)on the proliferation and apoptosis of human Tenon fibroblasts(HTFs)induced by transforming growth factor-β2 (TGF-β2). METHODS: The HTFs were identified by immunofluorescence analysis with Vimentin and keratin. HTFs with no other addiction was as normal control; H+T group: HTFs+5μg/L TGF-β2; H+T+NC SiRNA group: HTFs+5μg/L TGF-β2+50nmol/L negtive SiRNA; H+T+ILK SiRNA group: HTFs+5μg/L TGF-β2+50nmol/L ILK SiRNA. The ILK SiRNA were transfected into HTFs by lipofectamine 2000, then the cells were stimulated with 5μg/L TGF-β2. The protein expression of ILK were analyzed by Western Blot. The proliferation levels of HTFs were analyzed by CCK-8, the apoptosis of HTFs were analyzed by Hoechst 33342/PI double staining. RESULTS: The protein ILK were expressed in both TGF-β2 treated and control groups, and TGF-β2 up-regulated the expression of ILK, ILK SiRNA inhibited the protein expression of ILK(P2 treated group increased, and in ILK SiRNA group the cell proliferation rate was suppressed after exposured to ILK SiRNA for 48h(P2 stimulated group(P>0.05), compared with the normal control group, however in the ILK SiRNA group, we found lots of apoptosis cells and a few of necrotic cells(PCONCLUSION: The ILK SiRNA attenuates the abnormal proliferation of HTFs induced by TGF-β2, thereby enhancing the apoptosis of HTFs.

Background: Microparticles (MPs) are small extracellular plasma membrane particles shed by activated and apoptotic cells, which are involved in the development of atherosclerosis. Our previous study found that microRNA (miR)-19b encapsulated within endothelial MPs (EMPs) may contribute to the upregulation of circulating miR-19b in unstable angina patients. Hypoxia is involved in atherosclerosis as a critical pathological stimulus. However, it still remains unclear whether the increase of miR-19b levels in EMPs is related to hypoxia and if the effect of miR-19b – wrapped within EMPs – stimulates hypoxia on vascular endothelial cells. This study aimed to explore the changes of miR-19b in EMPs induced by hypoxia as well as their effects on endothelial cells. Methods: Human umbilical vein endothelial cells (HUVECs) were cultured in vitro and arranged to harvest EMPs in two parts: the first part consisted of EMPcontrol and EMPhypoxia and the second part included EMPvehicle, EMPNC mimic, and EMPmiR-19b mimic. Cell migration was detected by scratch migration and transwell chamber migration. Angiogenesis was assessed by tube formation assays. Furthermore, we predicted the target gene of miR-19b by bioinformatics analysis, and luciferase assay was used to verify the targeted gene of miR-19b. Data were analyzed by one-way analysis of variance. Student's t-test was used when two groups were compared. Results: Compared with EMPcontrol- and EMPhypoxia-inhibited migration of cells by scratch migration assay (80.77 ± 1.10 vs. 28.37 ± 1.40, P < 0. 001) and transwell chamber migration assay (83.00 ± 3.46 vs. 235.00 ± 16.52, P < 0.01), the number of tube formations was markedly reduced by 70% in the EMPhypoxia group (P < 0.001) in vitro analysis of HUVECs. Meanwhile, a strong inhibition of migration and tube formation of HUVECs in the presence of miR-19b-enriched EMPmiR-19b mimic was observed. This effect might be due to the delivery of miR-19b in EMPs. Transforming growth factor-β2 (TGFβ2) was predicted to be one of the target genes of miR-19b, and we further confirmed that TGFβ2 was a direct target gene of miR-19b using the luciferase assay. The expression of TGFβ2 in HUVECs was inhibited by treatment with EMPhypoxia and EMPmiR-19b mimic. Conclusions: MiR-19b in EMPs induced by hypoxia could reduce endothelial cell migration and angiogenesis by downregulating TGFβ2 expression, which may have inhibited the progression of atherosclerosis.

Age-related cataract (ARC) is a common cause of blindness in elderly individuals. Long non-coding RNA (lncRNA) myocardial infarction associated transcript (MIAT) has been reported to participate in various biological processes in a number of diseases; however, the biological mechanism underlying MIAT during ARC is not completely understood. The expression levels of MIAT, microRNA (miR)-181a and connective tissue growth factor (CTGF) were measured by reverse transcription-quantitative PCR. The protein expression levels of CTGF, α-smooth muscle actin, fibronectin, collagen type I, ERK, phosphorylated (p)-ERK, mitogen-activated protein kinase (MEK), and p-MEK were detected by western blotting. Cell viability and migration were assessed using MTT and Transwell assays, respectively. Moreover, a dual-luciferase reporter assay was performed to investigate the interaction between miR-181a and MIAT or CTGF. MIAT and CTGF were upregulated, while miR-181a was significantly downregulated in ARC tissues compared with normal tissues. MIAT or CTGF knockdown decreased cell viability, migration, epithelial-mesenchymal transition and extracellular matrix production in TGF-β2-treated SRA01/04 cells. It was hypothesized that miR-181a may be sponged by MIAT and may target CTGF. Furthermore, the miR-181a inhibitor reversed the inhibitory effect of MIAT knockdown on the progression of TGF-β2-treated SRA01/04 cells. Moreover, CTGF knockdown also reversed MIAT overexpression-mediated progression of TGF-β2-treated SRA01/04 cells. In addition, MIAT and CTGF regulated the activity of the ERK signaling pathway. The results suggested that MIAT may regulate the progression of ARC via the miR-181a/CTGF/ERK signaling pathway, which may serve as a novel therapeutic target for ARC. [ABSTRACT FROM AUTHOR]

Osteonecrosis of the femoral head (ONFH) is a pathological process that initially occurs in the weight-bearing field of the femoral head. Due to the unknown pathogenesis, this study was for the investigation of the effect of microRNA-141 (miR-141) targeting transforming growth factor-β2 (TGF-β2) on regulating osteoblast activity and osteoclast activity in steroid-induced ONFH. Tissues of ONFH and normal femoral head were collected for detecting the expression of miR-141 and TGF-β2. A rat model of ONFH was constructed by injection of hormones, and transfected with miR-141 inhibitors and overexpressed TGF-β2. The apoptosis of bone cells was detected by TUNEL staining. The expression of osteoprotegerin (OPG), osteoprotegerin ligand (OPGL), Bcl-2, Bax, Runx2, BMP2 and RANK were detected. Highly expressed miR-141 and lowly expressed TGF-β2 existed in femoral head tissues in ONFH. Inhibited miR-141 resulted in elevated TGF-β2 in femoral head tissues in ONFH of rats. Depressed miR-141 or overexpressed TGF-β2 inhibited the apoptosis of bone cells of rats with ONFH and induced elevated OPG, Bcl-2, BMP2, Runx2 and declined OPGL, Bax and RANK expression in the femoral head tissues of rats with ONFH. Altogether, we find that down-regulated miR-141 promotes osteoblast activity and inhibits osteoclast activity to ameliorate ONFH via up-regulated TGF-β2 expression. [ABSTRACT FROM AUTHOR]

AIM: To investigate the expression differences of transforming growth factor-β2 (TGF-β2), basic fibroblast growth factor (bFGF) and intercellular cell-adhesion molecule-1 (ICAM-1) in lens epithelial cells (LECs) of complicated cataract with silicone oil tamponade and age-related cataract. METHODS: Totally 150 eyes of 150 patients (aged 35 to 77y) were investigated, including 75 patients with complicated cataract after silicone oil tamponade and 75 patients with age-related cataract. The central piece of anterior capsules was collected during cataract surgery. TGF-β2, bFGF and ICAM-1 were detected in the 60 specimens of the two groups by immunohistochemistry. The expression levels of the three kinds of messenger ribonucleic acid (mRNA) were determined by real-time quantitative reverse transcription-polymerase chain reaction in the 90 specimens of the two groups. RESULTS: TGF-β2 was detected in the cytomembrane and cytoplasm of the LECs and bFGF was detected in the nucleus. ICAM-1 was positive in the cytomembrane of the LECs and the distribution of positive cells was uneven. The mRNA genes expression of the TGF-β2, bFGF and ICAM-1 was significant differences between the two groups and markedly increased in complicated cataract group (P

Ching-Long Chen,1,2 Yi-Hao Chen,1,2 Ming-Cheng Tai,2 Chang-Min Liang,2 Da-Wen Lu,1,2 Jiann-Torng Chen1,2 1Graduate Institute of Medical Science, National Defense Medical Center, Taipei, Taiwan; 2Department of Ophthalmology, Tri-Service General Hospital, National Defense Medical Center, Taipei, Taiwan Abstract: Proliferative vitreoretinopathy (PVR) is the main cause of failure following retinal detachment surgery. Transforming growth factor (TGF)-β2-induced epithelial-to-mesenchymal transition (EMT) plays an important role in the development of PVR, and EMT inhibition decreases collagen gel contraction and fibrotic membrane formation, resulting in prevention of PVR. Resveratrol is naturally found in red wine and has inhibitory effects on EMT. Resveratrol is widely used in cardioprotection, neuroprotection, chemotherapy, and antiaging therapy. The purpose of this study was to investigate the effects of resveratrol on TGF-β2-induced EMT in ARPE-19 cells in vitro. We found that resveratrol suppressed the decrease of zona occludens-1 (ZO-1) and caused an increase of alpha-smooth muscle actin expression in TGF-β2-treated ARPE-19 cells, assessed using Western blots; moreover, it also suppressed the decrease in ZO-1 and the increase of vimentin expression, observed using immunocytochemistry. Resveratrol attenuated TGF-β2-induced wound closure and cell migration in ARPE-19 cells in a scratch wound test and modified Boyden chamber assay, respectively. We also found that resveratrol reduced collagen gel contraction – assessed by collagen matrix contraction assay – and suppressed the phosphorylation of Smad2 and Smad3 in TGF-β2-treated ARPE-19 cells. These results suggest that resveratrol mediates anti-EMT effects, which could be used in the prevention of PVR. Keywords: resveratrol, epithelial-to-mesenchymal transition, proliferative vitreoretinopathy, transforming growth factor-β2, retinal pigment epithelial cells

METHODS: NF-κB p65 ASODN and NF-κB p65 missense oligodeoxynucleotide (MSODN) were designed and synthesized. Human lens epithelial cell line (HLE B-3) cells were prepared for study and divided into 7 groups. Control group was HLE B-3 cells cultured in vitro in dulbecco's modified eagle medium (DMEM). T1, T2, and T3 group were HLE B-3 cells cultured in vitro in DMEM with 10 ng/mL TGF-β2 for 6h, 12h, 24h respectively. A+T group was HLE B-3 cells cultured with 10 ng/mL TGF-β2 for 24h after transfected by NF-κB p65 ASODN for 24h. M+T group was HLE B-3 cells cultured with 10 ng/mL TGF-β2 for 24h after transfected by NF-κB p65 MSODN for 24h. The negative control group was HLE B-3 cells cultured with 10 ng/mL TGF-β2 for 24h after cultured with transfer agent (HiPerFect) for 24h. Cell morphology was observed at different time points using an inverted microscope. The expression of NF-κB p65 mRNA was detected with reverse transcription-polymerase chain reaction (RT-PCR), and the expression of α-smooth muscle actin (α-SMA) protein was assayed with ELISA. RESULTS: With the TGF-β2 stimulation prolongation, the expression of NF-κB p65 mRNA and α-SMA protein increased in T1, T2, T3 groups compared with the control group, and the difference was statistically significant (P0.05), but the difference among A+T group and other groups was statistically significant (P

Aim: To determine the in vitro protective effect of recombinant prominin-1 (Prominin-1)+microRNA-29b (P1M29) on N-methyl-D-aspartate (NMDA)-induced excitotoxicity in retinal ganglion cells (RGCs)., Methods: RGC-5 cells were cultured, and NMDA-induced excitotoxicity at the range of 100-800 µmol/L was assessed using the MTT assay. NMDA (800 µmol/L) was selected as the appropriate concentration for preparing the cell model. To evaluate the protective effect of P1M29 on the cell model, Prominin-1 was added at the concentration of 1-6 ng/mL for 48h, and the cell survival was investigated with/without microRNA-29b. After obtaining the appropriate concentration and time of P1M29 at 48h, real-time polymerase chain reaction (PCR) was utilized to detect the relative mRNA expression of vascular endothelial growth factor (VEGF) and transforming growth factor (TGF)-β2. Western blot detection was applied to measure the phosphorylation levels of protein kinase B (AKT) and extracellular regulated protein kinases (ERK) in RGC-5 cells after treatment with Prominin-1. Apoptosis study of the cell model was conducted by flow cytometry for estimating the anti-apoptotic effect of P1M29. Immunofluorescence analysis was used to analyze the expression levels of VEGF and TGF-β2., Results: MTT cytotoxicity assays demonstrated that P1M29 group had significantly higher cell survival rate than Prominin-1 group ( P <0.05). Real-time PCR data indicated that the expression levels of VEGF were significantly increased in both Prominin-1 and P1M29 groups compared NMDA and microRNA-29b group ( P <0.05), while TGF-β2 were significantly decreased in both microRNA-29b and P1M29 groups compared NMDA and Prominin-1 group ( P <0.05). Western blot results showed that both Prominin-1 and P1M29 groups significantly increased the phosphorylation levels of AKT and ERK compared to NMDA and microRNA-29b groups ( P <0.05). Flow cytometry analysis revealed that P1M29 could prevent RGC-5 cell apoptosis in the early stage of apoptosis, while immunofluorescence results showed that P1M29 group had higher expression of VEGF and lower expression of TGF-β2 with a stronger green fluorescence than NMDA group., Conclusion: Prominin-1 combined with microRNA-29b can provide a suitable therapeutic option for ameliorating NMDA-induced excitotoxicity in RGC-5 cells., (International Journal of Ophthalmology Press.)

The aim of the present study was to compare the efficacy and complications of two fixation techniques, namely dynamic hip screw (DHS) and proximal femoral nail antirotation (PFNA), in the treatment of osteoporotic femoral intertrochanteric fracture in elderly patients, and to detect changes in transforming growth factor β2 (TGF-β2) expression in the two groups. A total of 100 elderly patients with femoral intertrochanteric fracture were randomly divided into two groups that were treated with either DHS or PFNA. Peri-operative complications were observed in the patients and ELISA was used to detect TGF-β2 expression levels at 1, 7, 15 and 30 days after surgical treatment. The clinical efficacy and the incidence rate of complications at 3 months after the operation were compared. In comparison with the DHS group, the PFNA group had a shorter operation time, a lower bleeding volume and a shorter post-operative weight-bearing time. The contents of TGF-β2 in the two groups at 7 days after the operation were higher than those at 1 day, reached a peak at 15 days and had gradually decreased again at 30 days after the operation. The contents of TGF-β2 at 1, 7 and 15 days in the PFNA group were higher than those at the identical time-points in the DHS group (P<0.01). Regarding the clinical efficacy in the two groups at 3 months of post-surgery, the rate of excellent/good efficacy in the PFNA fixation group (90.0%) was higher than that in the DHS fixation group (74.0%). Of note, PFNA fixation had a higher clinical efficacy, a shorter operation time, less intra-operative trauma, a relatively faster fracture healing process and fewer complications in comparison with DHS fixation, and is therefore more suitable for treating osteoporotic femoral intertrochanteric fracture in the elderly. PFNA fixation is superior to DHS fixation, which may be associated with the higher level of TGF-β2 expression in comparison with that in the DHS group. [ABSTRACT FROM AUTHOR]

Transforming growth factor (TGF)‑β2‑induced epithelial‑mesenchymal transition (EMT) in human retinal pigment epithelium (RPE) cells has an important role in the pathophysiology of intraocular fibrotic disorders, which may cause vision impairment and blindness. Autophagy, an intracellular homeostatic pathway, contributes to the physiological and pathological processes of RPE. Furthermore, autophagy has previously been reported to function in the EMT process in numerous tissue and cell types. However, the association between autophagy and the EMT process in RPE cells has not yet been fully determined. The present study demonstrated that TGF‑β2‑treated human RPE cells (ARPE‑19 cell line) exhibited a significantly increased autophagic flux compared with control cells, as determined by western blot analysis of the protein levels of microtubule‑associated protein 1 light chain 3‑II and p62 (also termed sequestosome 1). Furthermore, it was demonstrated that autophagy activation enhanced the TGF‑β2‑induced EMT process in ARPE‑19 cells, and inhibition of autophagy by chloroquine administration attenuated TGF‑β2‑induced EMT, which was determined by analyzing the expression of mesenchymal and epithelial markers by reverse transcription‑quantitative polymerase chain reaction and/or western blotting. A transwell migration and invasion assays was also performed that demonstrated that autophagy activation by rapamycin enhanced TGF‑β2‑stimulated RPE cell migration and invasion, and inhibition of autophagy reduced TGF‑β2‑stimulated RPE cell migration and invasion. These results also demonstrated that autophagy activation enhanced the TGF‑β2‑induced EMT process in ARPE‑19 cells, and inhibition of autophagy attenuated TGF‑β2‑induced EMT. Overall, the results of the present study demonstrated that TGF‑β2‑induced EMT may be regulated by autophagy, thus indicating that autophagy may serve as a potential therapeutic target for the attenuation of EMT in intraocular fibrotic disorders. [ABSTRACT FROM AUTHOR]

The present study aims to investigate the association of transforming growth factor-β2 (TGF-β2) and matrix metalloproteinases (MMPs), MMP-2 and MMP-3, and tissue inhibitors of matrix metalloproteinases (TIMPs), TIMP-1, TIMP-2 and TIMP-3 in the aqueous humor of patients with high myopia or cataracts. The levels of TGF-β2 and MMPs/TIMPs were measured with the Luminex xMAP Technology using commercially available Milliplex xMAP kits. The association between TGF-β2 and MMPs/TIMPs levels was analyzed using the Spearman's correlation test. The levels of TGF-β2 were identified to be positively correlated with the levels of TIMP-1 and TIMP-3 (TIMP-1: r=0.334; P=0.007; TIMP-3: r=0.309; P=0.012). The levels of MMP-2, MMP-3 and TIMP-2 did not significantly correlate with TGF-β2 levels (P>0.05). A positive correlation was identified between TGF-β2 and TIMPs in the aqueous humor of human eyes with elongated axial length. It appears that TGF-β2 stimulates the expression of TIMPs as a compensatory reaction to the development of high myopia. [ABSTRACT FROM AUTHOR]

Allogeneic mesenchymal stem cells (MSCs) are a promising cell source for treating musculoskeletal injuries in horses. Effective and safe allogeneic therapy may be hindered, however, by recipient immune recognition and rejection of major histocompatibility complex (MHC)-mismatched MSCs. Development of strategies to prevent immune rejection of MHC-mismatched MSCs in vivo is necessary to enhance cell survival and potentially increase the efficacy and safety of allogeneic MSC therapy. The purposes of this study were to evaluate if transforming growth factor-β2 (TGF-β2) downregulated MHC expression on equine MSCs and to determine if TGF-β2 treatment altered the phenotype of MSCs. Equine bone marrow-derived MSCs from 12 horses were treated with 1, 5, or 10 ng/ml TGF-β2 from initial isolation until MHC expression analysis. TGF-β2-treated MSCs had reduced MHC I and MHC II surface expression compared to untreated controls. TGF-β2 treatment also partially blocked IFN-γ-induced upregulation of MHC I and MHC II. Constitutive and IFN-γ-induced MHC I and MHC II expression on equine MSCs was dynamic and highly variable, and the effect of TGF-β2 was significantly dependent on the donor animal and baseline MHC expression. TGF-β2 treatment did not appear to change morphology, surface marker expression, MSC viability, or secretion of TGF-β1, but did significantly increase the number of cells obtained from culture. These results indicate that TGF-β2 treatment has promise for regulating MHC expression on MSCs to facilitate allogeneic therapy, but further work is needed to maintain MHC stability when exposed to an inflammatory stimulus.

AIM:To investigate the morphological altering effect of transforming growth factor-β2 (TGF-β2) on untransfected human corneal endothelial cells (HCECs) in vitro.METHODS: After untransfected HCECs were treated with TGF-β2 at different concentrations, the morphology, cytoskeleton distribution, and type IV collagen expression of the cells were examined with inverted contrast light microscopy, fluorescence microscopy, immunofluorescence or Western Blot.RESULTS:TGF-β2 at the concentration of 3-15 μg/L had obviously alterative effects on HCECs morphology in dose and time-dependent manner, and 9 μg/L was the peak concentration. TGF-β2 (9 μg/L) altered HCE cell morphology after treatment for 36h, increased the mean optical density (PPCONCLUTION:TGF-β2 has obviously alterative effect on the morphology of HCECs from polygonal phenotype to enlarged spindle-shaped phenotype, in dose and time-dependence manner by inducing more, elongation and alignment of F-actin, rearrangement of microtubule and larger spread area of collagen type IV.

Objective The proliferation of Tenon’s capsule fibroblasts after glaucoma filtration surgery is a harmful element for operation failure. However, the underlying mechanism remains unclear. In this study, we evaluated the role of long non-coding RNA (lncRNA)-MEG3 in the proliferation of Glaucoma Tenon’s capsule fibroblasts (GTFs) with a model of transforming growth factor-β (TGF-β)-induced proliferation which has been used in previous studies to explore the mechanism underlying glaucoma surgery failure. Methods In vitro cell proliferation model was made by TGF-β2 (5 ng/mL)-stimulated GTFs obtained from patients with glaucoma filtration surgery, and cell viability and cell proliferation was assessed by MTT assay and [3H] thymidine incorporation assay respectively. Relative expression of MEG3 and Nrf2 mRNA was determined by quantitative real-time PCR. Nrf2 protein level was detected by western blot. Functional interaction between MEG3 and Nrf2 was examined by RNA pull down and RNA Binding Protein Immunoprecipitation (RIP). Results As detected in isolated Tenon’s capsule fibroblasts, expression level of both MEG3 and Nrf2 is decreased in GTFs compared with control fibroblasts (HTFs). Adenovirus mediated overexpression of MEG3 attenuates cell ability and cell proliferation, as well as contributed to upregulation of Nrf2 protein in TGF-β2-stimualted GTFs. MEG3 positively affects the expression of Nrf2 protein rather than Nrf2 mRNA. We found that this functional interaction of them depends on MEG3-Nrf2 protein formation by RNA pull down and RIP analysis and also is proved to be involved in TGF-β2-induced cell proliferation. Conclusion The above data suggested that the functional interaction between lncRNA-MEG3 and Nrf2 constitutes the mechanism by which TGF-β2 induces Tenon’s capsule fibroblasts proliferation after glaucoma filtration surgery via the direct binding of MEG3 to Nrf2. [ABSTRACT FROM AUTHOR]

Aim: To investigate the effect of all-trans retinoic acid (ATRA) on retinol dehydrogenase 5 (RDH5), matrix metalloproteinase-2 (MMP-2) and transforming growth factor-β2 (TGF-β2) transcription levels, and the effect of RDH5 on MMP-2 and TGF-β2 in retinal pigment epithelium (RPE) cells., Methods: After adult RPE cell line-19 (ARPE-19 cells) intervened with gradient concentrations of ATRA (0-20 µmol/L) for 24h, flow cytometry was used to detect the proliferation and apoptosis of cells in each group, and quantitative real-time polymerase chain reaction (qRT-PCR) was used to detect RDH5, MMP-2 and TGF-β2 mRNA expression. Then, after ARPE-19 cells transfected with three different siRNA targets for 48h, the RDH5 knockdown efficiency of each group and expression of MMP-2 and TGF-β2 mRNA within them was detected by qRT-PCR., Results: Flow cytometry results showed that ATRA could inhibit the proliferation of RPE cells and promote the apoptosis of RPE cells, and the difference of apoptosis was statistically significant when the ATRA concentration exceeded 5 µmol/L and compared with the normal control group ( P =0.027 and P =0.031, respectively). qRT-PCR results showed that ATRA could significantly inhibit the expression level of RDH5 mRNA ( P <0.001) and promote the expression of MMP-2 and TGF-β2 mRNA ( P =0.03 and P <0.001, respectively) in a dose-dependent manner, especially when treated with 5 µmol/L ATRA. The knockdown efficiency of RDH5 siRNA varies with different targets, among which RDH5 siRNA-435 had the highest knockdown efficiency, i.e. , more than 50% lower than that of the negative control group ( P =0.02). When RDH5 was knocked down for 48h, the results of qRT-PCR showed that the expressions of MMP-2 and TGF-β2 mRNA were significantly up-regulated ( P <0.001)., Conclusion: ATRA inhibits the expression of RDH5 and promotes MMP-2 and TGF-β2, and further RDH5 knockdown significantly upregulates MMP-2 and TGF-β2. These findings suggest that RDH5 may be involved in an epithelial-mesenchymal transition of RPE cells mediated by ATRA., (International Journal of Ophthalmology Press.)

AIM: To investigate if insulin can promote retinal pigment epithelial(RPE)cell proliferation and the secretion of transforming growth factor β2(TGF-β2). METHODS: Human RPE cells were exposed to different concentrations insulin at different times(0 to 48 hours). We used the MTS method to test the proliferation of RPE cells. And we used the ELISA method to text the secretion of TGF-β2 of RPE cells in various cases, and the Real-time PCR method to test the expressions of TGF-β2 mRNA. RESULTS: The MTS results showed that after 12 hours exposure of insulin RPE cells proliferated significantly(P

Sphingosylphosphorylcholine (SPC) is a bioactive lipid that binds to G protein-coupled-receptors and activates various signaling cascades. Here, we show that in renal mesangial cells, SPC not only activates various protein kinase cascades but also activates Smad proteins, which are classical members of the transforming growth factor-β (TGFβ) signaling pathway. Consequently, SPC is able to mimic TGFβ-mediated cell responses, such as an anti-inflammatory and a profibrotic response. Interleukin-1β-stimulated prostaglandin E2 formation is dose-dependently suppressed by SPC, which is paralleled by reduced secretory phospholipase A2 (sPLA2) protein expression and activity. This effect is due to a reduction of sPLA2 mRNA expression caused by inhibited sPLA2 promoter activity. Furthermore, SPC upregulates the profibrotic connective tissue growth factor (CTGF) protein and mRNA expression. Blocking TGFβ signaling by a TGFβ receptor kinase inhibitor causes an inhibition of SPC-stimulated Smad activation and reverses both the negative effect of SPC on sPLA2 expression and the positive effect on CTGF expression. In summary, our data show that SPC, by mimicking TGFβ, leads to a suppression of proinflammatory mediator production and stimulates a profibrotic cell response that is often the end point of an anti-inflammatory reaction. Thus, targeting SPC receptors may represent a novel therapeutic strategy to cope with inflammatory diseases.

An important role of transforming growth factor-β (TGF-β) in the development of regulatory T cells is well established. Although integrin-mediated activation of latent TGF-β1 is considered essential for the induction of regulatory T (Treg) cells by antigen-presenting cells (APCs), such an activation mechanism is not applicable to the TGF-β2 isoform, which lacks an integrin-binding RGD sequence in its latency-associated peptide. Mucosal and ocular tissues harbour TGF-β2-expressing APCs involved in Treg induction. The mechanisms that regulate TGF-β activation in such APCs remain unclear. In this study, we demonstrate that murine APCs exposed to TGF-β2 in the environment predominantly increase expression of TGF-β2. Such predominantly TGF-β2-expressing APCs use thrombospondin- 1 (TSP-1) as an integrin-independent mechanism to activate their newly synthesized latent TGF-β2 to induce Foxp3+ Treg cells both in vitro and in vivo. Expression of Treg induction by TGF-β2-expressing APCs is supported by a TSP-1 receptor, CD36, which facilitates activation of latent TGF-β during antigen presentation. Our results suggest that APC-derived TSP-1 is essential for the development of an adaptive regulatory immune response induced by TGF-β2-expressing APCs similar to those located at mucosal and ocular sites. These findings introduce the integrin-independent mechanism of TGF-β activation as an integral part of peripheral immune tolerance associated with TGF-β2-expressing tissues. [ABSTRACT FROM AUTHOR]

Purpose: Transforming growth factor-β2 (TGF-β2) is a potent inducer of posterior capsular opacification (PCO), a critical Smad-dependent event. This study was conducted to investigate the contributions of Smad2 and Smad3 to PCO development based on selective over-expression of either Smad2 or Smad3. Methods: We selectively activated the TGF-β/Smad pathway in cell lines transfected with expression plasmids containing Smad2 or Smad3. These cell lines were then analyzed to determine the individual contributions of Smad2 and Smad3 to TGF-β2 treatment response in an in vitro culture of HLE B-3 cells. The effects of Smad2 and Smad3 on cell viability were assessed by MTT and flow cytometry assay. A transwell assay was used to observe the role of Smad2 and Smad3 in the migration of HLE B-3 cells. Western blotting, real-time PCR, and immunocytofluorescence staining were performed to detect the accumulation of ECM proteins and EMT in response to selective Smad2 or Smad3 activation. The presence of soluble collagen I, and fibronectin in the culture medium supernatant were detected by ELISA. Results: Selective Smad3 activation via gene transfection enhanced TGF-β2-responsive growth inhibition and apoptosis. Transwell assay results showed that TGF-β2-induced cell migration was Smad2 dependent and Smad3 independent. Analysis by Western blot, RT-PCR and ELISA demonstrated that the determinant factor in ECM secretion was Smad3 signaling rather than Smad2 signaling. Western blot and RT-PCR showed that the loss of E-Cadherin and acquisition of α-SMA, the hallmark of epithelial-mesenchymal transition (EMT), were both reliant on Smad2 signaling. Immunocytofluorescence staining confirmed the role of Smad2 in the accumulation of α-SMA. Conclusions: Smad2 and Smad3 are both necessary for the formation of PCO. The discovery of additional TGF-β2/Smad signaling mechanisms may provide potential therapeutic targets to help combat PCO. [ABSTRACT FROM AUTHOR]

Context: Chronic exposure to ambient particulate matter pollution during childhood is associated with decreased lung function growth and increased prevalence of reported respiratory symptoms. The role of airway epithelium-derived factors has not been well determined. Objective: To determine if urban particulate matter (UPM) stimulates production of vascular endothelial growth factor (VEGF) and transforming growth factor-β2 (TGF-β2), and gene expression of mucin 5AC (MUC5AC) and interleukin-(IL)-8 by primary airway epithelial cells (AECs) obtained from carefully phenotyped healthy and atopic asthmatic school-aged children. Methods: Primary AECs from 9 healthy and 14 asthmatic children were differentiated in air--liquid interface (ALI) culture. The apical surface was exposed to UPM suspension or phosphate buffered saline (PBS) vehicle control for 96 h. VEGF and TGF-β2 concentrations in cell media at baseline, 48 and 96 h were measured via ELISA. MUC5AC and IL-8 expression by AECs at 96 h was measured via quantitative polymerase chain reaction. Results: Baseline concentrations of VEGF, but not TGF-β2, were significantly higher in asthmatic versus healthy cultures. UPM stimulated production of VEGF, but not TGF-β2, at 48 and 96 h; the magnitude of change was comparable across groups. At 96 h there was greater MUC5AC and IL-8 expression by UPM exposed compared to PBS exposed AECs. Conclusions: Induction of the pro-remodeling cytokine VEGF may be a potential mechanism by which UPM influences lung function growth in children irrespective of asthma status. Respiratory morbidity associated with UPM exposure in children may be related to increased expression of MUC5AC and IL-8. [ABSTRACT FROM AUTHOR]

Background The remodeling patterns in different types of chronic rhinosinusitis ( CRS) have rarely been compared, particularly the difference between eosinophilic and noneosinophilic CRS with nasal polyps ( CRSw NP). Moreover, whether there is a link between remodeling and inflammation remains controversial. Objective To directly compare the remodeling features of different CRS and to explore their relationship with inflammation in Chinese patients. Methods Histologic characteristics of surgical samples were analyzed in 33 controls, 72 eosinophilic and 76 noneosinophilic CRSw NP, and 72 CRS without nasal polyps ( CRSs NP) patients. Tissue samples from 38 controls, 26 eosinophilic and 26 noneosinophilic CRSw NP, and 32 CRSs NP patients were measured for mRNA and/or protein expression of profibrotic growth factors, metalloproteinases ( MMPs), tissue inhibitor of metalloproteinases ( TIMPs), hypoxia-inducible factor ( HIF)-1α, interleukin ( IL)-8, eosinophil cationic protein ( ECP), and myeloperoxidase ( MPO). Results The amount of collagen decreased, whereas the edema scores increased, from CRSs NP to noneosinophilic CRSw NP and to eosinophilic CRSw NP. Transforming growth factor ( TGF)-β2 protein levels were enhanced in CRSs NP compared with CRSw NP. TIMP-4 protein levels decreased in eosinophilic CRSw NP compared with noneosinophilic CRSw NP and CRSs NP. The number of neutrophils decreased from CRSs NP to noneosinophilic CRSw NP and to eosinophilic CRSw NP. ECP levels were only up-regulated in eosinophilic CRSw NP. ECP levels and neutrophil number correlated positively with the severity of edema and fibrosis, respectively. Neutrophils were the major sources of TGF-β2 in CRSs NP and noneosinophilic CRSw NP. Conclusion Distinct remodeling patterns are revealed for different types of CRS, particularly for eosinophilic and noneosinophilic CRSw NP. Tissue remodeling associates with inflammation in CRS. [ABSTRACT FROM AUTHOR]

Purpose: To evaluate the changes in intraocular cytokine after intravitreal bevacizumab (IVB) for uveitic cystoid macular edema (CME). Methods: The authors evaluated 9 eyes of 8 patients who underwent IVB for uveitic CME. The aqueous humorlevels of vascular endothelial growth factor (VEGF), interleukin (IL)-6, IL-8, tumor necrosis factor (TNF)-α, and transforming growth factor (TGF)-β2were measured using suspension array technology at baseline and I month after IVB. Results: The VEGF level was decreased to insignificant level (p = .008). TGF-β2 and TNF-α levels increased significantly (p = .008 and .021, respectively). IL-6 and IL-8 showed no significant change (p= .051 and.110, respectively). Conclusion: IVB resulted in a significant decrease of VEGF levels, which was associated with anatomical improvement of CME at 1 month. Compensatory elevations of proinflammatory cytokines (IL-6 and IL-8) after selective VEGF inhibition were not observed. Marked elevation of TGF-β2 after IVB seems to play an immunosuppressive role. [ABSTRACT FROM AUTHOR]

Background: Whether breast milk (BM) can protect against allergy has been studied extensively, with conflicting results. Variations in mothers' BM composition may explain some of the conflicting results. Our aim was to assess the impact of maternal allergy and probiotic intervention on BM food antibodies, transforming growth factor (TGF)-β2 and interleukin (IL)-10 and their impact on allergy development in children until the ages of 2 and 5. Methods: We measured total IgA, IgA antibodies to cow's milk (CM), casein, β-lactoglobulin and ovalbumin (OVA), TGF-β2 and IL-10 in 364 colostrum samples and 321 BM samples taken at 3 months from mothers participating in a prospective study evaluating the allergy-preventive effect of probiotics in a cohort with an increased risk for allergy. Results: CM, casein and OVA antibodies, TGF-β2 and IL-10 were detectable in most samples. Maternal allergy was associated with raised levels of IgA to casein (p = 0.04) and lower levels of TGF-β2 (p = 0.006) in mature BM. Probiotic supplementation was associated with increased IL-10 (p = 0.046) and decreased casein IgA antibodies (p = 0.027) in mature BM. High OVA IgA antibodies in colostrum were associated with the development of atopy by the age of 2, while low levels in mature BM were a significant risk factor for the development of eczema by the age of 2. TGF-β2 levels in BM constituted a risk for development of allergy by the age of 2. Conclusions: The immunologic composition of BM was only slightly affected by maternal atopy and could be altered by probiotic supplementation. Small effects of BM components on allergy development in children were evident. Copyright © 2012 S. Karger AG, Basel [ABSTRACT FROM AUTHOR]